Luminescence microscopy. Electron microscopy
This chapter will discuss the main principles, design, and application of a confocal laser scanning microscope (CLSM) using Leica devices as an example. The principle of CLSM is the registration of the light flux emanating from the focal plane of the lens when the focuses coincide, i.e., the detector diaphragm must be positioned so that its image exactly coincides with the focus of the light illuminating the object. Lasers and sensitive detectors are used as a light source to obtain an image.
The main feature of CLSM is the ability to obtain a layer-by-layer image of the object under study (for example, a cell) with high resolution and low noise. This is achieved by step-by-step scanning of an object with a focused beam of light from a coherent source or stage, using specific fluorescent probes and special methods for limiting light fluxes.
CLSM scanning systems can be classified as follows.
1. Beam scanning.
A) Mirror systems: single-mirror, double-mirror, resonant magnetoelectric.
B) Light-fiber systems: single-fiber, multi-fiber.
B) Lens scanning:
· piezoelectric movement of the lens along the X, Y axes;
· piezoelectric movement of the lens along the Z axis.
D) Acousto-optical beam deflectors: two acousto-optic deflectors for scanning along the X and Y axes.
D) Disk systems:
· single-spiral, single-sided and double-sided;
· multi-spiral single-sided and double-sided.
E) Combined systems: acousto-optical deflector along the Y-axis and scanning with a mirror along the X-axis.
2. Scanning with a table.
A) Stepper drive: scanning table with stepper motors along the X, Y, Z axes.
B) Combined drive: scanning stage with a stepper motor along the X, Y axes and a piezoelectric drive along the Z axis.
Rice. 5. Schematic diagram of the operation of CLSM.
1 - scanning table; 2 - test sample; 3, 7 - lenses; 4 - scanning device; 5 - beam splitter; 6 - beam of light from the laser; 8, 12 - image of points B and C; 9 - needle diaphragm; 10 - image of point A in the center of the needle diaphragm; 11 - radiation receiver; 13, 15 - glow of points B and C, located outside the focus of lens 3; 14 - glow of point A, located in the focus of lens 3.
The excitation light flux 6 from the laser source enters through the beam splitter plate 5, the scanning system 4 onto the lens 3 and is focused to point A of the plane of the test specimen (for example, a cell), which is in focus. We believe that intracellular structures are associated with fluorescent probes and beam focusing point A can be considered as a point source of light, the fluorescence flux from which through lens 3, beam splitter plate 5, lens 7 is focused in the plane of needle diaphragm 9 (“pinhole”) and recorded by photodetector 11 The illumination by the excitation flux of drug fragments lying outside the lens focus along the optical axis (points B and C) is lower than at point A. Consequently, the component of illumination of the detector target from points B and C can be significantly reduced. Fluorescence fluxes emanating from points B and C, which lie out of focus, are limited by a pinhole diaphragm and do not reach the detector, or a small part of them does. Thus, when scanning a preparation in the plane XY The detector registers a signal, the level of which is determined by the distance from the scanning plane along the Z coordinate. The alignment of the focus of lens 3 with the scanning plane and the focus of lens 7 with a needle diaphragm is reflected in the term “confocality”. Step-by-step movement of the scanning plane along the Z axis allows you to obtain a series of contrasting layer-by-layer images and reconstruct the internal three-dimensional structure (3-D) of the object under study. Image quality, in-plane resolution XY and along the Z axis depends on the quality of the optics, the quality of the scanning systems, the size and manufacturing accuracy of the pinhole diaphragm, the rigidity of the structure, the efficiency of the signal processing algorithms used, and the specificity of the fluorescent probes.
To determine the spatial resolution of the microscope, an image analysis of a point light source is performed. The image of a point source formed by a conventional lens is an Airy diffraction spot consisting of a bright central core and fainter outer rings.
Rice. 6. Airy diffraction spot.
Two point sources of equal brightness, the distance between them is d , are visible as two different points if the distance between the centers of the Airy circles exceeds the following value:
r A = XY =0.6 λ/NA,
where r A - the radius of the first dark ring in the Airy circle, λ is the wavelength of the light source in nm, NA is the numerical aperture.
This expression is called the Rayleigh criterion. It determines the resolution of the microscope in the sample plane (XY). In this case, the resolution limit is determined primarily by the wave nature of light, and therefore it is often simplified by considering NA=1. In CLSM imaging results in a slight decrease in the size of the Airy spot. The intensity of the Airy spot for a standard microscope decreases according to the law n -2, where n is the transverse displacement, and for CLSM the intensity decreases as n -4. This leads to an increase in the resolution of CLSM by 1.5 times. For a confocal microscope, the Rayleigh criterion has the form:
XY c r =0.4 λ/NA,
where XY c r is the resolution of the CLSM.
To assess the advantages of CLSM, we consider the instrumental function (the structure of the Airy spot) and analyze the resolution of the microscope in the axial direction (Z). The three-dimensional Airy spot (intensity distribution) is a three-dimensional hardware function (THF) that defines the image of an object. The TAF for a conventional microscope has a conical shape, expanding up and down from the center, while for a confocal microscope it has an elliptical shape and is less elongated in the axial direction.
Rice. 7. View of the three-dimensional hardware function of a point source of a conventional microscope - (a) and CLSM - (b).
The Rayleigh criterion is also applicable to determine the resolution of a microscope in the direction of the optical axis. For a conventional microscope, two point sources located at some distance along the optical axis can be resolved if the maxima of their Airy spots are at a distance:
Z r =2 λ/NA 2 ,
where Z r is the axial resolution of the microscope.
The energy distribution in the Airy spot for CLSM is narrower, and the resolution in the axial direction is 1.4 times higher. For a confocal microscope, the Rayleigh criterion has the form:
Z r c =1.4 λ/NA 2 ,
where Z r c is the axial resolution of the CLSM.
Modern CLSM have one main advantage - the ability to obtain thin optical sections. The following parameters affect the image quality when obtaining thin optical sections:
· confocal aperture size;
· numerical aperture of the lens NA;
· refractive index;
· absorption of light in the sample.
The decrease in light intensity as the thickness of the sample increases affects the resolution of the microscope along the optical Z axis. Resolution depends on the optics of the microscope and the sample. The thickness of the optical section in a confocal microscope is usually characterized by the width of the distribution at half-height of the intensity peak ∆z 1/2 = 0.65 μm. If the sample and detector are ideal, this parameter depends on the numerical aperture of the objective, the wavelength λ and the refractive index of the immersion medium n i .
Rice. 8. Dependence of the axial resolution of the microscope ∆z 1/2 on the numerical aperture of the lens.
In CLSM, an adjustable diaphragm is placed in front of the detector, changing the amount of light. Needle diaphragms are designed to create conditions for maximum or complete filtration of light entering the image formation plane from points that do not coincide with the focal plane or are located next to the analyzed element of the object in the focal plane. The size of the needle diaphragm is finite, and the lateral resolution of the device, the brightness of the illuminated elements of the specimen shifted relative to the focal plane along the Z axis, and the depth of field depend on it. The size of the diaphragm affects the thickness of the resulting section. The smaller the aperture, the closer the section thickness is to the theoretical limit (the width of the distribution at half-height of the intensity peak), and at very large apertures the ability to obtain a thin section becomes impossible.
As mentioned earlier, CLSM makes it possible to obtain an optical cross-section at a significant depth from the surface of the sample, with the important point being the refractive index and the depth issue. The passage of the incident and reflected beam through the sample affects the image quality. If the refractive indices of the immersion medium and the sample are close (the effect of the difference in the refractive indices of the immersion liquid and the object on the appearance of scattered light, which reduces the image contrast and acts as a spherical aberration effect), the light cone will converge. If the refractive indices differ, spherical aberration appears.
Rice. 9. Refractive indices of the immersion medium and sample.
a – image formation using an immersion lens without aberration; b – spherical aberration caused by the discrepancy between the indicators.
With different refractive indices, light rays coming at different distances from the optical axis are not focused to one point, which leads to a loss of image quality. If possible, the refractive indices of the sample and the objective should be matched. If the refractive indices of the sample and the immersion medium are different, the image of the object at depth is blurred along the optical axis, and the plane of focus is shifted. To increase penetration depth, the lens must have a large numerical aperture, such as an oil immersion lens. However, such lenses have a small maximum distance from the objective lens to the focal plane. The penetration depth is affected by the heterogeneity of the sample, which leads to a decrease in light intensity at great depths and the appearance of a shadow. Ideally, a sample to achieve maximum penetration depth and maximum resolution would have a refractive index equal to that of the lens, but this is a homogeneous sample, and such biological samples do not exist.
