laser scanning confocal microscopy experiments

Use our with the FV4000 microscope and achieve clear images of features and structures deep within your sample. Silicone oil has a refractive index close to that of live cells or tissue, greatly reducing the spherical aberration as compared to air, water, or other oils. With less aberration, you can achieve clearer images of your sample at depth. And silicone immersion oil does not dry out at 37 ℃ (98.6 °F), making it effective for long-term time-lapse imaging.

Take your confocal imaging to the next level and save time during data analysis. While the microscope’s signal-to-noise ratio is already very good, denoise can further reduce the noise for stunning, data-rich resonant images.

To speed up image analysis, you can pretrain an AI model so that the system can automatically segment your image data, greatly reducing the workload of this often time-consuming manual process. Then, further streamlines the analysis so that you can get your data quickly.

Improve your resonant scanner image quality by incorporating TruAI noise reduction. Although resonant scanner images are effective in capturing cellular dynamics at high speeds with low damage, this usually causes a compromise in the S/N ratio. TruAI noise reduction can improve these images without sacrificing time resolution using pre-trained neural networks based on the noise pattern of the SilVIR™ detectors. These pre-trained TruAI noise reduction algorithms can be used for on-the-fly processing as well as post processing.

Image analysis requires data extraction using segmentation techniques based on intensity value thresholds. However, this can be time-consuming and is affected by the sample conditions.

TruAI image segmentation using deep learning helps streamline image processing and minimize sample variables for more accurate image analysis. It enables you to segment very weak fluorescence images or tissues that are usually difficult to extract using the simple thresholding method.

The FV4000 microscope is engineered to be modular, making it easy for you to configure the system based on your applications and budget. You can start with a standard FV4000 and easily upgrade to multiphoton imaging by adding the as your research changes.

Application Notes

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Fluorescence Microscopy: A Concise Guide to Current Imaging Methods

Christian a. combs.

NHLBI Light Microscopy Facility, NIH, Building 10, Room 6N-309, 10 Center Dr. Bethesda, MD 20892, Phone: 301-496-3236, Fax: 301-480-1477, vog.hin.iblhn@csbmoc

Introduction

Fluorescence microscopy is a powerful tool for modern cell and molecular biologists and, in particular, neurobiologists. It provides a window into the physiology of living cells at sub-cellular levels of resolution. This allows direct visualization of the inner workings of physiological processes at a systems level context in a living cell or tissue. Fluorescence microscopy enables the study of diverse processes including protein location and associations, motility, and other phenomenon such as ion transport and metabolism. This versatility explains why thousands of papers a year are published using variants of fluorescent microscopy techniques. Many new techniques have been developed over the last decade which enable more comprehensive exploitation of light for biologic imaging. These advances include the wide-spread use of fluorescent proteins (for review see ( Shaner et al., 2005 ), the myriad number of new fluorophores available (for reviews see ( Eisenstein, 2006 ; Suzuki et al., 2007 ), the growth of the utility of the basic confocal microscope, the use of multi-photon microscopy to optically image far deeper into tissues, and the breaking of the diffraction limit for “super-resolution”. Many of the new advanced techniques are now being commercialized, opening their use to a large fraction of modern biologists. However, for the biologist inexperienced in light microscopy, matching the best technique to the biological experiment can prove to be difficult. Optimal use of fluorescence microscopy requires a basic understanding of the strengths and weaknesses of the various techniques as well an understanding of the fundamental trade-offs of the variables associated with fluorescent light collection.

In a very simple form, the ideal light microscopy experiment can be viewed as optimizing the competing properties of image resolution (in the XY or lateral direction as well as the Z or axial dimension), imaging speed (and/or acquisition time), and the amount of signal collected from the fluorescing sample ( Figure 1 ). This is bounded by the limits imposed by photo-bleaching and/or photo-toxicity. In many experiments, light levels at the diffraction limited spot (focused by the objective) can be very high. This can lead to destruction of the fluorophore and unwanted biological consequences leading to cell death or changes in the physiology of the cells or tissue being illuminated. Given these constraints, these variables are difficult to balance and require careful attention to detail and systematic empirical testing. On top of these basic variables other secondary variables also can become important such as the cost of the necessary equipment and the difficulty of the technique.

Diagram of some of the critical opposing factors in an imaging experiment. The best image is one that can balance these factors to obtain the necessary information while avoiding photobleaching or phototoxic effects. Table 1 outlines how these factors differ between the various commercialized microscopy techniques discussed in this work.

This review is intended to expand and build-upon the last review of fluorescence microscopy in this series ( Coling and Kachar, 1997 ) which provided a foundation for understanding fluorescence microscopy and the basics of immuno-labelling. In this review, knowledge of the basics of fluorescence microscopy (including wide-field microscopy) presented in that paper will be assumed. The object of this review is to provide non-expert microscopists a concise description and guide to select techniques that may have the widest appeal and that are, or will soon be, commonly used in most light microscopy core facilities or advanced biological research labs. The techniques to be reviewed encompass the most basic (such as wide-field fluorescence microscopy) to cutting edge techniques like Stimulated Emission Depletion (STED) microscopy. An emphasis will be placed on explaining the strengths and weaknesses of these techniques in terms of balancing the variables discussed in Figure 1 . A table at the end of this review will summarize this discussion and should serve as a quick guide for choosing the appropriate imaging modality from among the techniques discussed.

Wide-field Fluorescence Microscopy (WFFM) Techniques

In the most basic form (WFFM) involves exciting the fluorophore(s) in the sample of interest using a fluorescence light source, a microscope, excitation and emission filters, and an objective. The resulting emission light, of longer wavelength, is observed through the microscope eyepieces or by a camera followed by computer digitization (for reviews see ( Coling and Kachar, 1997 ; Inoue and Spring, 1997 ). Over the last decade developments in microscope and camera design, light filters, and in new techniques have greatly improved resolution and light collection for WFFM.

One of the most significant advancements has been the development of electron multiplied (EMCCD) and very low-noise, cooled CCD cameras. These cameras allow for fast detection of low-light fluorescence (EMCCD) or allow for the gradual accumulation of fluorescence signal to be integrated with little noise (cooled CCD) while still maintaining high resolution. These advancements allow for faster imaging and better contrast at low signal levels such as when the excitation light is purposefully minimized to prevent photo-bleaching or photo-toxicity.

In addition to cameras, wide-field microscopy has also been improved by better light filters, mirrors, and objectives. Commercially available filters, for instance from Chroma Inc (Rockinham, VT), (Omega Brattleboro, VT), or Semrock (Rochester, NY), have very high transmittance or reflection values enabled through new sputter-coating technologies. Also, these filters can have very sharp wavelength dependencies which enable multi-color discrimination. In the last decade, all of the major microscope companies (such as Leica, Nikon, Olympus, and Zeiss) have also improved microscope objectives. These new objectives have very flat fields (which decreases objective induced gradients in intensity across an image), long working distances with good resolving power, improved light transmission from the near UV to the infra-red, and are available in varieties that match the refractive index of the sample being imaged.

The main advantages of basic WFFM are that it is the least expensive technique, it provides good XY dimension resolution (the ability to distinguish fine detail in a specimen in the XY dimension), can provide very fast temporal resolution (particularly with the new EMCCD cameras), and in many cases requires the least amount of excitation light ( Table 1 ). XY resolution (R xy ) in wide-field microscopy is a function of the NA of the objective and the wavelength of the excitation light according to Ernst Abbe's diffraction limit expression:

Comparison of selected characteristics of commercially available microscope techniques discussed in this unit (Black boxes are best in category, grey are worst).

Were λ is the wavelength of the emission light, NA is the numerical aperture of the objective. For a high NA objective (NA 1.4) lens this limit is around 200nm. All of the techniques listed in Table 1 are approximately limited to this type of XY resolution except where super-resolution is indicated. The main disadvantage of basic WFFM is that that all of the emission light is integrated through the sample in the Z dimension. Therefore, it is difficult to tell where the fluorescence from a point in the sample originated in the Z-dimension. For samples that are thin or where Z-discrimination is not critical this may not be a limiting factor. For thick samples (such as live cells or tissues) where optical sectioning is critical or where out of focus light obscures details even in the XY plane, other techniques such as confocal or multi-photon microscopy may be more appropriate (see the following sections), although fluorescence deconvolution microscopy and structured light microscopy (SLM) are WFFM techniques that are commercially available.

Structured Light Microscopy (SLM) is a form of WFFM that enables optical sectioning. SLM works by inserting a moveable grid pattern into the optical path of the excitation light in the wide-field microscope. This produces a pattern in the images produced. The pattern is moved in the XY and even Z-dimension and the way that the detected fluorescence from the sample interacts with the pattern is then analyzed using a simple mathematical formula to create the optical sections ( Figure 2 ). Many commercial systems are available for SLM and also moveable grating patterns are available for those wishing to modify existing WFFM microscopes. In the last few years SLM has been shown capable of producing super-resolution images (≈ half the diffraction limit) ( Gustafsson, 2000 ; Schermelleh et al., 2008 ).

The basic principles of structured light microscopy are shown in panels A , B , and C. If an unknown pattern (such as a biological sample) represented in (A) is multiplied by a known regular illumination pattern (B) then a beat pattern (moiré fringes) will appear (C). The pattern is the difference between the sample and the regular illumination pattern and is course enough to be seen through the microscope even if the original pattern in the sample was not resolvable. By moving the grid and the sample in space and computationally processing the resulting data an image can be generated that has resolution at least 2× better than a conventional wide-field image. D and F. Confocal and structured light images respectively of the edge of a Hela cell showing the actin cytoskeleton. E and G show enlargements of the images in D and F. The apparent fiber diameters are 110-120nm in the structured light images compared to 280 to 300nm in the confocal image. Figure A, B, and C are reproduced with permission from ( Gustafsson, 2005 ) . Figures D and E are reproduced with permission from ( Gustafsson, 2000 ). Panels A-E were originally published in color and have been altered here to black and white.

The main advantage of SLM is that one can optically section using WFFM without the cost of expensive confocal systems and in some cases produce super-resolution images without the cost or technical complexity associated with other super resolution techniques such as Stimulated Emission Depletion Microscope (STED, discussed in a section later in the paper). The main disadvantage of this technique is that multiple images must be taken to provide optical sections. This can lead to photo-bleaching. Also the optical sectioning ability of SLM is negated if the sample moves while the different images are being captured (as would be the case in live cells).

Deconvolution fluorescence microscopy (DFM) is a form of WFFM. DFM requires prior knowledge of the point spread function (PSF, for more information on the PSF see ( Coling and Kachar, 1997 )) to allow optical sectioning. In this technique, multiple XY sections are imaged through the sample in the Z-dimension. The resulting stack of images, still lacking Z-dimension discrimination, are then analyzed using an empirical or idealized mathematical model of the PSF created by the microscope optics. This analysis results in a volumetric recreation of the sample in 3D space. DFM can also be used to enhance other techniques that are able to optically section such as confocal and two-photon microscopy (discussed below). Software for DFM is available commercially or through plug-ins for the free image analysis program ImageJ ( http://rsbweb.nih.gov/ij/ , NIH, Bethesda, MD). Although DFM is a powerful technique when used in capable hands (reviews include ( Boccacci and Bertero, 2002 ; Wallace et al., 2001 )), the novice user should be warned that optical sectioning is done indirectly using a mathematical model. Knowledge of the limitations of the model used for DFM is critical to understanding the images produced and interpretation of the data. Given these constraints it is beyond the scope of this paper and is not listed in Table 1 .