During scanning, the CLSM obtains a series of optical sections with regularly increasing depths from the surface of the sample. For most CLSMs, acquiring hundreds of 2D images takes only a few minutes. An optical image can be obtained in two ways:
1) obtaining a sequence of optical sections in the XY plane located at a distance ∆z from each other. Comparison of the coordinates of the centers of objects in different sections makes it possible to determine their orientation and length distribution.
Rice. 10. Study of three-dimensional structure in the XY plane.
2) obtaining a number of optical sections in the XZ plane located at a distance ∆y. If the sample is parallel to the section plane, then its section in the XZ plane will be almost circular; by comparing images in different XZ sections, the curvature of the object under study can be determined.
Rice. 11. Study of three-dimensional structure in the XZ plane.
Three-dimensional reconstruction of the objects under study using CLSM methods is aimed at solving two problems:
· visualization of a three-dimensional image of an object obtained by “assembling” its optical sections;
· quantitative analysis of the internal structures of an object.
Today there are quite a lot of companies producing CLSM. Innovations of CLSM manufacturing companies:
· 2002 Leica announced an acousto-optical beam splitter (AOBS), which allows efficient separation of the laser excitation beam and luminescence;
· 2002 Carl Zeiss began producing the LSM 510 META confocal microscope with an original photodetector that simultaneously records signals in 32 spectral channels;
· 2004 Zeiss creates the high-speed LSM 5 Live, which has a scanning speed 20 times higher than a conventional CLSM;
· Olympus has developed a device with two scanners, which makes it possible to more effectively use, for example, the FRAP technique;
· Nikon - creates a compact and inexpensive CLSM with a simplified design;
· 2007 Leica announced the release of a 4Pi-confocal microscope, improving axial resolution by 4 - 7 times.
Let's consider a CLSM device belonging to a new, more advanced generation of devices - TCS SP5 from Leica.
Rice. 12. Multiphoton/confocal broadband system TCS SP5 (inverted microscope).
Key elements of CLSM TCS SP5: AOTF – acoustic-optical tunable filter, AOBS – acousto-optical beam splitter, SP-Detector – spectral photometer sensor.
AOTF - Acoustic-Optical Tunable Filter - serves to minimize optical exposure and adjusts laser power depending on the sample and fluorochrome. Allows you to select the required wavelength and control the intensity of the excitation light. AOTF is an electrically tunable filter that operates on the principle of volumetric diffraction of a light beam by inhomogeneities of the refractive index. Such inhomogeneities arise when an ultrasonic acoustic wave is excited in birefringent crystals. With anisotropic diffraction in uniaxial crystals, there is a minimum ultrasound frequency at which the angles of incidence and diffraction coincide, and the so-called collinear acousto-optical interaction occurs.
Rice. 13. Acoustic-optical tunable filter.
AOBS - acousto-optical beam splitter, is an acousto-optical crystal - a tunable refractive device operating in reverse mode. What does using AOBS give you:
1. To obtain a clear, low-noise image, a high degree of light transmission is required. Reducing noise by averaging the image over many successive scans necessarily leads to photobleaching of the object being studied. AOBS's light transmittance is superior to most dichroic mirrors across the entire visible spectrum. Therefore, averaging over a smaller number of scans is required. As a result, the drug will last much longer;
2. Bright and clear images require as many photons as possible to pass from the object to the detector, which improves image quality. AOBS ensures registration of the widest possible fluorescence bands, that is, the maximum number of photons;
3. Low bleaching during image acquisition is important to protect the sample from fading and living objects from toxic fluorochrome photolysis products. The light transmission curves of AOBS have very steep slopes, which makes it possible to record the fluorescence of a fluorochrome as close as possible to its excitation band;
4. Any dye in the visible range can be excited, since the reflection can be adjusted individually;
5. The issue of multi-parameter fluorescence has been resolved: up to eight laser emission lines can be programmed, leaving enough space for recording fluorescence, while the frequencies can be adjusted;
6. The ratio of dyes, like the ratio of excitation of metabolite - samples, for example, for Ca 2+, membrane potential, pH value, should quickly switch during sequential scanning. AOBS switches in a few microseconds;
7. Image registration in reflected light is another possibility of use. AOBS allows the transmission of reflected excitation light to be individually adjusted;
8. ROI scanning (sequential scanning and specific area scanning) is also improved: different excitation modes are applicable to different areas during a single scan;
9. Large volume 3D recordings in sequential mode benefit from fast switching devices as speed increases system efficiency;
10. Fluorescence correlation spectroscopy (FCS) requires low background and low light scattering. Only AOBS effectively blocks nearby emission lines, for example, from Ar lasers;
11. Spectral recording (Lambda scan) provides an accurate spectrum since the light transmission of AOBS is “white”, meaning it does not introduce changes to the emission spectrum - a common problem when performing spectral scans on a dichroic mirror system;
12. True confocal optical sectioning requires spot illumination and spot detection of fluorescence. AOBS is suitable for use with spot scanning confocal devices;
13. Imaging in multiphoton mode or with ultraviolet excitation can be performed in parallel without errors or limitations. AOBS does not change the excitation of invisible lasers and does not change the fluorescence spectrum;
14. It is not possible to perform erroneous operation since AOBS is controlled in conjunction with excitation control using AOTF (Acousto-Optical Tunable Filter). If the excitation line is selected, then the AOBS is programmed according to it. The operator does not need to make decisions - the work is performed correctly and automatically;
15. There is no adjustment, since there are no moving elements inherent in filter drums and sliders. The crystal is firmly mounted and programming is done electronically;
16. There is no need for expensive additional equipment, such as filter cubes, dichroic mirror sliders, etc. Therefore, the cost of technical assistance for installing new optical elements is much lower.
Rice. 14. AOBS – acousto-optical beam splitter.
SP-Detector – spectral photometer sensor. Light from the sample, which is the sum of the induced fluorescence spectra, passes through a pinhole diaphragm, which forms a confocal optical section. Next, using a prism, this light is decomposed into a spectrum. When passing the first detector, the light passes through a slit photometer device consisting of two motorized curtains. These curtains cut off the edges of the spectrum on both sides of the range and direct them to the 2nd and 3rd order sensors. As a result, the spectrum is split simultaneously into five channels. As a result, when using an SP detector, radiation from various dyes in the sample is recorded.
Rice. 15. Spectral detector SP.
The SP detector allows lambda scanning: spectral images are accumulated to immediately analyze the characteristics of the dyes involved in the experiment.
June 4, 2013A significant part of the scientific research carried out at the Faculty of Biology of Moscow State University, and the successful implementation of closely related educational programs, are unthinkable without the use of the most modern microscopic technology. As part of the Development Program of Moscow University, an interdepartmental laboratory of confocal microscopy was created at the Faculty of Biology, fully equipped with the necessary equipment and operating effectively.
3T3 fibroblasts on the walls of the matrix macropore
Text: Tretyakov Artemy
What can it do?
By using confocal microscope you can obtain several images of virtual sections of a cell, or a three-dimensional model assembled from them. Such possibilities are provided by a laser, the beam of which can be focused at any point in the cell. To obtain an image, the object must be fluorescently active, that is, when hit by a laser beam, it (or rather, certain molecules in its composition) must emit light with a longer wavelength than that incident on it, which is captured by the microscope. The image is built precisely on the basis of this fluorescence.
Photo
How does it work?
“The current level of development of fundamental and applied interdisciplinary scientific research, as well as the tasks of training specialists with interdisciplinary competencies, require intensive development and modernization of the material and technical base” (Development Program of Moscow University, p. 5 ;).
In addition to the laser, the electronic-mechanical device also includes an optical filter that transmits the laser beam, but reflects longer wavelength light onto a diaphragm called “pinhole” (from the English Pinhole - a hole made by a pin, the literal translation perfectly characterizes the pinhole device), which cuts off all unnecessary background light and thereby reduces or completely negates the influence of the underlying optical layers on the clarity of the display of the scanned area of the cell. If the background light were not cut off by the pinhole, the optical layers would overlap each other and interfere with the viewer - as if he were looking through foliage into the distance. Behind the diaphragm there is a photodetector that digitizes the received information.
It is clear that such “spot” scanning takes time. To speed up this process, the confocal system uses another module called a spinning disk, which makes it possible to obtain an image of not just one point, but an entire line in one act of laser operation.
A patent for such a system was received in 1961 by MIT professor Marvin Minsky. Its active use began in the 80s, and now confocal microscopy is experiencing its heyday. The first microscope appeared in Russia in 2003, it was the Axiovert 200M LSM510 Meta. Others followed, and now there are several large laboratories in our country, including the interdepartmental laboratory at the Faculty of Biology of Moscow State University. The laboratory has five microscopes, including: Olympus FV10i, Zeiss LSM710, Nicon Eclipse Ti-EAL.
Confocal microscopy has achieved not only the highest contrast and three-dimensionality, it has also mastered the fourth dimension - time, making it possible to study changes occurring in cells. For these purposes, each installation is equipped with systems for maintaining their life. All this provides ample opportunities for researchers who successfully use these opportunities.