Modern Confocal Microscopy

The laser scanning confocal microscope (LSCM) remains a key piece of equipment in most imaging laboratories. Most modern LSCM systems offer a variety of advantages and are equipped with software to perform complex 3D (z-stack), 4D (z-stack over time), or even 5D (z-stack over time including spectral imaging) experiments. These microscopes often include software to facilitate data acquisition for complex methodologies such as spectral deconvolution, Fluorescence Recovery After Photobleaching (FRAP), and Fluorescence Resonance Energy Transfer (FRET). There have been many reviews written about confocal microscopy, but readers are encouraged to see the following texts for comprehensive information regarding all forms of confocal microscopy (as well as other microscopy techniques)( Hibbs, 2004 ; Pawley, 2006 ).

In the last few years, many changes have been made to improve these microscopes, but the fundamental design for optical sectioning remains largely unchanged. Figure 3 shows a simplified diagram of the light path of an LSCM. This figure shows that laser light is directed to the sample through collimating and beam steering optics, scanning mirrors (which sweep the laser beam over the field of view) and an objective that focuses the light to a diffraction limited spot in the sample. Emission light from the sample is directed to light sensing detector(s) (typically photomultiplier tubes, also known as PMT's) through a pinhole that is in the conjugate image plane to the point of focus in the sample. After spatial filtering by the pinhole, the light is sensed by the detectors, and a proportionate voltage is produced and amplified and converted into digital levels for image display and storage.

Basic architecture of a modern confocal microscope. Excitation light from laser is passed through the various collimating optics in a scan-head to either a variable dichroic mirror (Nikon, Zeiss, or Olympus) or an AOBS (Acousto-Optical Beam Splitter) (Leica) where it is reflected through the objective and focused to a point on the sample. Moveable mirrors in the scan-head before the objective scan the excitation beam over the sample, a point at a time, to build the image. Fluorescence emission light passes back through the objective, through the dichroic or AOBS to the light sensing PMT(s) (photo-multiplier tube). An aperture (pinhole) placed in the conjugate image plane to the point of focus in the sample allows only light from the focal plane to impinge on the sample and out-of-focus light is blocked. The pinhole can be made larger to allow for larger optical sectioning capability allowing more out of focus light to impinge on the PMT(s). In some models a diffraction grating or prism placed in the beam-path of the emission light can act as a variable band-pass filter or as a spectral detector if the polychromatic light is spatially spread on a number of PMTs.

At the heart of the confocal microscope is the pinhole. When placed in the conjugate image plane to the point of focus on the sample it enables optical sectioning ( Figure 3 ). The pinhole optically sections by acting as a barrier to light originating from other focal planes in the sample. Although the pinhole facilitates optical sectioning it must be understood that the axial resolution is still worse than the XY resolution (which is similar to WFFM). Axial resolution (R z ) in the confocal microscope is set by the expression:

were λ is the wavelength of the emission light, η is the refractive index of the mounting medium, and NA is the numerical aperture of the objective. At an intermediate emission wavelength when coupled with a pinhole and a high numerical aperture lens this would enable an ideal axial resolution of approximately 0.6 μm. In practical terms, axial resolution is likely to be between 0.6 and 1.0 μm. The difference between the XY and Z dimensions leads to a resolution limit that is ellipsoidal in shape in 3D space.

Most LSCM manufacturers also offer a spectral imaging option that will allow for either variable band-pass emission filtering or spectral detection on a per pixel basis. This works by placing either a diffraction grating or a prism in the light path before the PMT detector(s). In many cases, polychromatic (spectral) light is passed to a PMT array to detect a range of wavelengths either sequentially or simultaneously depending on the range of wavelengths desired. Although this option allows for more versatility and direct selection of the emission range it can come at the cost of less sensitive detection, due to the light loss in the additional optics required and in the spreading out of the light over a series of detectors to enable the spectral detection.

Table 1 shows the main advantages and disadvantages of LSCM. The main advantage of LSCM is that one may optically section while still doing complex experiments. Another advantage is the versatility of imaging capabilities and types of experiments one can perform. Most of these systems have multiple channels for multi-color, variable pinhole sizing for selecting the desired optical section thickness (usually sacrificing z-resolution for signal intensity), and software for variable ROI (region of interest) selection. Another example would be the ability to separate spectrally overlapping fluorescent proteins with spectral detection and spectral deconvolution methods. In addition, these systems, particularly in the inverted microscope configuration, can accommodate both live cells or fixed cells or tissue. Many manufacturers also provide options for small stage incubation systems. These systems allow long term experiments, particularly when coupled to automated acquisition software that enables auto-focusing algorithms in tandem with precise XYZ stage movement. Disadvantages of a modern LSCM system include the relatively low scan speed (as the beam must be swept through each pixel in the field of view), the relative price, and the amount of light impinging on the sample. The flexibility of the LSCM does mitigate many of the disadvantages, and in many instances, one can balance the imaging conditions among the variables listed in figure 1 to get the most out of a given experiment. For instance, if full-frame imaging speed is too slow to capture a physiological event in a live cell experiment one might use a small ROI to increase temporal resolution. Despite this flexibility, one concern always remains and should be considered when conducting LSCM and that is keeping the light levels low enough to avoid killing or bleaching the sample.

Another type of confocal microscopy is multipoint confocal microscopy, which includes Nipkow spinning disk, swept-field, and slit line scanning microscope systems. Each of these types of microscope systems shares the characteristic that multiple parts of the sample are imaged at once, thus increasing imaging speed. In the case of the Nipkow spinning disk and swept field systems, a sensitive camera (typically an EMCCD) is also employed. This allows for fast (usually tens to hundreds of milliseconds vs. the seconds timeframe of the LSCM), relatively low-light confocal imaging. Nipkow scanning systems have a drawback in that confocal sectioning can only occur with relatively high NA objective lenses and the pinhole size is fixed. In the case of the slit-scanning confocal microscopes, there is also a modest decrease in resolution for the X or Y dimension. All of these systems are usually less expensive than a LSCM system but can become relatively expensive if a very sensitive camera is also included.

Total Internal Reflection Fluorescence (TIRF) Microscopy

TIRF microscopy provides very good axial resolution (Z-direction, along the axis of illumination) to levels of approximately 200nm (for review see ( Toomre and Manstein, 2001 ). Not only is the axial resolution better than most other techniques but this also can greatly reduce background light (thus increasing the signal to noise ratio) that can obscure fine details. The setup for TIRF microscopy is very simple and is similar to wide-field microscopy except that it employs an oblique angle for the excitation light impinging on the sample. When the incidence angle is set to a critical angle relative to the coverslip, the excitation light is totally internally reflected ( Figure 5A ). This generates an electromagnetic field at the interface, called an evanescent wave, which excites fluorophores in nearly the same manner as conventional fluorescence excitation light. The key here is that the evanescent wave propagates only a short distance above the coverslip ( Figure 5B ). Therefore, only fluorescent molecules in close proximity to the coverslip are excited. Figure 5C shows a wide-field and a TIRF image of the fluorescence from EGFP labeled myosin in drosophila embryo hemocytes. As can be seen in the overlay, only myosin molecules in portions of the cell near the coverslip are excited, showing where the cell is closest to or touching the coverslip.

TIRF microscopy excites a shallow region above the coverslip using oblique laser excitation which is totally internally reflected and produces an evanescent wave for fluorophore excitation. A. Internal reflection. Light propagating through the periphery of a high numerical aperture objective (>1.38) is totally internally reflected by the coverslip and sent down the opposing side of the objective. B. Evanescent wave is formed w hen the critical angle θ C is reached and the light is the totally reflected. The reflection at the coverslip is due to the oblique angle of illumination and the mismatch of refraction index (n) between the oil and coverslip. Note that the evanescent wave only excites fluorophores where the cell attaches or is touching the coverslip. C and D (no D indicated) show a wide-field and TIRF image, respectively, of GFP tagged myosin V from Drosophila embryo hemocytes. Comparing the two images it is evident where the Myosin 5 is closest to the coverslip particularly in the bottom cell. Hemocytes courtesy of Amy Hong, NHLBI, NIH. Figure B was reproduced with permission from Mike Davidson (Florida State University and the National High Magnetic Field Laboratory) and the Molecular Expressions website.

As mentioned above, the decay of the evanescent wave is exponential to the distance above the coverslip. This relationship can be expressed as:

Where I(Z) represents the intensity at a given distance ( z ) from the coverslip, I(o) is the intensity at the coverslip, and d is the penetration depth. The penetration depth d decreases as the reflection angle (θ C , shown in Figure 5 ) of the incident beam grows larger. This value is also dependent on the illumination wavelength and on the refractive index of the media present at the interface. In a typical commercially available objective-based TIRF system, the reflection angle of the excitation light can be changed mechanically on a special illumination module attached to the epi-fluorescence port of a wide-field microscope. Turning of the micrometer changes the position of the beam traveling in the periphery of the objective back aperture, resulting in a change in the angle of the beam exiting the front element. Another requirement for the typical objective based TIRF system is that high numerical oil objectives (>1.4 NA) are required to generate the necessary reflection angles to establish the evanescent wave.

As is shown in Table 1 , the main advantage of TIRF is enhanced z-resolution. XY resolution benefits from a reduction in background fluorescence. Also, relative to other techniques such as confocal and two-photon, a commercial turn-key objective based TIRF microscope system is inexpensive. These systems only require a microscope, special illuminator(s), lasers, camera, and a high numerical aperture objective lens. The main disadvantage of TIRF is related to its main advantage in that only fluorophores in the first 200-300nm can be excited. This obviously limits imaging to near the coverslip but enable a z-resolution to the same depth as the penetration of the evanescent wave. Also, because the intensity of the evanescent wave decreases according to this relationship, fluorescence intensity will be a function of distance from the coverslip as well as the concentration of the fluorescent molecules. This makes quantification of depth from the coverslip or comparisons of molecular concentration difficult from TIRF images.

Two-Photon Fluorescence Microscopy (TPFM)

TPFM is a type of laser scanning microscopy that is particularly useful for imaging thick samples both in vitro and in vivo . It has been used to image hundred's of microns into tissues (for reviews see ( Diaspro et al., 2006 ; Svoboda and Yasuda, 2006 )). An example of this type of imaging is shown in figure 6C . Deep imaging is achieved by using pulsed near-infrared excitation light. Infrared light penetrates much deeper into tissue do to decreased scattering and absorption than the visible wavelengths used in standard confocal and wide-field microscopy. This technique is also good for limiting the excitation (and often photo-bleaching and possible photo-toxicity) to just one focal plane. This also has the added benefit of eliminating the need for a pinhole aperture for optical section as is used in confocal microscopy. In confocal microscopy the pinhole is used to reject out-of-focus emission light from reaching the photo-sensor (photo-multiplier tube or camera). In effect, the pinhole selects only a small portion of the emission light to achieve optical sectioning with much of the emission light “thrown away”. In TPFM it is the excitation pulse that provides the optical sectioning: therefore all of the light can be collected from the excited focal spot and none of the scattered or ballistic emission light photons need be wasted during collection. TPFM is a form of multi-photon imaging. Multi-photon imaging refers to techniques where more than one photon at a time is used to excite a fluorophore (other examples are Second Harmonic Generation (SHG) imaging and Coherent Anti-stokes Raman (CARS) microscopy. CARS and SHG are not fluorescence techniques and are outside the scope of this review).

Principals of two-photon fluorescence microscopy (TPFM). A shows a regular one-photon (e.g. confocal) and TPFM energy transitions in a Jablonski diagram. In TPFM two photons are absorbed nearly simultaneously to produce twice the energy. In this example GFP is excited with 960 nm light for TPFM and 488nm higher energy light for a confocal experiment. The emission is the same for both cases. It should be noted that TPFM absorption spectra for most fluorophores, including GFP, are very broad (in some cases hundreds of nanometers), and that the maximum is roughly a little less than twice the one-photon absorption maxima. B Two-photon fluorescence is generated in only one plane when a laser pulse train propagating through an objective is focused to a spot. Fluorescence is generated only at the point where the maximal photon crowding occurs and falls off from this plane at a rate of the fourth power from the center of the focal spot. C I n vivo TPFM image of a mouse neocortex genetically labelled with a chloride indicator. This image shows the remarkable depth to which TPFM imaging is possible. Figure C is reproduced with permission from ( Helmchen and Denk, 2005 ).

TPFM excitation is generated when a fluorophore absorbs two photons essentially simultaneously. This roughly doubles the amount of energy absorbed by the fluorescent molecules which drives their excited electrons to the same energy level as would the absorption of one photon at one-half the two-photon excitation wavelength ( Fig. 6A ). An example would be the excitation of GFP (typically excited around 488nm in a confocal experiment) at around 960nm using a pulsed laser. This is an oversimplification, as the actual TPFM absorption spectra for many fluorophores are over 100nm broad, and “selection rules” that govern the relative strengths of absorption bands vary between one-photon and two-photon excitation, but in most cases that is a good starting point for guessing where the maximal TPFM excitation occurs. The broad spectral absorption range of the typical two-photon fluorophore allows for multiple fluorophores in a sample to be excited at one wavelength simultaneously. Corresponding emission wavelengths for each fluorophores are then separated in different channels with the appropriate set of dichroic and emission filters or with spectral detection. The inherent optical sectioning ability of two-photon excitation occurs due to increased probability of two-photon absorption that occurs at the diffraction limited spot due to spatial energy crowding ( Figure 6B ). This can be seen in the equation for time averaged two-photon fluorescence intensity (I f ):

Where δ 2 is the two photon cross section for the fluorophore, η is the quantum yield of the fluorophore, P is the intensity (power) of the excitation light, τ P is the pulse width of the excitation pulses, f P is the repetition rate of the laser, NA is the numerical aperture of the objective, h and c are Plank's constant and the speed of light respectively, and λ exc is the wavelength of the excitation light ( Diaspro et al., 2006 ). In fact, the probability of two-photon absorption decreases as the fourth power of distance away from this focal region along the z-axis (NA dependence) and increases as the square of the intensity (mW of power are typically required). Another variable is the temporal pulse width, τ P , of the excitation light pulse as it reaches the sample. In general, short pulse widths (on the order of 100fs) are optimal for two-photon excitation.

Commercially available turn-key confocal systems usually consist of a modified point-scanning confocal microscope which includes a Ti:Sapphire pulsed laser (often automatically tunable over a broad range of wavelengths) and non-descanned detector channel(s). The non-descanned detector is a PMT mounted closer to the sample where the emission does not travel back through the scan-head. Since no pinhole is necessary, this configuration can be employed to reduce light losses that would occur if the emission light passed back through the scan-head. Typically, commercially available pulsed lasers produce approximately 100fs pulses at rate of 80mHz. Dispersion in the optics of the microscope and objective will lengthen these pulse widths by at least a factor of two. Recently, commercial lasers optionally include an additional unit for pre-compensation of this dispersion which can reduce the pulse length at the sample (which restores the two-photon fluorescence efficiency).

In summary, as is shown in table 1 , the main advantage of TPFM is the depth of imaging (hundreds of microns) into the sample. Another important advantage is that the bleaching and phototoxicity are limited to the focal plane, however in the focal plane the damage can be greater due to the higher light intensities (mW compared to μW in confocal) needed for TPFM. TPFM typically requires the same time frame for acquisition as traditional CLSM (on the order of 1s/frame). One big disadvantage is cost due to the need for a point-scanning microscope and a tunable pulsed Ti:Sapphire laser. This cost increases substantially if one also adds a pre-compensation unit to correct dispersion in excitation pulse lengths or selects higher power lasers whose gain enables lasing at the hard to tune to regions approaching 700nm or somewhat above 1000nm.

Stimulated Emission Depletion (STED) Fluorescence Microscopy

STED microscopy, developed by Stefan Hell and colleagues, is a relatively new super-resolution technique that has been shown to improve fluorescence microscopy resolution by approximately an order of magnitude over traditional diffraction limited techniques such as LSCM. STED can produce optical resolution to levels that were previously thought possible only with electron microscopy, and it has been used to examine key biological processes that no other technique could have examined ( Kellner et al., 2007 ; Willig et al., 2006b ). STED improves resolution by a direct reduction in the emission spot size by using a second laser beam (the STED beam) ( Figure 7 and explained below). It is important to note that the improvement in resolution is done directly without the need for post processing and mathematical redistribution of the light, as is done in deconvolution microscopy or by combining multiple images that has taken with respect to a moveable grid pattern, as is done in structured light microscopy. STED is so straight-forward that to the user this seems like a normal point-scanning technique such as LSCM or TPFM. In fact, Leica Microsystems (Wetzlar, Germany) has now commercialized this system on their current point-scanning microscope stand.

Technical principals of Stimulated Emission Depletion (STED) microscopy. A . The combination of the normal excitation beam with the phase modulated STED beam produces a sub-diffraction emission spot. The images on the right in ( A) show the doughnut pattern produced by the phase modulation of the STED beam. This beam when overlapped with the diffraction-limited excitation spot quenches emission where the beams overlap leaving the middle, sub-diffraction sized, spot for spontaneous fluorescence. B . Comparison of confocal (left) and STED (right) images reveals a marked increase in resolution by STED since more labeled particles are visualized. Scale bar, 500 nm. Figure reproduced with permission from ( Willig et al., 2006b ).

Figure 7A shows the setup for the STED technique. In the commercially available system (Leica Microsystems), two pulsed (picosecond time-domain) lasers are included. One excites the fluorophore as would normally occur in a LSCM experiment. The second, longer wavelength laser is used for the patterned quenching of the focal spot (STED beam). Specialized optics in the scanhead spatially shapes the phase of the STED beam wave-front to form a doughnut pattern (with a sharply decreased laser intensity at the central portion of the doughnut) at the focal spot. The STED wavelength must be red shifted (longer wavelength) such that it does not overlap the absorption spectrum of the fluorophore but does overlap with its emission spectrum. In this way, it quenches (forces the excited electrons into a lower energy state without giving off fluorescent light) the emission of the fluorophore in the area of the spot where the STED beam overlaps the excitation beam. This reduces the ultimate emitting region to that of the middle of the doughnut. The size of this region is related to the power of the STED beam according to the following equation:

where R is the lateral (XY) resolution, λ is the wavelength of the excitation light, NA is the numerical aperture of the objective, and ζ describes the intensity of the STED beam. Stefan Hell and colleagues have reported limiting the excitation size to around 30nm, or almost the size of a few fluorescent molecules ( Willig et al., 2007 ).

The main disadvantages of the STED approach are the cost of the system and the amount of power that impinges on the sample ( Table 1 ). The cost of the system is relatively high because two pulsed lasers are needed in addition to the already expensive laser scanning microscope system and very sensitive emission detectors (avalanche photo-diodes) required ( fast electronics and are sensitive in the far-red portion of the emission spectrum). Another disadvantage is the amount of power that is used for a STED system is high (tens of mW for the second beam). Since there is the potential for destruction of the probe or sample, only very photo-stable probes can be used. The list of probes that have been used to date are LDS721, certain ATTO dyes (AttoTech), and fluorescent proteins ( Willig et al., 2006a ). While the current commercial system can only improve lateral resolution, axial resolution can also be improved ( Hell, 2007 ). It remains unclear how deep into tissue this technique remains effective. It is likely that that the loss of coherence of the shape of the STED beam with depth is a limiting factor and will be tissue dependant. In general, STED is most effective for fixed tissue as movement or diffusion of the fluorescent marker during scanned imaging will negate any gains in resolution.

Final Considerations

Many of the microscope systems available from manufacturers have become very easy to run. While this provides easy image acquisition for many of the techniques listed in Table 1 , the danger remains that an incomplete understanding of the fundamental physics and limitations of the techniques can result in wrong, incomplete, or biased data (as recently noted in ( Pearson, 2007 ) ). It should be noted that Table 1 is only a rough guide to the commercially available fluorescence microscopy techniques and the reader should consult other sources to provide more complete understanding. There are many reviews available that are not listed here and many good web based resources such as Molecular Expressions: Exploring the World of Optics and Microscopy ( http://www.microscopy.fsu.edu/ ), The Molecular Probes Handbook ( http://probes.invitrogen.com/handbook/ ) and the Confocal Listserv ( http://listserv.acsu.buffalo.edu/cgi-bin/wa?A0=CONFOCAL&D=0&F=P&T=0 ). One final note is that one must also be able to analyze the data one has collected. There are many good commercially available image processing software packages available as well as the comprehensive free program ImageJ ( http://rsbweb.nih.gov/ij/ ). Last, the mention of any company producing microscopy related products in this work is in no way intended as an endorsement of them by the National Institutes of Health or the author.

Maximum projection reconstruction from confocal images obtained through a 65 μm stack of mouse cerebellum labeled with a combination of fluorescent proteins. In the online color version of this image one can see the unique colors produced and spectrally detected by the genetic combinations of individual fluorescent proteins which the authors label as XFP's. These colors were used to trace and map the various synaptic circuits. This figure was reproduced with permission from ( Livet et al., 2007 ). This figure was originally published in color, and can be seen online in color, but has been altered for the print version of this article in black and white.

Acknowledgments

The author wishes to thank Dr. Jay Knutson, Dr. Paul Jobsis, and Dr. Aleksandr Smirnov for useful discussions and critical reading of this manuscript. The author also thanks Ethan Taylor and Alan Hoofring for help with many of the figures in this work. The author also thanks those who permitted reprinting of some of the figures in this article. Those authors are cited in the figure legends and permission was granted by the publishers. This work was supported by the intramural research program of the National Institutes of Health and the National Heart Lung and Blood Institute.

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• Published: 19 September 2024

A cuproptosis nanocapsule for cancer radiotherapy

• You Liao   ORCID: orcid.org/0000-0003-3765-4529 1 , 2   na1 ,
• Dongmei Wang   ORCID: orcid.org/0000-0002-5995-631X 1 , 2   na1 ,
• Chenglu Gu 1 , 2 ,
• Xue Wang 1 , 2 ,
• Shuang Zhu   ORCID: orcid.org/0000-0002-3962-0587 1 , 2 ,
• Ziye Zheng 3 ,
• Fuquan Zhang 3 ,
• Junfang Yan   ORCID: orcid.org/0000-0002-0101-5947 3 &
• Zhanjun Gu   ORCID: orcid.org/0000-0003-3717-2423 1 , 2  

Nature Nanotechnology ( 2024 ) Cite this article

Metrics details

• Biomaterials
• Nanotechnology in cancer

Residual tumours that persist after radiotherapy often develop acquired radiation resistance, increasing the risk of recurrence and metastasis while providing obstacles to re-irradiation. Using samples from patients and experimental mice, we discovered that FDX1 and LIAS, key regulators of cuproptosis, were up-regulated in residual tumours following radiotherapy, conferring the increased sensitivity to cuproptosis. Therefore, we proposed a novel radiosensitization strategy focused on cuproptosis, using a copper-containing nanocapsule-like polyoxometalate as a paradigm. In an initial demonstration, we showed that the nanocapsule released copper ions in a controlled manner upon exposure to ionizing radiation. Furthermore, radiation-triggered cuproptosis overcame acquired radiation resistance even at clinically relevant radiation doses and activated a robust abscopal effect, with a 40% cure rate in both radioresistant and re-irradiation tumour models. Collectively, targeting cuproptosis is a compelling strategy for addressing acquired radiation resistance, optimizing the local antitumour effects of radiotherapy while simultaneously activating systemic antitumour immunity.