And, since we are talking about the laboratory of the Faculty of Biology of Moscow State University, the research carried out in this laboratory should be mentioned as examples.
Silk and web
Confocal microscopy has achieved not only the highest contrast and three-dimensionality, it has also mastered the fourth dimension - time, making it possible to study changes occurring in cells.
One of the interesting works is the creation of biodegradable prostheses for the restoration of blood vessels and other hollow organs. In the simplest cases, such tissue-engineered structures look like tubes or films that are placed on the site of damage and thus act as a “patch.” They can be made from different materials, including silk fibroin from silkworm cocoons. The advantage of this material is that it is stable and forms a flexible and durable structure of the prosthesis. The prosthesis itself looks like a film only when viewed with the naked eye; in fact, it is a matrix - a mesh multilayer base, a frame that imitates the structure of living tissue. This scaffold helps the new cells get into the correct position, ensuring their even distribution. As a result, the damaged organ wall is restored, and the unnecessary frame undergoes biodegradation. But it is not so easy to check how evenly the cells are distributed throughout the framework, and whether they live well in the deep layers of the matrix (whether there are any difficulties in gas exchange and the exchange of metabolic products), and whether they change morphology when penetrating inside it. It was at this stage that the use of a microscope (Zeiss Axiovert 200M LSM 510) greatly simplified the task. Mainly due to the ability to examine an object without destruction, as well as due to the ability of the microscope to look inside the matrix to a depth of 600 microns.
Photo
Similar work was carried out with bone tissue, the matrix for which was made both from the same fibroin and from spidroin, a protein originally discovered in the body of the spider and used by it to weave webs. In laboratory conditions, the protein is produced not by spiders at all, but by yeast, into whose genome a slightly modified spider gene is introduced, responsible for the synthesis of this protein.
Not so simple
But, if the occurrence of damage in tissues is a simple and understandable process, other pathologies are not always clear. And before starting effective treatment and, especially, preventing their development, it is necessary to find out the mechanism of their occurrence. These include atherosclerosis - a disease of the arteries, as a result of which lipids accumulate in the vessel wall, or more precisely, in the intima - its inner layer, making the wall thicker, which ultimately can even lead to complete blockage of the vessel. What causes this disease is not entirely clear, just as it is not clear what role is played by immunocompetent cells, in places where they accumulate, atherogenesis soon begins. Complete answers to these questions have not yet been found, but the study of atherogenesis continues, although there are much fewer publications on this topic than on the topic of tissue-engineered prostheses.
Photo
Fates of molecules
The interdepartmental laboratory of confocal microscopy is regularly used in their work by more than 20 undergraduate and graduate students from almost 10 departments.
The studies described above mainly monitor the state of cells. But the capabilities of a confocal microscope are not limited to this; it is quite capable of tracking even the fate of an individual molecule in a cell, as long as it has fluorescent activity. To do this, molecules are usually labeled with fluorochromes - special dyes that have this activity. Fluorochromes exist as individual molecules, and their labeling involves chemically binding the fluorochrome to the molecule of interest to the researcher. After this, such labeled molecules enter the cell and their further fate is monitored using a microscope. In this way, for example, the activity and movement in the cell of ricin and viscumin were compared. Both substances are protein toxins, both are of plant origin and consist of two parts - subunits, one of which is responsible for binding to the cell and penetrating inside, and the other for inactivating the ribosome, which ultimately leads to cell death. The practical benefit is again associated with the treatment of another dangerous disease - cancer. This clear “separation of responsibilities” between subunits makes it possible to replace the first subunit, for example, with an antibody. The result is an “immunotoxin” that acts only on certain cells to which this antibody is suitable. There are two main problems. First: choosing an antibody that will approach cancer cells and prevent the toxin from penetrating healthy ones. And second: choosing a toxin that, when converted into an immunotoxin, will remain highly effective.
Photo: Primary culture of heart cells of newborn rat pups: A - control, B - treated with 20 µM isoproterenol for 24 hours. 1 - TMRE staining of mitochondria; 2 - phase contrast. Scale 10 µm. (Smirnova T.A., Saprunova V.B. “Adaptive changes in mitochondria of cultured cardiomyocytes of newborn rats under the influence of isoproterenol.” International conference “Receptors and intracellular signaling” MAY 24-26, 2011. Pushchino).
Education
The list of works carried out using confocal microscopy can be continued for a long time. This effective method, which also allows one to work with living cells, is in demand in almost any scientific research. And therefore, preparing students to work in this laboratory plays a huge role. It is regularly attended by more than 20 undergraduate and graduate students engaged in course work, pre-course work and diploma work.
Embryologists, molecular biologists, cytologists, bioengineers, immunologists, and zoologists work at the facilities.
The Laboratory of Confocal Microscopy was founded in 2004.
Head of the laboratory – associate professor M.M. Moisenovic
Young specialists A.A. Ramonova and A.Yu. Arkhipova work in the laboratory
Currently, the laboratory has 2 confocal systems installed:
- Axiovert 200M with confocal attachment LSM510 META (Carl Zeiss, Germany)
- Confocal laser scanning system, manufactured by NIKON CORPORATION (Japan) - an inverted medical and biological microscope for laboratory research Eclipse with accessories: with a Ti-E stand, with a TIRF illuminator, with an A1 confocal module and a spinning disk-based confocal module.
All news "
Margolin 389p.
Optical microscopy used all the achievements of both technology and technology, as well as information and computer technologies. This led to significant improvements in existing equipment and methods for its use, which, in turn, led to the emergence of new methods, in particular, confocal microscopy. A confocal microscope differs from a classical optical microscope in that at each moment in time an image of one point of an object is recorded, and a full image is constructed by scanning (moving the sample or rearranging the optical system). Thus, the principle of scanning electron microscopy is implemented in a unique form, which makes it possible to record and process the signal from each individual point for as long as desired.
In a classical microscope, light from various points of the sample enters the photodetector. In a confocal microscope, in order to record light from only one point, a small diaphragm is placed after the objective lens in such a way that the light emitted by the analyzed point passes through the diaphragm and will be recorded, and the light from the remaining points is mainly blocked by the diaphragm, like this shown in Fig. 7.28.
Rice. 7.28. Scheme of beam transmission in a confocal optical microscope
Another feature is that the illuminator does not create uniform illumination of the field of view, but focuses the light onto the analyzed point. This can be achieved by placing a second focusing system behind the sample, but this requires that the sample be transparent. In addition, objective lenses are usually expensive, so using a second focusing system for illumination is of little preference. An alternative is to use a beam splitter so that both incident and reflected light are focused by a single lens. This scheme also makes adjustment easier.
Let us now consider how and how quantitatively the contrast changes when using confocal microscopy. Since light passes through the lens twice in a confocal microscope, the point blur function (hereinafter denoted PSF) will be the product of the independent probabilities that a photon will hit a point with its coordinates or a photon will be detected from this point.
If we use the Rayleigh criterion for resolution, it turns out that the resolution in a confocal microscope increases, but not significantly. For a confocal microscope we have an expression for resolution r:
While for a conventional microscope:
However, the main advantage of a confocal microscope is not an increase in resolution in the sense of the Rayleigh criterion, but a significant increase in contrast. In particular, for a conventional PSF in the focal plane, the ratio of the amplitude at the first side maximum to the amplitude at the center is 2%, and for a confocal microscope this ratio will be 0.04%. It follows from this that a dim object with an intensity, for example, 200 times less than that of a bright object, cannot be detected in a conventional microscope, although the distance between objects may be significantly greater than the distance prescribed by the Rayleigh criterion. At the same time, such an object should be well recorded in a confocal microscope.
An important parameter is the size of the apertures in the focal plane of the irradiating and collecting lenses. The image of the aperture in the object plane determines from which areas the light is detected by the photodetector. Obviously, reducing the aperture size leads to a decrease in the amount of light transmitted, increases the noise level and ultimately can negate any achieved contrast benefits. Thus, the question arises about the optimal choice of aperture size and a reasonable compromise.
An aperture with a hole size smaller than the Airy spot simply results in a loss of intensity and has no effect on resolution. A single-spot Airy aperture allows maximum use of the resolving power of the objective lens. However, a diaphragm with an opening size approximately 3 to 5 times larger than the Airy spot appears to be the most suitable compromise. It should be understood that the size discussed here refers to the size of the image in the object plane, and therefore the actual size of the aperture hole depends on the magnification of the lens. Specifically, when using a 100x lens, an aperture with a 1 mm aperture will project into the object plane into a circle of 10 µm radius.
The development of the idea of confocal microscopy was the development of a confocal laser scanning microscope (KJICM), which was caused by the need for more sensitive and metrologically rigorous methods for analyzing the shape and spatial structure of observed objects. A schematic diagram of the CLSM with the main functional connections is shown in Fig. 7.29.