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Acknowledgements

We greatly acknowledge the financial support from the National High Level Hospital Clinical Research Funding (2022-PUMCH-B-052, to F.Z., J.Y. and Z.G.), Strategic Priority Research Program of Chinese Academy of Sciences (XDB36000000, to Z.G.), National Basic Research Program of China (2021YFA1201200 and 2020YFA0710702, to Z.G.), the Directional Institutionalized Scientific Research Project, which relies on the Beijing Synchrotron Radiation Facility of Chinese Academy of Sciences (E12982U810 to Z.G.), the National Natural Science Foundation of China (22375205, to Z.G.) and the Beijing Natural Science Foundation (2222087, to Z.G.).

Author information

These authors contributed equally: You Liao, Dongmei Wang.

Authors and Affiliations

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China

You Liao, Dongmei Wang, Chenglu Gu, Xue Wang, Shuang Zhu & Zhanjun Gu

College of Materials Science and Optoelectronic Technology, University of Chinese Academy of Sciences, Beijing, China

Department of Radiation Oncology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

Ziye Zheng, Fuquan Zhang & Junfang Yan

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Contributions

Y.L. and Z.G. conceived and designed the experiments. Y.L. and D.W. performed the experiments. Y.L. and D.W. synthesized and characterized PWCu polyoxometalates. Y.L. and D.W. performed the cell and animal experiments. C.G., X.W., S.Z. and Z.Z. assisted with the mouse radiation experiment. Y.L., D.W. and Z.G. analysed the data. Y.L. and Z.G. contributed materials and analysis tools. Z.G., J.Y. and F.Z. supervised the project. Y.L., D.W., J.Y. and Z.G. wrote the paper. Y.L. and D.W. revised the paper. All authors discussed the results and edited and approved the paper before submission.

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Correspondence to Junfang Yan or Zhanjun Gu .

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Nature Nanotechnology thanks Xin Jiang, Fudi Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended data fig. 1 up-regulation of fdx1 and lias in experimental tumours after ebrt..

a , Treatment scheme of tumours from mouse models before and after IR irradiation. b , Representative confocal images of tumour lesions before and after IR irradiation (scale bar, 50 μm). c , d , Normalized fluorescence signals of FDX1 and LIAS (CT26, n  = 5 mice per group; Hepa 1-6, n  = 3 mice per group; HeLa, n  = 3 mice per group; MDA-MB-231, n  = 4 mice per group). Data in c , d are shown as mean ± s.d. P values were calculated by two-tailed unpaired t -test ( c , d ). The experiments shown in b were repeated independently at least two times with similar results. Panel a created with BioRender.com .

Source data

Extended data fig. 2 the intratumoral distribution of pwcu after intratumoral injection..

a , 3D reconstruction images of synchrotron radiation micro-CT. b , H&E staining images (scale bar, 500 μm) and LA-ICP-TOFMS element mapping images (scale bar, 500 μm) of tumour tissue. LA-ICP-TOFMS, laser ablation-inductively coupled plasma-time of flight mass spectrometry. The experiments in b were repeated independently at least two times with similar results.

Extended Data Fig. 3 Comparison of radiosensitizing effect between HfO 2 and PWCu.

a , Schematic diagram of orthotopic 4T1-R breast cancer establishment and radiotherapy. b , c , Growth curves of tumours. Results are expressed as mean tumour volume ± s.d. with % TGI and P value relative to NT (on day 23) as determined by two-tailed unpaired t -test. n  = 10 biologically independent mice per group. CR, complete response. d , Tumour growth inhibition rates of 2 Gy × 3 IR on 4T1-R tumours. n  = 10 biologically independent mice per group. e , Representative photographs of tumours. Data in d are represented as box and whiskers (centre line, median; box limits, 25th and 75th percentiles; whiskers, minimum and maximum). The P value was calculated by two-tailed unpaired t -test ( d ).

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Supplementary Figs. 1–27, Tables 1–5 and Methods.

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Liao, Y., Wang, D., Gu, C. et al. A cuproptosis nanocapsule for cancer radiotherapy. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-024-01784-1

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Accepted : 08 August 2024

Published : 19 September 2024

DOI : https://doi.org/10.1038/s41565-024-01784-1

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Principles and practices of laser scanning confocal microscopy

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• 1 Department of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, Wisconsin 53706, USA. [email protected]
• PMID: 11131973
• DOI: 10.1385/MB:16:2:127

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging applications at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstructions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice.

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• Open access
• Published: 17 September 2024

Impact of different activation procedures on sodium hypochlorite penetration into dentinal tubules after endodontic retreatment via confocal laser scanning microscopy

• Betul Gunes 1 ,
• Kübra Yeşildal Yeter 1 &
• Yasin Altay 2  

BMC Oral Health volume  24 , Article number:  1103 ( 2024 ) Cite this article

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Infected dentinal tubules are a possible source of bacteria that are responsible for the failure of root canal treatment. Therefore, disinfection of dentinal tubules by increasing the penetration of the irrigation solution is important for success in retreatment cases. This study utilized confocal laser scanning microscopy (CLSM) to assess and compare the impact of XPR, ultrasonic irrigation (UI) and sonic activation (SA) on NaOCl penetration into dentinal tubules following endodontic retreatment.

A total of forty mandibular premolars were enrolled in this investigation. Following root canal preparation up to ProTaper X3 file (30/0.07), root canals were obturated with gutta-percha and bioceramic root canal sealer with single cone technique. The root canal filling materials were removed using ProTaper nickel-titanium rotary retreatment files until the working length was reached. The retreatment procedure was finalized using the ProTaper Next X4 (40/0.06). The teeth were divided into four groups based on the irrigation activation technique: control (conventional needle irrigation), SA, UI and XPR. During the final irrigation procedure, Rhodamine B dye was introduced to 5% NaOCl for visualization via CLSM. Subsequent to image acquisition, the maximum penetration, penetration percentage, and penetration area were calculated. Data were statistically analyzed using the Kruskal-Wallis, Friedman, and Bonferroni Dunn multiple comparison tests through R software ( p  < 0.05).

In the middle third, UI yielded a significantly higher penetration percentage than the control group ( p  < 0.05). The UI and XPR groups showed increased penetration percentages in the coronal and middle thirds compared with the apical third ( P  < 0.05). Maximum penetration was notably reduced in the apical third than in comparison with the coronal and middle thirds in all groups ( p  < 0.05). In the control, SA and XP groups, the penetration area was ranked in descending order as coronal, middle and apical ( p  < 0.05). Conversely, in the ultrasonic group, the penetration area was significantly lower in the apical third than in the middle and coronal thirds ( p  < 0.05).

Conclusions

UI enhanced the penetration percentage in the middle third of the root compared with that in the control group. XPR and SA showed no significant effect on NaOCl penetration following retreatment.

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Introduction

Endodontic treatment failures can result from various factors, including coronal leakage, missed canals and procedural errors; a systematic review was reported that the success rate of primary root canal treatment ranged between 68% and 85% [ 1 ]. In such cases, clinicians must select an appropriate retreatment option to ensure long-term success [ 2 ]. The primary choice for addressing endodontic failures is non-surgical endodontic retreatment, involving the complete removal of existing filling materials and the eradication of microorganisms from the root canal system [ 3 ]. However, the complete removal of root canal filling materials is the most challenging step in non-surgical retreatment. Residual root canal filling materials can impede the penetration of irrigation solutions and root canal sealers into dentinal tubules [ 4 , 5 , 6 , 7 ], potentially leading to incomplete healing or new inflammation. Numerous methods have been proposed for root canal retreatment, using hand and/or rotary instruments with or without different solvents. However, neither instruments nor solvents can entirely eliminate root canal filling material [ 8 , 9 , 10 ]. Therefore, supplementary procedures are recommended to reduce the remaining root canal filling material [ 11 , 12 , 13 , 14 ].

Infected dentinal tubules are a possible source of bacteria responsible for post-treatment diseases [ 15 ]. Therefore, the complete removal of root canal filling material and the disinfection of dentinal tubules are vital for long-term success in retreatment cases. Previous studies have primarily focused on the impact of supplementary procedures such as sonic, ultrasonic and laser assisted irrigation activation techniques, as well as specially designed instruments (XP Endo Finisher R), on the removal of root canal filling material from the root canal walls [ 9 , 10 , 11 , 12 , 13 , 14 ].

Ultrasonic irrigation (UI) generates cavitation and acoustic streaming that enhances cleaning of the root canal wall surface by increasing the shear stress [ 16 ]. Prior researches have investigated the effectiveness of UI in removing root canal filling materials during retreatment [ 17 , 18 , 19 ] and enhancing the penetration of irrigation solutions into dentinal tubules during primary root canal treatment [ 12 , 20 , 21 ]; these studies suggest that UI is a viable method for both procedures. Notably, irrigation activation using sonic devices operates at lower frequencies than that using UI devices. Nevertheless, existing studies examining the efficacy of sonic activation (SA) in promoting the penetration of irrigation solutions during primary root canal treatment have produced conflicting results [ 18 , 19 ].

Recently, the XP-endo Finisher R (XPR) (FKG, La Chaux-de-Fonds, Switzerland) file, crafted from a MaxWire alloy with a core diameter of ISO #30 (distinct from its precursor, the XP-endo Finisher (XPF), which has an ISO #25 core diameter) and featuring no taper, has been introduced. XPR can alter its shape by adjusting its temperature during the transition from the martensite to the austenite phase. At body temperature, XPR adopts a spoon-shaped configuration, enabling it to eliminate debris and root canal filling material from otherwise inaccessible regions without modifying the canal shape [ 22 , 23 ]. A recent systematic review and meta-analysis of in vitro studies involving XPF and XPR concluded that both files offer benefits in removing root canal filling materials from the root canal walls [ 24 ]. Notably, to the best of our knowledge, no previous study has examined the impact of XPR on the penetration of sodium hypochlorite (NaOCl) following the removal of root canal filling materials.

The objective of this in vitro study was to compare the effects of XPR, UI, and SA on the penetration of NaOCl into the dentinal tubules after retreatment using confocal laser scanning microscopy (CLSM). The null hypothesis proposed that there would be no significant difference in the penetration of NaOCl into the dentinal tubules between the groups.

Materials and methods

Ethical compliance of this in vitro study was granted by the University Ethics Committee (18/24.09.2019). The sample size was calculated based on a previous study [ 25 ] with a similar methodology in the literature. As a result of the power analysis performed using G*Power 3.1 Software (Heinrich Heine University, Düsseldorf, Germany), following these input conditions: effect size = 0.58; α err = 0.05; power = 0.95, total sample size was determined to be at least 33. Forty freshly extracted human mandibular premolars with a single root and root canal, confirmed through radiographs, fully developed roots and no signs of root cracks or resorption, were selected for this study. The teeth were preserved in a saline solution at room temperature until they underwent the experimental procedure. Prior to root canal preparation teeth were decoronated at 16 mm from the apex for standardization.

Preparation and obturation of the root canals

A #10 K-file was introduced into the root canal until the tip of the file was visible from the apex; then, 1 mm was subtracted from this length to determine the working length for each specimen. Root canals were prepared using X1, X2 and X3 ProTaper Next rotary system files (Dentsply Maillefer, Balleigues, Switzerland) following the manufacturer’s recommendations. During the preparation, the root canals were irrigated with 5% NaOCl. Final irrigation was carried out using 17% ethylenediaminetetraacetic acid (EDTA), 5% NaOCl and distilled water. After the final irrigation procedure, the root canals were dried with paper points and filled with a bioceramic-based root canal sealer (BioRoot RCS; Septogon, Saint Maur Des Fosses, France) and #25/0.06 tapered ProTaper X3 gutta-percha (Dentsply Maillefer, Ballaigues, Switzerland) using the single-cone obturation technique. Coronal access to the root canal was sealed using temporary filling material (Cavit G; 3 M Espe, Seefeld, Germany). Specimens were stored at 37 °C under 100% humidity for 2 weeks to allow the sealer to completely set.

Retreatment of root canal filling materials

After removing the temporary filling material, the coronal 3 mm of the root canal filling was removed using a Gates-Glidden #2. Retreatment of the root canal filling materials was performed using D1, D2 and D3 ProTaper retreatment files until the working length was reached. The retreatment procedure was finalized using the ProTaper Next X4 (40/0.06). No solvent was used during retreatment procedure. Root canals were irrigated with NaOCl during the retreatment procedure. The specimens were randomly divided into four groups ( n  = 10) according to the final irrigation activation procedure.

Conventional needle irrigation (control)

The root canals were irrigated with 17% EDTA and 5% NaOCl solutions for 1 min each. For irrigation, a 31-gauge closed end, two-sided vented irrigation needle (NaviTip Sideport; Ultradent Products. Inc., South Jordan, UT, USA) was placed in the canal 2 mm short of the working length and used in a back-and-forth motion.

The root canals were irrigated with 17% EDTA and activated with a sonic irrigation activation device (EndoActivator, Dentsply Advanced Endodontics, Santa Barbara, CA, USA) using a 25/0.04 polymer tip placed 2 mm short of the working length. Activation was performed for 20 s at 10,000 cpm according to the manufacturer’s recommendations. This procedure was repeated three times for a total of activation time 1 min. The same procedure was repeated to activate the 5% NaOCl solution.

Root canals were irrigated with 17% EDTA and then activated using an ultrasonic device (Newtron P5 XS BLED, Satelec/Acteon, Merignac, France) with an Irrisafe 20/0.01 tip (Satelec/Acteon, Merignac, France) placed 2 mm short of the working length. Activation was carried out for 20 s at a power setting of 6, following the manufacturer’s recommendations. This procedure was repeated three times for a total of activation time of 1 min. The same procedure was repeated to activate the 5% NaOCl solution.

Root canals were irrigated with 17% EDTA and then activated with the XP Endo Finisher R (30/0.00) retreatment file at 800 rpm and with a torque of 1 Ncm. Activation involved a 20-s up-and-down motion over a range of 7–8 mm, following the manufacturer’s recommendations. This procedure was repeated three times for a total of activation time of 1 min. The same procedure was repeated to activate the 5% NaOCl solution. Notably, the XPR file is activated at body temperature, and preparations for this group was carried out in a 37 °C water bath.

A 0.1% rhodamine fluorescent dye was added to the NaOCl solution before the irrigation procedure to enable the examination of solution penetration using CLSM. During the final irrigation procedure, each solution was applied at a volume of 2.5 ml for each specimen. At the conclusion of the final irrigation procedure, the root canals were irrigated with 2.5 ml of distilled water. All endodontic procedures were performed by an experienced endodontist.

Assessment of penetration of NaOCl

Following the final irrigation procedure, the specimens were embedded in auto-polymerizing acrylic, and ∼  1 mm thick horizontal sections were obtained at 3 mm (apical), 8 mm (middle) and 12 mm (coronal) from the apex using a diamond cutting disc (Isomed1000, Buehler, Lake Bluff, IL) under water cooling. The apical, middle and coronal thirds were scanned using CLSM at x5 magnification with a laser wave-length of 561 nm (LSM 800; Zeiss, Jena, Germany) (Fig. 1 ). Subsequently, after acquiring the images, the following three parameters were calculated for each third.

Representative confocal laser scanning images of the coronal, middle and apical root thirds of each experimental group

1) Maximum penetration depth : This parameter was defined as the distance from the root canal wall to the deepest point of penetration at four standardized points with 90° angles. The total value of these four measurements was divided by 4 to calculate the mean maximum penetration depth (μm) [ 26 ] (Fig.  2 a).

( a ) Measurement of maximum dentinal tubule penetration at four standardized points. ( b ) Measurement of sealer penetration (yellow lines) and circumference of the root canal wall (white line) to calculate percentage of sealer. ( c ) Measurement of the area of penetrations

2) Penetration percentage : The penetration percentage was calculated by dividing the length to which the irrigation solution penetrated the dentinal tubules along the root canal walls by the circumference of the root canal wall and multiplying this result by 100 (as %) [ 25 ] (Fig.  2 b).

3) Area of penetration : To calculate the area of penetration, the total area covered by penetration of the irrigation solution was measured (μm 2 ) [ 27 ] (Fig.  2 c).

The investigator who performed the measurements on the CLSM images was blinded to the treatment groups. The images were analyzed using the Zeiss Zen software 2.3 (Carl Zeiss).

Statistical analysis

The study was conducted with an average of 10 replicates, following a 4 × 3 factorial experimental design. The analysis aimed to determine whether a statistical difference existed between the means of the activation methods (Control, SA, UI and XPR) and the root thirds (apical, middle and coronal). Statistical analysis was performed using the Anderson Darling test to assess normal distribution and Levene test to evaluate the homogeneity of group variances. However, these tests did not meet the prerequisites for parametric tests ( P  < 0.005). The Kruskal–Wallis test was employed to compare the medians of the activation methods in each root third, whereas the Friedman test was used for the median comparisons of the root third for each activation method. Statistical analyses were performed using R software (R Core Team, 2020). To determine which activation method and root third exhibited statistically significant differences between medians, the Bonferroni–Dunn multiple comparison test was applied at a significance level of 5%.

The mean, standard error and p - values obtained by comparing the data of root regions for each experimental group are shown in Table  1 . When examining each activation method for the different root thirds, the SA and control groups had no significant impact on the penetration percentage in all root thirds ( p  > 0.05). In contrast, the UI and XPR groups displayed a higher penetration percentage in the coronal and middle thirds than in the apical third ( p  < 0.05). The maximum penetration value was significantly lower in the apical third than in the coronal and middle thirds in all groups ( p  < 0.05). In the control, sonic and XP groups, the irrigation solution penetration area was ranked from highest to lowest as coronal, middle and apical, respectively ( p  < 0.05). However, in the ultrasonic group, the penetration area was significantly smaller in the apical third than in the middle and coronal thirds ( p  < 0.05).

The mean, standard error and p - values obtained by comparing the data of experimental groups for each root region are shown in Table  2 . When comparing the irrigation activation methods for each root third, no statistically significant differences were observed in the penetration area, penetration percentage, or maximum penetration parameters in the apical and coronal thirds ( p  > 0.05). In the middle third, the UI group exhibited a higher percentage of penetration than the control group ( p  < 0.05), whereas the penetration area and maximum penetration parameters were statistically similar for all experimental groups ( p  > 0.05).

After primary root canal therapy, the presence of bacteria within the dentinal tubules is considered as a primary cause of apical periodontitis [ 15 ]. Thus, effective irrigation during and after the removal of root canal filling materials is crucial to achieving optimal disinfection in retreatment cases. In this study, we evaluated the impact of various final irrigation activation techniques on the penetration of NaOCl into the dentinal tubules using CLSM. According to our results, UI demonstrated a significantly higher penetration percentage in the middle third of the root than in the control and SA group, where the UI and XPR performed similar. In the apical root third, NaOCl exhibited less penetration than in the coronal and middle thirds, except for the SA and control groups, where these changes remained statistically insignificant. As a result, we partially rejected null hypothesis.

It has been reported that the failure of endodontic treatment may be due to the persistence of microorganisms in the root canal system, especially in the apical part and even in well-treated teeth [ 28 ]. The persistence of microorganisms in the root canal system after retreatment may cause periradicular inflammation to continue. Therefore, optimum disinfection of the root canal system is important to increase the success rate of retreatment. In a previous study examining bacterial penetration in root filled teeth, bacteria were most frequently detected in the cervical third and inner dentin adjacent to the root canal [ 29 ]. In our study, we aimed to evaluate the penetration of NaOCl after retreatment by calculating the penetration percentage parameter around the root canal circumference and the maximum penetration and penetration area parameters in the region between the root canal and edge of the root in the horizontal root section for the coronal, middle and apical thirds.

Previous studies examining the effects of different activation systems on irrigation penetration solution into dentinal tubules primarily focused on primary root canal treatment [ 17 , 19 , 30 , 31 , 32 ]. These studies varied in terms of specimen type (human or bovine teeth), irrigation solution (NaOCl, CHX, Irritol), irrigation activation system (UI, SA, Laser-assisted activation) and observation method (Stereomicroscope, CLSM), making direct comparisons challenging. To the best of our knowledge, this study is the first to evaluate the penetration of irrigation solutions into dentinal tubules after retreatment. Therefore, directly comparing the results of our study with those reported in the literature is not appropriate.

According to our results, the only difference between the activation methods was observed between the percentage of penetration of the irrigation solution in UI and control groups in the middle third of the root. UI increased the penetration percentage of NaOCl compared with that in the control group. This result is in accordance with the results of the study of Akcay et al. [ 19 ] that observed irrigation penetration during primary root canal treatment. The UI provides cavitation and acoustic streaming [ 16 ] and produces sufficient shear forces to dislodge debris in instrumented canals [ 33 ]. Greater cleanliness of the canal walls may result in increased irrigation penetration. The effect of SA on the penetration of irrigation solutions has been studied, but the results are controversial [ 19 , 31 , 33 ]. Some studies reported that SA was superior to conventional needle irrigation, but these studies evaluated different types of irrigation solutions [ 31 , 33 ]. Similar to our study, Akcay et al. [ 19 ] reported no significant difference between SA and conventional needle irrigation and the same irrigation solution was used in our study. SA does not have the ability to allow sufficient streaming because of its low energy level and long wavelength [ 34 ]. The efficacy of XPR in the removal of root canal filling materials has been previously studied [ 11 , 23 , 35 , 36 ]. The results of these studies demonstrated that XPR was more effective than UI and SA for the removal of root canal filling materials. However, our results do not directly correlate with those of the aforementioned studies. In this study, no statistically significant differences were observed among XPR, UI and SA.

While bacterial penetration can be seen in every region of the root canal, more bacterial presence can be seen in the apical third because it is more difficult to reach this region via preparation or irrigation procedures [ 37 ]. The importance of bacterial presence in the apical region in root canal treatment failure was previously emphasized [ 28 ]. Studies that evaluated the penetration of irrigation solution into dentinal tubules showed lower penetration values for the apical third than for the middle and coronal thirds [ 19 , 31 ]. This result can be explained by the presence of narrower, sclerotic and fewer dentinal tubules at the apical root third [ 38 ]. Furthermore, the flow of the irrigation solutions to the apical third may be inefficient at removing the smear layer and debris [ 39 ]. Consistent with these studies, our results showed less penetration for the apical third than for the middle and coronal thirds for the UI and XPR groups for all of the parameters. In the SA and control groups, the apical root third showed less penetration than the middle and coronal thirds in terms of the penetration area and maximum penetration parameters. However, the SA and control groups showed similar percentages of penetration in all of the root thirds. The percentage of penetration, a circumferential measurement of the root canal wall, may be more affected by dentin tubule density. The inability to establish standardization in specimens may have revealed this contrasting result. In terms of clinical significance, future studies on the penetration of irrigation solution penetration should focus on alternative methods to enhance the penetration of NaOCl in the apical region.

The penetration of irrigation solutions into the dentinal tubules was observed using various microscopy techniques, including stereomicroscopy, scanning electron microscopy (SEM) and CLSM [ 17 , 19 , 31 , 33 , 40 , 41 ]. Detailed images of dentinal tubules can be obtained using CLSM, providing observations and measurements of penetration into the dentinal tubules in a single image along the circumference of the root canal walls [ 41 ]. Moreover, CLSM is a non-destructive method and does not dehydrate the specimen [ 42 ]. In contrast, SEM can visualize the dentinal tubules in only one plane, necessitating the further reconstruction of high magnification images to obtain a detailed final image. Acquiring images via SEM involves additional procedures, such as coating the specimens with gold and working under a vacuum, which can be time-consuming and can introduce artefacts during the preparation of the specimens [ 43 ]. Stereomicroscopy, on the other hand, is not capable of providing sufficiently detailed images of the dentinal tubules. Given these considerations, CLSM imaging was chosen as the preferred method for evaluating NaOCl penetration into the dentinal tubules in this study.