The main feature of CLSM is the possibility of layer-by-layer imaging of the object under study with high resolution and low noise level. This is achieved by step-by-step scanning of the object with a focused beam of light from a coherent source or by moving the stage using special fluorescent probes and special methods for limiting light fluxes.
Rice. 7.29. Block diagram of KJICM:
1 - scanning table; 2 - test sample; 3, 6 - lenses; 4 - scanning device; 5 - beam splitter plate; 7, 9 - needle diaphragms; 8 - radiation receiver; 10 - laser; 11 - Control block; 12 - computer; 13 - axis scanning drive z.
The use of a pinhole diaphragm in CLSM, the dimensions of which are coordinated with the microscope magnification and wavelength, makes it possible to increase the resolution by more than 10%. It is obvious that the resolution of CLSM and, accordingly, the capabilities of analyzing fine structures can exceed the similar capabilities of a conventional microscope by no more than 40% under conditions of scanning a sample with a thin beam. The resolution of KLCM depends on the microscopy method and lighting. KLCM resolution is determined both optical system and electronic path information processing. Therefore, in the design of the KLCM, its circuits, parameters such as the resolution of the optical system, scanning step, detector characteristics must be coordinated, and optimal processing algorithms and appropriate software must be selected.
In general, the depth of field of KLCM depends on the aperture, wavelength, coherence of light sources and the size of the pin diaphragm. The needle diaphragm is the main design element that distinguishes KLCM from other types of microscopes. Needle diaphragms are designed to create conditions for maximum or complete filtration of light entering the image formation plane from points that do not coincide with the focal plane or are located next to the analyzed element of the object in the focal plane.
Selecting the optimal needle diaphragm diameter is important to obtain the required device characteristics. Relationships for estimating the lateral resolution and depth of field of KJICM are obtained under the assumption that the needle diaphragm has a small aperture, being a luminous point. In reality, the size of the needle diaphragm is finite and the transverse resolution of the device and the brightness of the illuminated elements of the sample, shifted relative to the focal plane along the axis, depend on it z, and depth of field. With small needle diaphragm diameters, the luminous flux becomes low, which reduces the signal-to-noise ratio and reduces contrast. At larger diameters, the effectiveness of the needle diaphragm is reduced by reducing the aperture.
Basic concept
Confocal point sensor principle from the Minsk patent
The principle of confocal microscopy was patented in 1957 by Marvin Minsky and seeks to overcome some of the limitations of traditional wide-field fluorescence microscopes. In a conventional (i.e. wide field) fluorescence microscope, the entire sample is flooded uniformly with light from the light source. All parts of the sample in the optical path are excited at the same time and the resulting fluorescence is detected using a photodetector microscope or cameras, including the large out-of-focus background part. In contrast, a confocal microscope uses an illumination point (see point spread function) and a tiny hole in an optically coupled plane at the front of the detector to defocus the signal—the name "confocal" comes from this configuration. Once light emitted by fluorescence very close to the focal plane can be detected, the image at optical resolution, particularly in the depth direction of the sample, is much better than that of wide-field microscopes. However, since most of the fluorescence light from the sample is blocked by the puncture, this increased resolution comes at the expense of reduced signal intensity - so long exposures are often required. To compensate for this signal drop after puncture The light intensity is detected using a sensitive detector, usually a photomultiplier tube (PMT) or an avalanche photodiode, converting the light signal into an electrical signal that is recorded by a computer.
Once one point in the sample is illuminated at a time, 2D or 3D images are required to be scanned over a regular raster (ie, a rectangular pattern of parallel scan lines) in the sample. The beam is scanned across the sample in a horizontal plane using one or more (servo-controlled) oscillating mirrors. This scanning method typically has low reaction lag and scanning speed can be varied. Slow scanning provides a better signal-to-noise ratio, resulting in better contrast and higher resolution.
The achievable focal plane thickness is determined primarily by the wavelength of the light used, divided by the numerical aperture of that lens, but also by the optical properties of the sample. Fine optical sectioning makes these types of microscopes particularly good at 3D imaging and surface profiling of samples.
Consecutive slices make up a "Z-stack", which can either be processed by certain software to create a 3D image, or it is combined into a 2D stack (predominantly the maximum pixel intensity is taken; other common methods include the use of standard deviation or pixel stacking).
Confocal microscopy provides the capacity for direct, non-invasive, serial optical sectioning of intact, fat and living specimens with a minimum of sample preparation and little improvement in lateral resolution. Biological samples are often treated with fluorescent dyes to make selected objects visible. However, the actual dye concentration can be kept low to minimize disruption to biological systems: some instruments can track individual fluorescent molecules. In addition, transgenic techniques can create organisms that produce their own chimeric fluorescent molecules (such as fusion of GFP, green fluorescent protein, with a protein of interest). Confocal microscopes operate on the principle of point excitation in a sample (diffraction limited to point) and detection of the resulting fluorescent signal point. The pinhole on the detector provides a physical barrier that blocks out-of-focus fluorescence. Only the focus, or central spot of the Airy disk, is recorded. Raster scan the sample at a single point, while allowing thin optical sections to be collected by simply changing the Z-focus. The resulting images can be stacked to produce a 3D image of the sample.
Methods used for horizontal scanning
Four types of confocal microscopes are commercially available:
Confocal laser scanning microscopes use multiple mirrors (usually 2 or 3 scans linearly along the x- and y-axis) to scan the laser onto the sample and "descan" the image through a fixed pinhole and detector.
Benefits
CLSM is widely used in many biological scientific disciplines, from cell biology and genetics to microbiology and developmental biology. It is also used in quantum optics and nanocrystalline imaging and spectroscopy.
Biology and Medicine
An example of a stack of confocal microscopy images showing the distribution of actin filaments throughout a cell.
Clinically, CLSM is used in the evaluation of various ocular diseases, and is particularly useful for imaging, qualitative analysis, and quantification of endothelial cells in the cornea. It is used to localize and identify the presence of filamentous fungal elements in the corneal stroma in cases of keratomycosis, allowing rapid diagnosis and thus early establishment of definitive therapy. Research into CLSM techniques for endoscopic procedures (endomicroscopy) also shows promise. In the pharmaceutical industry, it has been recommended to monitor the manufacturing process of thin pharmaceutical film forms to control the quality and uniformity of drug distribution.
Optics and crystallography
CLSM is used as a data retrieval mechanism in some 3D optical data storage systems and has helped determine the age of the Magdalene papyrus.
Options and improvement
Improved axial resolution
The point spread function is a point ellipsoid, several times as long as it is wide. This limits the axial resolution of the microscope. One method to overcome this is 4π microscopy, where incident and emitted light or can interfere with both the top and bottom of the sample to reduce the volume of the ellipsoid. Alternative technique confocal microscopy theta. In this technique, the cone of illuminating light and the detection light are positioned at an angle to each other (best results when they are perpendicular). The intersection of two handicap functions gives a much smaller effective sample volume. From this evolved a single-plane illumination microscope. Additionally, deconvolution can be used using an experimentally derived point spread function to remove out-of-focus light, improving contrast in both axial and lateral planes.
Super resolution
There are confocal variants that achieve resolution below the diffraction limit, such as stimulated emission depletion microscopy (STED). Besides this technique, a wide variety of other (non-confocal based) super-resolution techniques are available like palm, (e)STORM, SIM cards, and so on. They all have their advantages, such as ease of use, resolution and the need for special equipment, buffer or fluorophore.
Low Temperature Performance
For imaging samples at low temperatures, two main approaches have been used, both laser scanning confocal microscopy-based architectures. One approach is to use a continuous flow cryostat: only the sample is kept at low temperature and is optically addressed through a transparent window. Another possible approach is to place part of the optics (especially an objective microscope) in a cryogenic storage Dewar flask. This second approach, although more cumbersome, guarantees better mechanical stability and avoids losses due to the window.
Images
Partial profile of the surface of a 1-Euro coin, measured using Nipkow disk confocal microscopy.
Reflection of data for 1-euro coin.
story
Beginning: 1940-1957
First confocal scanning The microscope was built by Marvin Minskow in 1955 and a patent was filed in 1957. Scanning a focal plane illumination point was achieved by moving the stage. No scientific publication was presented, and no images made of it were preserved.
Tandem scanning microscope
Scheme of Petran's tandem scanning microscopy. A red bar has been added to indicate Nipkow disk.
In 1960, the Czechoslovakian Mojmir Petran Faculty of Medicine at Charles University in Pilsen developed the Tandem scanning microscope, the first commercialized confocal microscopy. It was sold to a small company in Czechoslovakia and in the United States by Tracor-North (later NORAN) and used a rotating Nipkow disk to generate multiple micro-hole excitations and emissions.