Every effort was made to select teeth that met the inclusion criteria. Notably, aging can lead to dentinal tubule sclerosis, especially in the apical third. This sclerosis can alter the number and diameter of the dentinal tubules, directly influencing the penetration of the irrigation solutions into them [ 44 ]. This variation can introduce considerable variability in experimental results and should be recognized as a limitation. Furthermore, to maintain standardization, the number and volume of irrigants, as well as the duration of the irrigation and activation procedures, were kept consistent for all experimental groups.

In this in vitro study, the following conclusions were drawn within the established limitations: in the middle third of the root, UI increased the penetration percentage compared with the control group, while XPR and SA showed no notable effect on NaOCl penetration following retreatment. Future studies are needed to explore the impact of various irrigation activation techniques on penetration of irrigation solutions into dentinal tubules after the retreatment procedure to provide definitive insights.

Data availability

The data of this research are available upon reasonable request from the corresponding author.

Abbreviations

• Confocal laser scanning microscopy

Ethylenediaminetetraacetic acid

Sodium hypochlorite

• Sonic activation

Scanning electron microscopy

• Ultrasonic irrigation

XP-endo Finisher

XP endo Finisher R

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Acknowledgements

This study was supported by the Eskisehir Osmangazi University Scientific Research Projects Coordination Unit (Project number: 202111002).

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Faculty of Dentistry, Department of Endodontics, Eskisehir Osmangazi University, Eskişehir, 26040, Turkey

Betul Gunes & Kübra Yeşildal Yeter

Faculty of Agriculture, Department of Animal Science Biometry and Genetic, Eskisehir Osmangazi University, Eskişehir, Turkey

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B. G.: Conceptualization, Investigation, writing- reviewing&editing. K. Y. Y.: Conceptualization, writing-original draft preparation, writing- reviewing&editing, visualization, project administration. Y. A.: Formal analyses, writing- reviewing&editing.

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Correspondence to Kübra Yeşildal Yeter .

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Ethical compliance of this in-vitro study was approved by the Eskişehir Osmangazi University Ethics Committee (18/24.09.2019). All participants gave informed consent to participate.

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Gunes, B., Yeter, K.Y. & Altay, Y. Impact of different activation procedures on sodium hypochlorite penetration into dentinal tubules after endodontic retreatment via confocal laser scanning microscopy. BMC Oral Health 24 , 1103 (2024). https://doi.org/10.1186/s12903-024-04891-6

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DOI : https://doi.org/10.1186/s12903-024-04891-6

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ⓘ Geversite

ⓘ Heazlewoodite

ⓘ Hollingworthite

ⓘ Isoferroplatinum

ⓘ Kashinite

ⓘ Magnesiochromite

ⓘ Magnetite

ⓘ var: Titanium-bearing Magnetite

ⓘ Millerite

ⓘ Pargasite

ⓘ Pentlandite

ⓘ 'Prassoite'

ⓘ Pyrrhotite

ⓘ Sperrylite var: Platarsite

ⓘ Tetra-auricupride

ⓘ Tetraferroplatinum

ⓘ Tolovkite

ⓘ Tulameenite

Rock types recorded nearby (within 20 km)

ⓘ Chromitite

ⓘ Clinopyroxenite

Regions in mindat.org that contain this locality

Regions in GADM Database

• Places - European, Western and Northern Russia

YEKATERINBURG: FACTORIES, URAL SIGHTS, YELTSIN AND THE WHERE NICHOLAS II WAS KILLED

Sverdlovsk oblast.

Sverdlovsk Oblast is the largest region in the Urals; it lies in the foothills of mountains and contains a monument indicating the border between Europe and Asia. The region covers 194,800 square kilometers (75,200 square miles), is home to about 4.3 million people and has a population density of 22 people per square kilometer. About 83 percent of the population live in urban areas. Yekaterinburg is the capital and largest city, with 1.5 million people. For Russians, the Ural Mountains are closely associated with Pavel Bazhov's tales and known for folk crafts such as Kasli iron sculpture, Tagil painting, and copper embossing. Yekaterinburg is the birthplace of Russia’s iron and steel industry, taking advantage of the large iron deposits in the Ural mountains. The popular Silver Ring of the Urals tourist route starts here.

In the summer you can follow in the tracks of Yermak, climb relatively low Ural mountain peaks and look for boulders seemingly with human faces on them. You can head to the Gemstone Belt of the Ural mountains, which used to house emerald, amethyst and topaz mines. In the winter you can go ice fishing, ski and cross-country ski.

Sverdlovsk Oblast and Yekaterinburg are located near the center of Russia, at the crossroads between Europe and Asia and also the southern and northern parts of Russia. Winters are longer and colder than in western section of European Russia. Snowfalls can be heavy. Winter temperatures occasionally drop as low as - 40 degrees C (-40 degrees F) and the first snow usually falls in October. A heavy winter coat, long underwear and good boots are essential. Snow and ice make the sidewalks very slippery, so footwear with a good grip is important. Since the climate is very dry during the winter months, skin moisturizer plus lip balm are recommended. Be alert for mud on street surfaces when snow cover is melting (April-May). Patches of mud create slippery road conditions.

Yekaterinburg

Yekaterinburg (kilometer 1818 on the Trans-Siberian Railway) is the fourth largest city in Russia, with of 1.5 million and growth rate of about 12 percent, high for Russia. Located in the southern Ural mountains, it was founded by Peter the Great and named after his wife Catherine, it was used by the tsars as a summer retreat and is where tsar Nicholas II and his family were executed and President Boris Yeltsin lived most of his life and began his political career. The city is near the border between Europe and Asia.

Yekaterinburg (also spelled Ekaterinburg) is located on the eastern slope of the Ural Mountains in the headwaters of the Iset and Pyshma Rivers. The Iset runs through the city center. Three ponds — Verkh-Isetsky, Gorodskoy and Nizhne-Isetsky — were created on it. Yekaterinburg has traditionally been a city of mining and was once the center of the mining industry of the Urals and Siberia. Yekaterinburg remains a major center of the Russian armaments industry and is sometimes called the "Pittsburgh of Russia.". A few ornate, pastel mansions and wide boulevards are reminders of the tsarist era. The city is large enough that it has its own Metro system but is characterized mostly by blocky Soviet-era apartment buildings. The city has advanced under President Vladimir Putin and is now one of the fastest growing places in Russia, a country otherwise characterized by population declines

Yekaterinburg is technically an Asian city as it lies 32 kilometers east of the continental divide between Europe and Asia. The unofficial capital of the Urals, a key region in the Russian heartland, it is second only to Moscow in terms of industrial production and capital of Sverdlovsk oblast. Among the important industries are ferrous and non-ferrous metallurgy, machine building and metalworking, chemical and petrochemicals, construction materials and medical, light and food industries. On top of being home of numerous heavy industries and mining concerns, Yekaterinburg is also a major center for industrial research and development and power engineering as well as home to numerous institutes of higher education, technical training, and scientific research. In addition, Yekaterinburg is the largest railway junction in Russia: the Trans-Siberian Railway passes through it, the southern, northern, western and eastern routes merge in the city.

Accommodation: There are two good and affordable hotels — the 3-star Emerald and Parus hotels — located close to the city's most popular landmarks and main transport interchanges in the center of Yekaterinburg. Room prices start at RUB 1,800 per night.

History of Yekaterinburg

Yekaterinburg was founded in 1723 by Peter the Great and named after his wife Catherine I. It was used by the tsars as a summer retreat but was mainly developed as metalworking and manufacturing center to take advantage of the large deposits of iron and other minerals in the Ural mountains. It is best known to Americans as the place where the last Tsar and his family were murdered by the Bolsheviks in 1918 and near where American U-2 spy plane, piloted by Gary Powers, was shot down in 1960.

Peter the Great recognized the importance of the iron and copper-rich Urals region for Imperial Russia's industrial and military development. In November 1723, he ordered the construction of a fortress factory and an ironworks in the Iset River Valley, which required a dam for its operation. In its early years Yekaterinburg grew rich from gold and other minerals and later coal. The Yekaterinburg gold rush of 1745 created such a huge amount of wealth that one rich baron of that time hosted a wedding party that lasted a year. By the mid-18th century, metallurgical plants had sprung up across the Urals to cast cannons, swords, guns and other weapons to arm Russia’s expansionist ambitions. The Yekaterinburg mint produced most of Russia's coins. Explorations of the Trans-Baikal and Altai regions began here in the 18th century.

Iron, cast iron and copper were the main products. Even though Iron from the region went into the Eiffel Tower, the main plant in Yekaterinburg itself was shut down in 1808. The city still kept going through a mountain factory control system of the Urals. The first railway in the Urals was built here: in 1878, the Yekaterinburg-Perm railway branch connected the province's capital with the factories of the Middle Urals.

In the Soviet era the city was called Sverdlovsk (named after Yakov Sverdlov, the man who organized Nicholas II's execution). During the first five-year plans the city became industrial — old plants were reconstructed, new ones were built. The center of Yekaterinburg was formed to conform to the historical general plan of 1829 but was the layout was adjusted around plants and factories. In the Stalin era the city was a major gulag transhipment center. In World War II, many defense-related industries were moved here. It and the surrounding area were a center of the Soviet Union's military industrial complex. Soviet tanks, missiles and aircraft engines were made in the Urals. During the Cold War era, Yekaterinburg was a center of weapons-grade uranium enrichment and processing, warhead assembly and dismantlement. In 1979, 64 people died when anthrax leaked from a biological weapons facility. Yekaterinburg was a “Closed City” for 40 years during the Cold Soviet era and was not open to foreigners until 1991

In the early post-Soviet era, much like Pittsburgh in the 1970s, Yekaterinburg had a hard struggle d to cope with dramatic economic changes that have made its heavy industries uncompetitive on the world market. Huge defense plants struggled to survive and the city was notorious as an organized crime center in the 1990s, when its hometown boy Boris Yeltsin was President of Russia. By the 2000s, Yekaterinburg’s retail and service was taking off, the defense industry was reviving and it was attracting tech industries and investments related to the Urals’ natural resources. By the 2010s it was vying to host a world exhibition in 2020 (it lost, Dubai won) and it had McDonald’s, Subway, sushi restaurants, and Gucci, Chanel and Armani. There were Bentley and Ferrari dealerships but they closed down

Transportation in Yekaterinburg

Getting There: By Plane: Yekaterinburg is a three-hour flight from Moscow with prices starting at RUB 8,000, or a 3-hour flight from Saint Petersburg starting from RUB 9,422 (direct round-trip flight tickets for one adult passenger). There are also flights from Frankfurt, Istanbul, China and major cities in the former Soviet Union.

By Train: Yekaterinburg is a major stop on the Trans-Siberian Railway. Daily train service is available to Moscow and many other Russian cities.Yekaterinburg is a 32-hour train ride from Moscow (tickets RUB 8,380 and above) or a 36-hour train ride from Saint Petersburg (RUB 10,300 and above). The ticket prices are round trip for a berth in a sleeper compartment for one adult passenger). By Car: a car trip from Moscow to Yekateringburg is 1,787 kilometers long and takes about 18 hours. The road from Saint Petersburg is 2,294 kilometers and takes about 28 hours.

Regional Transport: The region's public transport includes buses and suburban electric trains. Regional trains provide transport to larger cities in the Ural region. Buses depart from Yekaterinburg’s two bus stations: the Southern Bus Station and the Northern Bus Station.