The Czechoslovak patent was filed in 1966 by Petran and Milan Hadravský, a Czechoslovak colleague. The first scientific publication with data and images obtained with this microscope was published in the journal Science in 1967, authored by M. David Egger of Yale University and Petran. A footnote to this article mentions that Petran designed the microscope and supervised its construction, and that he was, in part, a "research fellow" at Yale University. A second edition from 1968 described the theory and technical details of the instrument and had Hadravský and Robert Galambos, group leader at Yale University, as additional authors. A US patent was issued in 1970. It was filed in 1967.
1969: First confocal laser scanning microscopy
In 1969 and 1971, M. David Egger and Paul Davidovits of Yale University, published two papers describing the first confocal laser scanning microscopy. This was a scanner spot, meaning only one spot illumination was generated. It uses epi-illumination-reflection microscopy to observe neural tissue. A 5 mW helium-neon laser with a wavelength of 633 nm reflected light from a translucent mirror towards the target. The goal was a simple lens with a focal length of 8.5 mm. Unlike all previous and most recent systems, the sample was scanned by moving this lens (the scanning target), which causes the focal point to move. The reflected light returned to the translucent mirror, the transmitted part was oriented by another lens to point detection, behind which the photomultiplier tube was placed. The signal was visualized using a CRT oscilloscope; the electron beam was transferred simultaneously to the target. A special device made it possible to take Polaroid photographs, three of which were featured in a 1971 publication.
The authors reflect on fluorescent dyes for in vivo studies. They cite the Minsky patent, thanks to Steve Baer, then a doctoral student at the Albert Einstein School of Medicine in New York, where he developed a confocal line scanning microscope, who proposed using a laser with a "Minsky microscope" and thanks to Galambos, Hadravsky and Petran for discussions. leading to the development of his microscope. The motivation for their development was that in Tandem scanning microscopy only a 10 -7 fraction of the illuminating light is involved in generating the image in part of the eye. Thus, image quality was not sufficient for most biological studies.
1977-1985: Spot scanners with lasers and scene scanning
In 1977, Colin JR Sheppard and Tony Wilson described confocal epi-laser-illuminated, scanning stage and photomultiplier tube detectors. The stage could be moved along the optical axis (Z-axis), allowing optical serial sections.
In 1979, Fred Brakenhoff and his colleagues showed that the theoretical benefits of optical sectioning and improved resolution were actually achievable in practice. In 1985, this group became the first to publish compelling confocal microscopy images that could answer biological questions. Soon after, many more groups began to use confocal microscopy to answer scientific questions that still remained a mystery due to technological limitations.
In 1983, IJ Cox und S. Sheppard of Oxford published the first work on a computer-controlled confocal microscope. The first commercial laser scanning microscope, the stage-scanner SOM-25 was offered by Oxford Optoelectronics (following several TAKE-frame acquisitions by BioRad) beginning in 1982. It was based on the Oxford group's design.
Since 1985: Laser point scanners with beam scanning
In the mid-1980s, William Bradshaw Amos and John Graham White and colleagues working in the Laboratory of Molecular Biology in Cambridge built the first confocal beam scanning microscope. The sample stage does not move, instead illuminating the spot, allowing for faster image acquisition: four images per second with 512 lines each. Intermediate images are greatly exaggerated, due to the beam path being 1-2 meters long, allowing the use of a conventional iris diaphragm as a "pinhole", with a diameter of ~1 mm. The first micrographs were taken by prolonged exposure to film before the addition of the digital camera. Further improvement allowed scaling into preparation for the first time. Zeiss around the same time led to the commercial CLSM distributed by the Swedish company Sarastro. The enterprise was acquired in 1990 by Molecular Dynamics, but CLSM was eventually discontinued. In Germany, Heidelberg Instruments, founded in 1984, developed CLSM, which was originally meant for industrial applications rather than biology. This document was transferred in 1990 to Leica Lasertechnik. Zeiss was already a non-confocal flying spot laser scanning microscope on the market, which was upgraded to a confocal one. The 1990 report noted "some" manufacturer confocals listed: Sarastro, Technical Instruments, Meridian Instruments, Bio-Rad, Leica, Tracor-Nordic and Zeiss.
In 1989, Fritz Karl Preikschat, with his son Ekhard Preikschat, invented the laser diode scanning microscope for particle size analysis. He and Ekhard Preikschat co-founded Lasentec to commercialize. In 2001, Lasentec was acquired by Mettler Toledo (NYSE: MPD). About ten thousand systems have been installed worldwide, mainly in the pharmaceutical industry to provide in-situ control of the crystallization process in large purification systems.
- Two-photon excitation microscopy: Although they use appropriate technology (both laser scanning microscopes), multiphoton fluorescence microscopes are not strictly confocal microscopes. Term confocal arises due to the presence aperture V conjugate focal plane(confocal). This aperture is usually missing in multiphoton microscopes.
- Total internal reflection fluorescence microscope (TIRF) o
confocal microscopy- Virtual CLSM (Java-based)
- Animation and explanation on different types of microscopes, including fluorescence and confocal microscopes. (Université Paris Sud)
Basic Concepts
Although modern instruments differ significantly from the earliest versions, the confocal imaging principle pioneered by Marvin Minsky and patented in 1957 is used in all modern confocal microscopes. In traditional wide-field microscopes, the entire sample is illuminated by a mercury or xenon light source, and the image is either observed visually or projected onto an image recorder or photographic film. The method of image formation with a confocal microscope is fundamentally different. Illumination is accomplished by scanning the entire surface of the sample with one or more focused beams of light, usually from a laser arc source. The illuminated area of the sample is focused by a lens and then scanned using a computer-controlled scanning device. The sequence of light rays from the sample is detected through a pinhole diaphragm (or in some cases a slit diaphragm) by a photomultiplier tube (PMT), whose output is converted into an image displayed on a computer. Although unstained specimens can be observed by light reflected from them, they are usually marked with one or more fluorescent dyes.
Image acquisition methods
Confocal microscopy uses a variety of imaging techniques to examine a huge number of different types of samples. All of them are based on the technical ability to obtain high-definition images, called optical sections, in a sequence of relatively thick sections or the entire sample (whole mount). The optical slice is the basic element of the image. The images themselves are obtained by observing bonded and stained samples under single, dual, triple and multi-wavelength illumination modes, and the images generated using different lighting and staining techniques will be accurately correlated with each other. It is possible to obtain images of a living cell and a temporally unfolded sequence of images (registration of images in a given time interval), and numerical processing methods applied to image sequences allow the creation of an integrated whole image from a series of z-axis images and three-dimensional images of samples, as well as representation three-dimensional data in time sequence, that is, a four-dimensional image. Early confocal microscopes imaged using reflected light, but in fact, a laser scanning confocal microscope can be used to image using any transmitted light source commonly used in microscopy.
Creating an Image
The procedures for sample preparation and imaging with confocal microscopes are essentially those that have been developed over the years in traditional wide-field microscopy. In biomedicine, the main application of confocal microscopy is the imaging of bound or living cells and tissues, which are typically stained with one or more fluorescent tags. There are a large number of different fluorescent dyes that can be incorporated into relatively simple protocols and used to stain specific cellular organelles and structures. Among the huge variety of dyes available, there are, for example, dyes for the nucleus, Golgi apparatus, endoplasmic reticulum, mitochondria, as well as dyes such as fluorescent phalloidin, which indicates polymerized actin in cells. Regardless of the sample preparation method used, the main advantage of confocal microscopy is the flexibility in image presentation and analysis that results from the simultaneous acquisition of multiple images and their digital presentation on a computer.
Critical aspects of confocal microscopy
Quantitative 3D imaging in fluorescence microscopy is often complicated by artifacts introduced during sample preparation due to controlled and uncontrolled experimental quantities or microscope positioning and layout issues. This article, written by Dr. James B. Pauley, systematizes the most common external factors that often obscure results obtained in wide-field fluorescence and confocal microscopy. Topics discussed include the laser system, alignment of optical components, objective magnification, bleaching artifacts, aberrations, immersion oil, coverslip thickness, quantum efficiency, and sample environment.
Aberrations in multicolor confocal microscopy
Design improvements have simplified confocal microscopy to the point that it has become a common tool for cell biology research. But as confocal microscopes have become more powerful, greater demands have been placed on their optics. In fact, optical aberrations that cause minor image defects in wide-field microscopy can have devastating effects in confocal microscopy. Unfortunately, the stringent optical requirements of confocal microscopy are often hidden by optical systems that guarantee sharp images even with a weak microscope. Optical manufacturers produce many different microscope lenses designed for specific applications. This article shows how objective trade-offs impact confocal microscopy.
Three-color imaging in confocal microscopy
A laser scanning confocal microscope (LSCM) is commonly used to obtain digital images of fluorescent samples labeled with one, two, and three labels. The use of red, green, and blue (RGB) colors is most informative for representing the light distribution of up to three fluorescent cell marks when a complementary color is used for each relative position and when the images of different colors form a single three-color pattern. In this section, we will look at a simplified version of a recently published method for obtaining three-color confocal images using the popular image processing program, Adobe Photoshop. In addition, several applications for creating a three-color imaging protocol for confocal image presentation are discussed. It is worth keeping in mind that these numerical methods are not limited to LSCM images and can be applied to digital images imported into Photoshop from other sources.
Basics of Confocal Reflectance Microscopy
Confocal reflectance microscopy can be used to obtain additional information about a sample with relatively little additional effort because the techniques require minimal sample preparation and equipment changeover. In addition, with confocal reflectance microscopy, information from unstained tissues is as readily available as data obtained from stained samples that reflect light. This method can also be combined with more common fluorescence imaging techniques. Examples of recent applications include recording unstained cells within a population of fluorescently stained cells and observing interactions between fluorescently stained cells growing on an opaque structured substrate.
Confocal Image Gallery
The Nikon MicroscopyU Confocal Image Gallery is a sequence of digital images obtained using a Nikon PCM-2000 confocal microscope combined with an Eclipse E-600 upright microscope. A sequence of images of optical sections in different planes of the sample was obtained by scanning along the optical axis of the microscope. The sequence is represented by an interactive Java application that allows you to either “play” a series of slices automatically or scroll them back and forth like slides.
Laser scanning confocal microscopy
Several techniques have been developed to overcome the phenomenon of poor contrast inherent in images of thick specimens typically produced by microscopes. Confocal and deconvolution techniques produce significantly better images of medium-thick samples (5 to 15 microns). Images of the thickest specimens (20 microns or more) are degraded by exposure to high ambient light from out-of-focus areas and are perhaps best obtained by confocal microscopy techniques. Using this tutorial, samples are presented as a series of optical sections along the z-axis using a virtual confocal microscope.
Reflectance confocal microscopy
Use this tutorial to explore individual surface layers of integrated circuits. Digital images for guidance were acquired using a Nikon Optiphot C200 reflectance confocal microscope. For each series, a sequence of optical sections was recorded along the z axis as the microscope penetrated and focused deep into (in 1-micrometer increments) the mosaic of circuits on the surface of the silicon crystal.
Basic provisions
Compared to traditional microscopy, confocal microscopy has several advantages, including a shallow penetration depth into the sample under study, the absence of background illumination, and the ability to obtain a series of optical sections of thick samples. In biomedicine, the main application of confocal microscopy is the imaging of bound or living cells and tissues, which are typically labeled with one or more fluorescent tags.
Rice. 1. Scheme of ray path in confocal microscopy
When imaging fluorescent samples with a traditional wide-field microscope, secondary luminescence emitted by the sample from areas outside of the study often affects the clarity of the image of in-focus features. This is especially problematic for specimen thicknesses greater than 2 micrometers. Confocal microscopy provides modest improvements in both axial and in-plane resolution; But it is the ability to eliminate the background light that occurs in fluorescently stained samples of large thickness that has caused a recent surge in popularity of this research method. Being relatively easy to operate, most modern confocal microscopes have become part of the basic equipment in many multi-user imaging systems. Since the resolution achieved by a laser scanning confocal microscope (LSCM) is slightly better than a traditional wide-field optical microscope, but still significantly lower than the resolution of a transmission electron microscope, it, in a sense, has become a bridge between the two most common research methods. Figure 1 shows a schematic diagram of the passage of light in a basic configuration confocal microscope.
In traditional wide-field microscopes, the entire sample is illuminated with a mercury or xenon light source, and the image is either observed visually or projected onto an imager or photographic film. The method of obtaining an image with a confocal microscope is fundamentally different. The sample is illuminated by scanning it with one or more focused beams, usually a laser (Figure 2). The images obtained by scanning the sample are thus called optical sections. This terminology refers to a non-invasive testing technique in which images are obtained using focused light rather than by physically dissecting the sample.
Rice. 2. Wide-field and spot scanning of samples
Confocal microscopy has greatly simplified the study of living specimens, making it possible to obtain data in three dimensions (z-series) and improving the process of imaging multistained specimens. Figure 3 compares a traditional episcopic fluorescence image with a confocal image of the same sections of a butterfly pupa whole mount with propidium iodide-stained epithelium. An impressive increase in resolution and, as a consequence, sharpness of the image of nuclei in an LSCM image is evident, due to the elimination of out-of-focus fluorescent emission.
Laser scanning confocal microscope (LSCM)
LSCM is now the most common version of confocal microscopes used in biomedicine. In the introduction, special attention is paid to LSCM, since the design and structure of these microscopes allows even novice users to work with them. Other design solutions have occupied their own special niches in biology. For any model or modification of a confocal microscope, most of the rules for sample preparation apply, with minor modifications, as do other techniques based on optical sectioning, such as deconvolution and multiphoton techniques.
Development of confocal microscopy
The invention of the confocal microscope is credited to Marvin Minsky, who created a working microscope in 1955. The development of confocal microscopy was largely driven by the desire to observe biological processes in living tissue (in vivo), and Minsky's goal was to image the neural network in an unstained preparation of a living brain. The principles of confocal microscopy, pioneered by Minsky and patented in 1957, are used in all modern confocal microscopes. Figure 1 explains the confocal principle, as applied to epifluorescence microscopy, which forms the basis of all modern confocal systems used in fluorescence imaging. In the original configuration, Minsky used a pinhole (diaphragm) placed opposite a zirconium arc light source used as a pinhole light source.
Rice. 3. Butterfly wing epithelium
Light from a point source was focused in the form of a point by a lens on a given focal plane in the sample and, passing through it, was focused by a second lens on a second pinhole (pinhole), which was in focus with the first (they were cofocal, i.e., confocal). The beams passing through the second pinhole hit a low-noise photomultiplier tube, which generated a signal depending on the brightness of the light coming from the sample. A second pinhole cut off light coming from regions above or below the focal plane in the sample from the photomultiplier tube. The use of spatial filtering to eliminate out-of-focus light and flare when working with samples thicker than the focal plane is a key principle of confocal microscopy. In his work, Minsky also described a reflectance microscope with a single lens and a dichromatic mirror, the design of which became the basis of systems used today.
To obtain a confocal image, it is necessary to scan the sample with light focused to a point. In the original device, assembled by Minsky, the light beam was stationary, and the sample itself moved on a vibrating stage. The immobility of the scanning beam relative to the optical axis of the microscope was an advantage of this installation, since it eliminated most optical defects that could distort the image. However, when studying biological samples, this could cause fluctuations and distortion, ultimately leading to a loss of resolution and image clarity. Moreover, when moving the stage and sample, it is impossible to perform any manipulations, such as, for example, microinjections of fluorescently stained cells.
But, regardless of the method of scanning the sample, it is necessary to obtain its image. And Minsky's original design did not produce a real image because the output from the photomultiplier tube was fed to a long-persistence oscilloscope used by the military, which had no recorder. Minsky later wrote that the underwhelming quality of his images was not due to the low resolution of the microscope itself, but to the low resolution of the oscilloscope display. It is now clear that due to a lack of technology, Minsky could not fully demonstrate the full potential of the confocal method, especially when imaging biological structures. He pointed out that this may be the reason why confocal microscopy was not immediately accepted by the very demanding community of biologists, for whom the quality of the resulting images was always a priority. At that time, they had at their disposal light microscopes with excellent optics, allowing them to observe and photograph brightly colored histological sections on highly sensitive color film. In modern confocal microscopes, images are generated from signals coming from a photomultiplier tube or captured by a digital charge-coupled device camera, processed directly by a computer imaging system, and displayed on a high-resolution screen and image documentation device of superior quality. A diagram of a modern laser scanning confocal microscope is shown in Figure 4.
Rice. 4. Scheme of a modern laser scanning confocal microscope
The basic optics of an optical microscope have not changed fundamentally for decades, since the final resolution of the instrument is determined by the wavelength, objective lens, and properties of the sample itself. Dyes used to enhance the contrast of samples and other optical microscopy techniques have improved significantly over the past 20 years. The rise and improvement of the confocal method was a consequence of the revival of optical microscopy, caused in large part by the success of modern technologies. Many of the technological advances that could have benefited Minsky's design are gradually becoming available (and affordable) to biologists and other microscopists. Among them, stable multi-frequency lasers used as improved point light sources, improved dichromatic mirrors, sensitive low-noise photodetectors, high-speed microcomputers with enhanced capabilities (due to the availability of high-capacity memory), sophisticated imaging software, high-resolution monitors and digital printers.These technologies developed independently and have been gradually integrated into confocal imaging systems since 1955. For example, digital image processing techniques were first successfully used in the early 1980s by researchers at the Woods Hole Oceanographic Institute. Using what they termed “video microscopes,” they were able to obtain images of the cellular structure of microtubes smaller than the theoretical resolution limit of an optical microscope. The apparent increase in resolution is made possible by digital optimization of images captured by a high-sensitivity super-silicon (SIT) video camera coupled to a digital image processor. Cellular structures were visualized using differential interference contrast (DIC) optics and further digital image processing.