Regional Transport: According the to Association for Safe International Road Travel (ASIRT): “Public transportation is well developed. Overcrowding is common. Fares are low. Service is efficient. Buses are the main form of public transport. Tram network is extensive. Fares are reasonable; service is regular. Trams are heavily used by residents, overcrowding is common. Purchase ticket after boarding. Metro runs from city center to Uralmash, an industrial area south of the city. Metro ends near the main railway station. Fares are inexpensive.

“Traffic is congested in city center. Getting around by car can be difficult. Route taxis (minivans) provide the fastest transport. They generally run on specific routes, but do not have specific stops. Drivers stop where passengers request. Route taxis can be hailed. Travel by bus or trolleybuses may be slow in rush hour. Trams are less affected by traffic jams. Trolley buses (electric buses) cannot run when temperatures drop below freezing.”

Entertainment, Sports and Recreation in Yekaterinburg

The performing arts in Yekaterinburg are first rate. The city has an excellent symphony orchestra, opera and ballet theater, and many other performing arts venues. Tickets are inexpensive. The Yekaterinburg Opera and Ballet Theater is lavishly designed and richly decorated building in the city center of Yekaterinburg. The theater was established in 1912 and building was designed by architect Vladimir Semyonov and inspired by the Vienna Opera House and the Theater of Opera and Ballet in Odessa.

Vaynera Street is a pedestrian only shopping street in city center with restaurants, cafes and some bars. But otherwise Yekaterinburg's nightlife options are limited. There are a handful of expensive Western-style restaurants and bars, none of them that great. Nightclubs serve the city's nouveau riche clientele. Its casinos have closed down. Some of them had links with organized crime. New dance clubs have sprung up that are popular with Yekaterinburg's more affluent youth.

Yekaterinburg's most popular spectator sports are hockey, basketball, and soccer. There are stadiums and arenas that host all three that have fairly cheap tickets. There is an indoor water park and lots of parks and green spaces. The Urals have many lakes, forests and mountains are great for hiking, boating, berry and mushroom hunting, swimming and fishing. Winter sports include cross-country skiing and ice skating. Winter lasts about six months and there’s usually plenty of snow. The nearby Ural Mountains however are not very high and the downhill skiing opportunities are limited..

Sights in Yekaterinburg

Sights in Yekaterinburg include the Museum of City Architecture and Ural Industry, with an old water tower and mineral collection with emeralds. malachite, tourmaline, jasper and other precious stone; Geological Alley, a small park with labeled samples of minerals found in the Urals region; the Ural Geology Museum, which houses an extensive collection of stones, gold and gems from the Urals; a monument marking the border between Europe and Asia; a memorial for gulag victims; and a graveyard with outlandish memorials for slain mafia members.

The Military History Museum houses the remains of the U-2 spy plane shot down in 1960 and locally made tanks and rocket launchers. The fine arts museum contains paintings by some of Russia's 19th-century masters. Also worth a look are the History an Local Studies Museum; the Political History and Youth Museum; and the University and Arboretum. Old wooden houses can be seen around Zatoutstovsya ulitsa and ulitsa Belinskogo. Around the city are wooded parks, lakes and quarries used to harvest a variety of minerals. Weiner Street is the main street of Yekaterinburg. Along it are lovely sculptures and 19th century architecture. Take a walk around the unique Literary Quarter

Plotinka is a local meeting spot, where you will often find street musicians performing. Plotinka can be described as the center of the city's center. This is where Yekaterinburg holds its biggest events: festivals, seasonal fairs, regional holiday celebrations, carnivals and musical fountain shows. There are many museums and open-air exhibitions on Plotinka. Plotinka is named after an actual dam of the city pond located nearby (“plotinka” means “a small dam” in Russian).In November 1723, Peter the Great ordered the construction of an ironworks in the Iset River Valley, which required a dam for its operation. “Iset” can be translated from Finnish as “abundant with fish”. This name was given to the river by the Mansi — the Finno-Ugric people dwelling on the eastern slope of the Northern Urals.

Vysotsky and Iset are skyscrapers that are 188.3 meters and 209 meters high, respectively. Fifty-story-high Iset has been described by locals as the world’s northernmost skyscraper. Before the construction of Iset, Vysotsky was the tallest building of Yekaterinburg and Russia (excluding Moscow). A popular vote has decided to name the skyscraper after the famous Soviet songwriter, singer and actor Vladimir Vysotsky. and the building was opened on November 25, 2011. There is a lookout at the top of the building, and the Vysotsky museum on its second floor. The annual “Vysotsky climb” (1137 steps) is held there, with a prize of RUB 100,000. While Vysotsky serves as an office building, Iset, owned by the Ural Mining and Metallurgical Company, houses 225 premium residential apartments ranging from 80 to 490 square meters in size.

Boris Yeltsin Presidential Center

The Boris Yeltsin Presidential Center (in the city center: ul. Yeltsina, 3) is a non-governmental organization named after the first president of the Russian Federation. The Museum of the First President of Russia as well as his archives are located in the Center. There is also a library, educational and children's centers, and exposition halls. Yeltsin lived most of his life and began his political career in Yekaterinburg. He was born in Butka about 200 kilometers east of Yekaterinburg.

The core of the Center is the Museum. Modern multimedia technologies help animate the documents, photos from the archives, and artifacts. The Yeltsin Museum holds collections of: propaganda posters, leaflets, and photos of the first years of the Soviet regime; portraits and portrait sculptures of members of Politburo of the Central Committee of the Communist Party of various years; U.S.S.R. government bonds and other items of the Soviet era; a copy of “One Day in the Life of Ivan Denisovich” by Alexander Solzhenitsyn, published in the “Novy Mir” magazine (#11, 1962); perestroika-era editions of books by Alexander Solzhenitsyn, Vasily Grossman, and other authors; theater, concert, and cinema posters, programs, and tickets — in short, all of the artifacts of the perestroika era.

The Yeltsin Center opened in 2012. Inside you will also find an art gallery, a bookstore, a gift shop, a food court, concert stages and a theater. There are regular screenings of unique films that you will not find anywhere else. Also operating inside the center, is a scientific exploritorium for children. The center was designed by Boris Bernaskoni. Almost from the its very opening, the Yeltsin Center has been accused by members of different political entities of various ideological crimes. The museum is open Tuesday to Sunday, from 10:00am to 9:00pm.

Where Nicholas II was Executed

On July, 17, 1918, during this reign of terror of the Russian Civil War, former-tsar Nicholas II, his wife, five children (the 13-year-old Alexis, 22-year-old Olga, 19-year-old Maria and 17-year-old Anastasia)the family physician, the cook, maid, and valet were shot to death by a Red Army firing squad in the cellar of the house they were staying at in Yekaterinburg.

Ipatiev House (near Church on the Blood, Ulitsa Libknekhta) was a merchant's house where Nicholas II and his family were executed. The house was demolished in 1977, on the orders of an up and coming communist politician named Boris Yeltsin. Yeltsin later said that the destruction of the house was an "act of barbarism" and he had no choice because he had been ordered to do it by the Politburo,

The site is marked with s cross with the photos of the family members and cross bearing their names. A small wooden church was built at the site. It contains paintings of the family. For a while there were seven traditional wooden churches. Mass is given ay noon everyday in an open-air museum. The Church on the Blood — constructed to honor Nicholas II and his family — was built on the part of the site in 1991 and is now a major place of pilgrimage.

Nicholas and his family where killed during the Russian civil war. It is thought the Bolsheviks figured that Nicholas and his family gave the Whites figureheads to rally around and they were better of dead. Even though the death orders were signed Yakov Sverdlov, the assassination was personally ordered by Lenin, who wanted to get them out of sight and out of mind. Trotsky suggested a trial. Lenin nixed the idea, deciding something had to be done about the Romanovs before White troops approached Yekaterinburg. Trotsky later wrote: "The decision was not only expedient but necessary. The severity of he punishment showed everyone that we would continue to fight on mercilessly, stopping at nothing."

Ian Frazier wrote in The New Yorker: “Having read a lot about the end of Tsar Nicholas II and his family and servants, I wanted to see the place in Yekaterinburg where that event occurred. The gloomy quality of this quest depressed Sergei’s spirits, but he drove all over Yekaterinburg searching for the site nonetheless. Whenever he stopped and asked a pedestrian how to get to the house where Nicholas II was murdered, the reaction was a wince. Several people simply walked away. But eventually, after a lot of asking, Sergei found the location. It was on a low ridge near the edge of town, above railroad tracks and the Iset River. The house, known as the Ipatiev House, was no longer standing, and the basement where the actual killings happened had been filled in. I found the blankness of the place sinister and dizzying. It reminded me of an erasure done so determinedly that it had worn a hole through the page. [Source: Ian Frazier, The New Yorker, August 3, 2009, Frazier is author of “Travels in Siberia” (2010)]

“The street next to the site is called Karl Liebknecht Street. A building near where the house used to be had a large green advertisement that said, in English, “LG—Digitally Yours.” On an adjoining lot, a small chapel kept the memory of the Tsar and his family; beneath a pedestal holding an Orthodox cross, peonies and pansies grew. The inscription on the pedestal read, “We go down on our knees, Russia, at the foot of the tsarist cross.”

Books: The Romanovs: The Final Chapter by Robert K. Massie (Random House, 1995); The Fall of the Romanovs by Mark D. Steinberg and Vladimir Khrustalëv (Yale, 1995);

See Separate Article END OF NICHOLAS II factsanddetails.com

Execution of Nicholas II

According to Robert Massie K. Massie, author of Nicholas and Alexandra, Nicholas II and his family were awakened from their bedrooms around midnight and taken to the basement. They were told they were to going to take some photographs of them and were told to stand behind a row of chairs.

Suddenly, a group of 11 Russians and Latvians, each with a revolver, burst into the room with orders to kill a specific person. Yakob Yurovsky, a member of the Soviet executive committee, reportedly shouted "your relatives are continuing to attack the Soviet Union.” After firing, bullets bouncing off gemstones hidden in the corsets of Alexandra and her daughters ricocheted around the room like "a shower of hail," the soldiers said. Those that were still breathing were killed with point black shots to the head.

The three sisters and the maid survived the first round thanks to their gems. They were pressed up against a wall and killed with a second round of bullets. The maid was the only one that survived. She was pursued by the executioners who stabbed her more than 30 times with their bayonets. The still writhing body of Alexis was made still by a kick to the head and two bullets in the ear delivered by Yurovsky himself.

Yurovsky wrote: "When the party entered I told the Romanovs that in view of the fact their relatives continued their offensive against Soviet Russia, the Executive Committee of the Urals Soviet had decided to shoot them. Nicholas turned his back to the detachment and faced his family. Then, as if collecting himself, he turned around, asking, 'What? What?'"

"[I] ordered the detachment to prepare. Its members had been previously instructed whom to shoot and to am directly at the heart to avoid much blood and to end more quickly. Nicholas said no more. he turned again to his family. The others shouted some incoherent exclamations. All this lasted a few seconds. Then commenced the shooting, which went on for two or three minutes. [I] killed Nicholas on the spot."

Nicholas II’s Initial Burial Site in Yekaterinburg

Ganina Yama Monastery (near the village of Koptyaki, 15 kilometers northwest of Yekaterinburg) stands near the three-meter-deep pit where some the remains of Nicholas II and his family were initially buried. The second burial site — where most of the remains were — is in a field known as Porosyonkov (56.9113628°N 60.4954326°E), seven kilometers from Ganina Yama.

On visiting Ganina Yama Monastery, one person posted in Trip Advisor: “We visited this set of churches in a pretty park with Konstantin from Ekaterinburg Guide Centre. He really brought it to life with his extensive knowledge of the history of the events surrounding their terrible end. The story is so moving so unless you speak Russian, it is best to come here with a guide or else you will have no idea of what is what.”