The classification of confocal microscope designs is usually based on the method of scanning the sample. There are two main scanning methods: stage scanning and illumination beam scanning; and at least two ways to scan the beam. Minsky's original instrument was based on a stage scanning system driven by a primitive tuning fork generator, which produced an image rather slowly. Modern confocal installations with stage scanning, which have gone far beyond their prototypes, are used mainly in materials science, for example, in the production of microcrystals. Systems based on this principle have recently become popular in biomedical fields where DNA analysis is carried out on microcrystals.
A more practical alternative for imaging biological systems is to scan a stationary sample with a beam. This principle underlies many measuring systems, the improvement of which has led to the emergence of today's popular research microscopes. We do not go into the technical details of confocal microscopy in this introduction, but essentially it uses two fundamentally different beam scanning techniques: multi-beam scanning and single-beam scanning. Single-beam scanning is the most common today, and it is this method that is used in LSCM. Here, the beam is scanned using computer-controlled mirrors driven by galvanometers at a speed of one frame per second. To achieve faster scanning, approximately at the video frame rate, some systems use an acousto-optic device or oscillating mirrors. An alternative method uses two beams for near real-time scanning, usually using a variation of a spinning Nipkow disk. These systems resulted from the modification and refinement of tandem scanning microscopes (TSMs) to create more efficient models for imaging fluorescently stained samples. Figure 5 shows such an advanced system with paired Nipkow disks and microlenses to improve sensitivity to faint fluorescent emission for real-time imaging.
Rice. 5. Optical design based on Nipkow disks
Today, in confocal microscopy, there are two alternative methods for obtaining optical sections: the deconvolution method and multiphoton. They differ technically, but like confocal methods, they are based on traditional optical microscopy. Deconvolution is based on computational algorithms for calculating and removing the information that comes from out-of-focus areas when creating an image. This technique has become very convenient due to efficient algorithms and high-performance minicomputers. Multiphoton microscopy uses the same scanning system as LSCM, but does not require a pinhole diaphragm in the receiver. There is no need for it, since the laser excites the fluorochrome mark only at the focal point, thereby eliminating out-of-focus radiation. When observing living tissue, this method has additional advantages, namely: reduced photobleaching, since the amount of energy transmitted by the laser beam and absorbed by the tissue of the sample is reduced.
The traditional optical microscope is the basis on which the LSCM is built. Instead of a tungsten or mercury lamp, a laser is used, which is coupled to a sensitive photomultiplier tube (PMT) and a computer that controls scanning mirrors and other scanning devices and facilitates the collection and presentation of images. The obtained data is stored on digital media and can be processed using numerous software packages, either on the computer of the system itself or on some other one.
According to the design of the LSCM, illumination and signal reception (registration) are limited to a point on the sample with a diffraction limit. The microscope lenses bring the illumination spot into focus, and the scanning device scans the sample with this spot under computer control. Signals from the sample's luminous spots enter a photomultiplier tube through a pinhole (or in some cases a slit) diaphragm, and the output signals from the photomultiplier are formed into an image and visually reproduced by a computer. Although unstained samples can be observed by light reflected from the sample, they are usually stained with one or more fluorescent dyes. One of the most common LSCMs, described in the literature around 1990, was developed in response to a fundamental problem faced by biological researchers. Many structures and individual macromolecules within immunofluorescently stained embryos cannot be visualized with a traditional epifluorescence microscope after the two-cell stage, since the volume of the embryo remains approximately the same as the number of cells increases. This means that with a denser arrangement of cells, the luminescence from cells outside the focal plane increases, which leads to a deterioration in image resolution.
Rice. 6. Nikon laser scanning confocal microscope configuration
A group of researchers working on this problem found that none of the confocal systems available at the time met their requirements. At that time, stage scanning microscopes were too slow. It took approximately 10 seconds to create a single image, and multibeam scanning instruments were not yet practical for fluorescence imaging. The LSCM was designed to meet the requirements of traditional epifluorescence microscopy and, along with others being developed at the same time, became the prototype for the complex systems now offered to the biomedical community by various companies. An example of a system used today (Nikon E1000) is shown in Figure 6.
In specially designed devices, the thickness of the optical sections can vary with changes in the diameter of the pinhole in front of the photodetector. Compared to other designs with a fixed pinhole size, this additional feature is extremely flexible when imaging biological structures. The image can be enlarged without loss of resolution by reducing the scanned area of the sample and placing the scanned information in a data array of the same size for storage and visual presentation (magnification changes in a similar way in a scanning electron microscope). This gives a single lens a zoom interval, which can be extremely useful when visualizing rare or fleeting events that might be missed or lost when changing lenses.
With the sophisticated and flexible capabilities of LSCM now offered commercially at reasonable prices, confocal microscopy has exploded in popularity in recent years, with many multi-user laboratories choosing this equipment over electron microscopes. The advantage of confocal microscopy is the relative ease with which high-quality images of samples prepared for traditional optical microscopy can be obtained and a wide range of applications in various fields of research.
The first generation of LSCMs worked well with fixed samples, but they failed to control the light energy of the lasers, which too often resulted in fatal destruction of the living sample unless serious precautions were taken. Despite these limitations, the images of the recorded samples were of such high quality that the confocal approach was unconditionally accepted by specialists. Subsequent generations of instruments have improved every aspect of the imaging process. In addition to this, the new devices have become much more ergonomic and easier to use, so that adjustments, changing filter combinations, and adjusting laser power using a computer have become much easier and faster. It is now possible to image with three fluorochromes simultaneously and with even more in sequence. Thanks to improved and more reliable software, faster computers, larger disk drives, and falling prices for random access storage devices, image processing has also advanced significantly.
Imaging Modes
The main application of a confocal microscope is to obtain images of various types of thick samples. The advantage of the confocal method stems from the ability to create images of the sample as a sequence of individual optical sections with high definition and resolution. In this case, several visualization modes are used; Each of them is based on an optical slice as the basic unit of image.
Rice. 1. Optical sections labeled with three marks
Individual optical sections
The optical section is the basic imaging unit in confocal microscopy techniques. Images of bound and stained samples can be obtained in single-, dual-, triple- and multi-wavelength illumination modes, while images of multi-colored samples will be combined with each other (if lenses with adequate chromatic aberration correction are used). Additional registration is usually performed using digital image processing methods. Most laser scanning confocal microscopes (LSCMs) require approximately 1 second to acquire an optical section, although images from multiple optical sections are typically averaged to improve the signal-to-noise ratio. The time it takes to obtain an image, of course, depends on the pixel size of the image and the speed of the system computer. To store a typical 8-bit image of 768x512 pixels, about 0.3 Mbit of memory will be required.
The optical sections shown in Figure 1 were obtained simultaneously by excitation light at three different wavelengths (488, 568 and 647 nanometers) using a single krypton/argon laser as the radiation source. As a sample, the imaginal disc of a Drosophila wing at the third instar is presented, in which three genes involved in wing formation are labeled. The three genes presented and the corresponding fluorochrome tags are: (a) vestigial (fluorescein - 496 nanometers); (b) wingless (lissamine rhodamine - 572 nanometers); and © CiD (cyanine 5 - 649 nanometers). A composite image of the three spatially represented gene domains that form the wing is located at the bottom right (image (d)).
Recording at a specified time interval and visualizing a living cell
Time-lapse studies of living cells have gained new impetus due to the increased resolution of LSCM. Previously, studies of the movements of cellular structures were carried out using 16 mm photographic film and an intervalometer with a clock mechanism connected to a camera, later using a video recorder with a time-lapse function, an optical disk recorder or a video digitization board. Now, using LSCM, it is possible to obtain optical sections at certain, preset intervals in real time.
Imaging living tissue using LSCM is much more difficult than imaging bound samples and is not always practical because the sample may not withstand the viewing conditions. Table 1 summarizes some factors that must be considered when observing living and associated cells using LSCM. Some samples are simply physically impossible to place on the microscope stage, or they will not be able to remain alive on it during the entire observation period. The phenomenon or structure being studied may not be within the field of view of the lens. For example, the Drosophila wing imaginal discs develop too deep in the larva to be observed; and after dissection, they cannot develop in culture. Therefore, today the only available method to observe gene expression in tissues of this type is to dissect the larva, tie and stain imaginal discs taken from samples at different stages of development.