In 1991, the acid-burned remains of Nicholas II and his family were exhumed from a shallow roadside mass grave in a swampy area 12 miles northwest of Yekaterinburg. The remains had been found in 1979 by geologist and amateur archeologist Alexander Avdonin, who kept the location secret out of fear that they would be destroyed by Soviet authorities. The location was disclosed to a magazine by one his fellow discovers.

The original plan was to throw the Romanovs down a mine shaft and disposes of their remains with acid. They were thrown in a mine with some grenades but the mine didn't collapse. They were then carried by horse cart. The vats of acid fell off and broke. When the carriage carrying the bodies broke down it was decided the bury the bodies then and there. The remaining acid was poured on the bones, but most of it was soaked up the ground and the bones largely survived.

After this their pulses were then checked, their faces were crushed to make them unrecognizable and the bodies were wrapped in bed sheets loaded onto a truck. The "whole procedure," Yurovsky said took 20 minutes. One soldiers later bragged than he could "die in peace because he had squeezed the Empress's -------."

The bodies were taken to a forest and stripped, burned with acid and gasoline, and thrown into abandoned mine shafts and buried under railroad ties near a country road near the village of Koptyaki. "The bodies were put in the hole," Yurovsky wrote, "and the faces and all the bodies, generally doused with sulfuric acid, both so they couldn't be recognized and prevent a stink from them rotting...We scattered it with branches and lime, put boards on top and drove over it several times—no traces of the hole remained.

Shortly afterwards, the government in Moscow announced that Nicholas II had been shot because of "a counterrevolutionary conspiracy." There was no immediate word on the other members of the family which gave rise to rumors that other members of the family had escaped. Yekaterinburg was renamed Sverdlov in honor of the man who signed the death orders.

For seven years the remains of Nicholas II, Alexandra, three of their daughters and four servants were stored in polyethylene bags on shelves in the old criminal morgue in Yekaterunburg. On July 17, 1998, Nicholas II and his family and servants who were murdered with him were buried Peter and Paul Fortress in St. Petersburg along with the other Romanov tsars, who have been buried there starting with Peter the Great. Nicholas II had a side chapel built for himself at the fortress in 1913 but was buried in a new crypt.

Near Yekaterinburg

Factory-Museum of Iron and Steel Metallurgy (in Niznhy Tagil 80 kilometers north of Yekaterinburg) a museum with old mining equipment made at the site of huge abandoned iron and steel factory. Officially known as the Factory-Museum of the History of the Development of Iron and Steel Metallurgy, it covers an area of 30 hectares and contains a factory founded by the Demidov family in 1725 that specialized mainly in the production of high-quality cast iron and steel. Later, the foundry was renamed after Valerian Kuybyshev, a prominent figure of the Communist Party.

The first Russian factory museum, the unusual museum demonstrates all stages of metallurgy and metal working. There is even a blast furnace and an open-hearth furnace. The display of factory equipment includes bridge crane from 1892) and rolling stock equipment from the 19th-20th centuries. In Niznhy Tagil contains some huge blocks of malachite and

Nizhnyaya Sinyachikha (180 kilometers east-northeast of Yekaterinburg) has an open air architecture museum with log buildings, a stone church and other pre-revolutionary architecture. The village is the creation of Ivan Samoilov, a local activist who loved his village so much he dedicated 40 years of his life to recreating it as the open-air museum of wooden architecture.

The stone Savior Church, a good example of Siberian baroque architecture. The interior and exterior of the church are exhibition spaces of design. The houses are very colorful. In tsarist times, rich villagers hired serfs to paint the walls of their wooden izbas (houses) bright colors. Old neglected buildings from the 17th to 19th centuries have been brought to Nizhnyaya Sinyachikha from all over the Urals. You will see the interior design of the houses and hear stories about traditions and customs of the Ural farmers.

Verkhoturye (330 kilometers road from Yekaterinburg) is the home a 400-year-old monastery that served as 16th century capital of the Urals. Verkhoturye is a small town on the Tura River knows as the Jerusalem of the Urals for its many holy places, churches and monasteries. The town's main landmark is its Kremlin — the smallest in Russia. Pilgrims visit the St. Nicholas Monastery to see the remains of St. Simeon of Verkhoturye, the patron saint of fishermen.

Ural Mountains

Ural Mountains are the traditional dividing line between Europe and Asia and have been a crossroads of Russian history. Stretching from Kazakhstan to the fringes of the Arctic Kara Sea, the Urals lie almost exactly along the 60 degree meridian of longitude and extend for about 2,000 kilometers (1,300 miles) from north to south and varies in width from about 50 kilometers (30 miles) in the north and 160 kilometers (100 miles) the south. At kilometers 1777 on the Trans-Siberian Railway there is white obelisk with "Europe" carved in Russian on one side and "Asia" carved on the other.

The eastern side of the Urals contains a lot of granite and igneous rock. The western side is primarily sandstone and limestones. A number of precious stones can be found in the southern part of the Urals, including emeralds. malachite, tourmaline, jasper and aquamarines. The highest peaks are in the north. Mount Narodnaya is the highest of all but is only 1884 meters (6,184 feet) high. The northern Urals are covered in thick forests and home to relatively few people.

Like the Appalachian Mountains in the eastern United States, the Urals are very old mountains — with rocks and sediments that are hundreds of millions years old — that were one much taller than they are now and have been steadily eroded down over millions of years by weather and other natural processes to their current size. According to Encyclopedia Britannica: “The rock composition helps shape the topography: the high ranges and low, broad-topped ridges consist of quartzites, schists, and gabbro, all weather-resistant. Buttes are frequent, and there are north–south troughs of limestone, nearly all containing river valleys. Karst topography is highly developed on the western slopes of the Urals, with many caves, basins, and underground streams. The eastern slopes, on the other hand, have fewer karst formations; instead, rocky outliers rise above the flattened surfaces. Broad foothills, reduced to peneplain, adjoin the Central and Southern Urals on the east.

“The Urals date from the structural upheavals of the Hercynian orogeny (about 250 million years ago). About 280 million years ago there arose a high mountainous region, which was eroded to a peneplain. Alpine folding resulted in new mountains, the most marked upheaval being that of the Nether-Polar Urals...The western slope of the Urals is composed of middle Paleozoic sedimentary rocks (sandstones and limestones) that are about 350 million years old. In many places it descends in terraces to the Cis-Ural depression (west of the Urals), to which much of the eroded matter was carried during the late Paleozoic (about 300 million years ago). Found there are widespread karst (a starkly eroded limestone region) and gypsum, with large caverns and subterranean streams. On the eastern slope, volcanic layers alternate with sedimentary strata, all dating from middle Paleozoic times.”

Southern Urals

The southern Urals are characterized by grassy slopes and fertile valleys. The middle Urals are a rolling platform that barely rises above 300 meters (1,000 feet). This region is rich in minerals and has been heavily industrialized. This is where you can find Yekaterinburg (formally Sverdlovsk), the largest city in the Urals.

Most of the Southern Urals are is covered with forests, with 50 percent of that pine-woods, 44 percent birch woods, and the rest are deciduous aspen and alder forests. In the north, typical taiga forests are the norm. There are patches of herbal-poaceous steppes, northem sphagnous marshes and bushy steppes, light birch forests and shady riparian forests, tall-grass mountainous meadows, lowland ling marshes and stony placers with lichen stains. In some places there are no large areas of homogeneous forests, rather they are forests with numerous glades and meadows of different size.

In the Ilmensky Mountains Reserve in the Southern Urals, scientists counted 927 vascular plants (50 relicts, 23 endemic species), about 140 moss species, 483 algae species and 566 mushroom species. Among the species included into the Red Book of Russia are feather grass, downy-leaved feather grass, Zalessky feather grass, moccasin flower, ladies'-slipper, neottianthe cucullata, Baltic orchis, fen orchis, helmeted orchis, dark-winged orchis, Gelma sandwart, Krasheninnikov sandwart, Clare astragalus.

The fauna of the vertebrate animals in the Reserve includes 19 fish, 5 amphibian and 5 reptile. Among the 48 mammal species are elks, roe deer, boars, foxes, wolves, lynxes, badgers, common weasels, least weasels, forest ferrets, Siberian striped weasel, common marten, American mink. Squirrels, beavers, muskrats, hares, dibblers, moles, hedgehogs, voles are quite common, as well as chiropterans: pond bat, water bat, Brandt's bat, whiskered bat, northern bat, long-eared bat, parti-coloured bat, Nathusius' pipistrelle. The 174 bird bird species include white-tailed eagles, honey hawks, boreal owls, gnome owls, hawk owls, tawny owls, common scoters, cuckoos, wookcocks, common grouses, wood grouses, hazel grouses, common partridges, shrikes, goldenmountain thrushes, black- throated loons and others.

Activities and Places in the Ural Mountains

The Urals possess beautiful natural scenery that can be accessed from Yekaterinburg with a rent-a-car, hired taxi and tour. Travel agencies arrange rafting, kayaking and hiking trips. Hikes are available in the taiga forest and the Urals. Trips often include walks through the taiga to small lakes and hikes into the mountains and excursions to collect mushrooms and berries and climb in underground caves. Mellow rafting is offered in a relatively calm six kilometer section of the River Serga. In the winter visitor can enjoy cross-mountains skiing, downhill skiing, ice fishing, dog sledding, snow-shoeing and winter hiking through the forest to a cave covered with ice crystals.

Lake Shartash (10 kilometers from Yekaterinburg) is where the first Ural gold was found, setting in motion the Yekaterinburg gold rush of 1745, which created so much wealth one rich baron of that time hosted a wedding party that lasted a year. The area around Shartash Lake is a favorite picnic and barbecue spot of the locals. Getting There: by bus route No. 50, 054 or 54, with a transfer to suburban commuter bus route No. 112, 120 or 121 (the whole trip takes about an hour), or by car (10 kilometers drive from the city center, 40 minutes).

Revun Rapids (90 kilometers road from Yekaterinburg near Beklenishcheva village) is a popular white water rafting places On the nearby cliffs you can see the remains of a mysterious petroglyph from the Paleolithic period. Along the steep banks, you may notice the dark entrance of Smolinskaya Cave. There are legends of a sorceress who lived in there. The rocks at the riverside are suited for competitive rock climbers and beginners. Climbing hooks and rings are hammered into rocks. The most fun rafting is generally in May and June.

Olenii Ruchii National Park (100 kilometers west of Yekaterinburg) is the most popular nature park in Sverdlovsk Oblast and popular weekend getaway for Yekaterinburg residents. Visitors are attracted by the beautiful forests, the crystal clear Serga River and picturesque rocks caves. There are some easy hiking routes: the six-kilometer Lesser Ring and the 15-kilometer Greater Ring. Another route extends for 18 km and passes by the Mitkinsky Mine, which operated in the 18th-19th centuries. It's a kind of an open-air museum — you can still view mining an enrichment equipment here. There is also a genuine beaver dam nearby.

Among the other attractions at Olenii Ruchii are Druzhba (Friendship) Cave, with passages that extend for about 500 meters; Dyrovaty Kamen (Holed Stone), created over time by water of Serga River eroding rock; and Utoplennik (Drowned Man), where you can see “The Angel of Sole Hope”., created by the Swedish artist Lehna Edwall, who has placed seven angels figures in different parts of the world to “embrace the planet, protecting it from fear, despair, and disasters.”

Image Sources: Wikimedia Commons

Text Sources: Federal Agency for Tourism of the Russian Federation (official Russia tourism website russiatourism.ru ), Russian government websites, UNESCO, Wikipedia, Lonely Planet guides, New York Times, Washington Post, Los Angeles Times, National Geographic, The New Yorker, Bloomberg, Reuters, Associated Press, AFP, Yomiuri Shimbun and various books and other publications.

Updated in September 2020

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