Table 1. Observation of bound and living cells using LSCM
Successful observation and imaging of living cells requires extreme care throughout the entire process. Maintaining acceptable conditions on the microscope stage is a must. The damage caused to a cell by laser beam irradiation can accumulate over repeated scanning, so this exposure should be kept to the minimum necessary and sufficient to obtain an image. Antioxidants, such as ascorbic acid, are typically added to the culture medium to reduce the oxygen released when fluorescent molecules are irradiated with excitation light and promote the formation of cell-killing free radicals. It is usually necessary to conduct extensive preliminary control experiments to evaluate the effect of irradiation on fluorescently stained cells, carefully ensuring that all imaging parameters are consistent with the observations being made. Following test images, the viability of live specimens must be assessed. Embryos, for example, must continue to develop normally throughout the observation process, so any abnormalities caused by radiation exposure or fluorochromes must be detected. Figure 2 shows a time-lapse photograph of a Drosophila embryo fluorescently stained with calcium green. A series of images shows changes in the distribution of fluorescent glow over time.
Each cell type requires its own measures to maintain their viability during observation. For some insect cells, maintaining room temperature and having a sufficiently large volume of suitable medium is sufficient. However, most cell types require a heated stage and sometimes a perfusion chamber to maintain proper carbon dioxide balance while on the stage. Selecting the type of cells for which the observation conditions using LSCM are least “hostile” will help to avoid many experimental problems. Improvements in modern confocal instruments have resulted in a significant reduction in potential problems. Increased quantum efficiency, larger numerical aperture (brightness) of objectives, and the use of less toxic cell dyes have led to confocal microscopy becoming a practical method for analyzing living cells. It is necessary to strive for the use of lower power lasers, while at the same time allowing image acquisition and processing to be performed as quickly as possible. If the pinhole aperture is increased to speed up image collection and recording (compared to observing non-living samples), subsequent deconvolution can sometimes restore lost image quality.
Rice. 2. Shooting at a given time interval
Many physiological processes and events occur too quickly to be captured by most LSCMs, which average one image per second for imaging. LSCMs using acousto-optical devices and slit diaphragms are faster than galvanometer-excited point scanning systems and are more practical for physiological studies. These faster setups combine good spatial and temporal resolution, which can reach 30 frames per second at full screen resolution, or close to video image speed. In slower pinhole scanning microscopes, temporal resolution can only be increased by reducing the sample scanning area. If full spatial resolution is required, the frame rate must be reduced, resulting in a loss of temporal resolution. Confocal systems, which use disk or oscillating mirror scanning, are also capable of imaging fast physiological processes or other transient events.
Z-series and 3D images
A Z-series is a sequence of optical sections of a sample taken at different levels in a plane perpendicular to the optical axis (z-axis). Z-series images are obtained by matching step-by-step changes in the microscope's fine focusing and then acquiring an image at each step. Step-by-step focus movement is usually performed by a computer-controlled stepper motor, which changes the focus by a predetermined amount. Using a computer macro program, you can acquire and save an image, refocus the microscope to a specified depth in the sample, acquire and save a second image, refocus again in a new plane, and so on until the programmed number of images is acquired.
The desired images can be taken from the z-series obtained by shooting a selected area of the sample and processed by special software for subsequent detailed examination of specific cells of interest. The Z-series can be thought of as a photomontage of images, as in Figure 3. This type of combination and display of images, as well as many other image manipulations, is a standard feature of modern image-manipulation software packages. The images in Figure 3 are a selection from a larger series with even more frequent z-steps. The green glow identifies the peripheral nervous system of a Drosophila embryo stained with the 22C10 antibody.
Rice. 3. Z-series of optical sections
From a series of several hundred optical sections of a sample produced by LSCM, it can be difficult to get an idea of the entire complex of interconnected structures. However, the z-series, once registered, is an ideal material for subsequent three-dimensional representation of the sample using volumetric imaging techniques. This approach is now widely used to clarify the relationship between tissue structure and function in medicine and biology. It is important to set the correct sample z-scan step determined by the step of the focus changing motor; in this case, the image will reflect the actual depth of the sample.
As long as the sample remains stationary while being observed, the z-series images produced by the LSCM will be perfectly recorded, and when stored digitally, they will be relatively easily converted into a 3D representation of the sample. Figure 4 compares a single optical section (a) with a z-series projection (b) and illustrates the value of this technique in imaging the peripheral nervous system of a Drosophila embryo labeled with the 22C10 antibody.
The stepper motor pitch set by the microscope operator is related to the optical section thickness, but may have other values. The thickness of the optical section is tied to the thickness of the sample section observed through the microscope, and depends on the lens and the diameter of the pinhole diaphragm. In some cases, however, the focal pitch may be the same as the optical slice thickness, and this can lead to confusion.
Once the z-series file is received, it is sent to be processed by a 3D reconstruction program specifically designed for processing confocal images. Such programs are extremely fast when used on graphic stations, but can also be successfully used on a personal computer or the graphic station of the confocal microscope itself, with a sufficiently fast processor and large RAM. Using these programs, you can create both individual three-dimensional representations of a sample, and a sequence of representations that replace each other, composed of different types of sample, which produces the effect of rotation or other spatial transformations and gives a better perception of the three-dimensional properties of the sample. The program allows you to vary the length, depth, make volumetric measurements, and also interactively change special image parameters, such as sample transparency, to highlight different structures at different levels of the sample.
Rice. 4. Optical section and z-series projection
Another way to represent a series of optical slices taken from a sequence of images acquired at a given time interval is a three-dimensional representation in which the z-axis has the function of the time axis. This approach is useful in visualizing physiological changes during organism development. An example of the application of this method was to clarify the dynamics of changes in calcium concentration during the development of sea urchin embryos. Color coding of optical sections taken at different depths is a simple way to represent 3D data. In practice, a color (usually red, green or blue) is assigned to each optical section taken at different depths of the sample, and the color images are then combined and the desired effect is achieved by changing the colors using an image processing program.
4D Imaging
With LCSM, dynamic phenomena occurring in living tissues or during the preparation of living tissue cultures and reflected in a sequence of images taken over a given time interval can be represented in four dimensions, with time as the fourth dimension. Z-series, obtained at regular intervals, are four-dimensional data sets: three spatial dimensions (x, y and z) and time as the fourth, which can be observed using a 4D viewer. Such programs allow you to compose and play stereo pairs taken at different points in time as a movie, or, alternatively, process and present reconstructed three-dimensional images taken at different points in time, like an edited film.
X-Z images
If a lateral view of the specimen is desired, such as a vertical section through the epithelial layer, an x-z section can be made in one of two ways. A side view can be obtained by scanning along a single line of the sample (x-axis) at different depths (z-axis), controlling the change in focus with a stepper motor, and then combining the entire series of slices into a single image. Another method is to use the section plane option in a 3D rendering program, where the side view is extracted from an existing z-series of optical slices. When imaging the butterfly wing epithelium in Figure 5, the laser scanned along a single line (the horizontal black line in the left image) penetrating the sample at different z-coordinates, or depths. The x-z image shown in Figure 5 was generated and presented by a confocal imaging system. The wing epithelium consists of two epithelial layers, but since the fluorescence intensity decreases with increasing depth of penetration of the laser beam into the sample, only the upper layer is clearly visualized.
Rice. 5. Image in X-Z plane
Creating an image in reflected light
All early confocal microscopes operated in reflected, or backscattered, light. Using reflected light, many samples can be observed unstained in confocal microscopy, or they can be labeled with highly reflective dyes such as immunogold or silver halide microcrystals. An advantage of reflected light observations, especially for living tissue, is that the sample is not subject to photobleaching. But some types of dyes can weaken the laser beam. Another potential problem is that some microscopes may experience internal reflections from optical elements along the optical beam path. For multibeam versions of LSCMs and slit-diaphragm LSCMs, the problem of reflected light does not exist, and in those microscopes that do, the use of polarizers, imaging of artifact-free areas and offset from the optical axis help reduce the problem.
Transmitted light imaging
Any of the transmitted light imaging modes commonly used in microscopy can be used in LFCM, including phase contrast, differential interference contrast (DIC), dark field, or polarized light. The light passing through the sample hits the transmitted light receiver, the signal of which, through a fiber-optic light guide, is sent to one of the photomultipliers in the scanning head of the microscope. Transmitted light and confocal epifluorescence images can be captured simultaneously using the same illuminator beam, ensuring accurate registration. By merging or synthesizing images using appropriate software, the exact location of labeled cells in the tissue can be reflected. Some studies suggest the following meaningful approach: combine a non-confocal transmitted light image with one or more confocal fluorescence images of labeled cells from the same sample. This approach will allow, for example, to determine the spatial and temporal aspects of the migration of a subpopulation of labeled cells within a population of unlabeled cells over several hours or even years.
Today, a color transmitted light receiver is already widely used, which accepts transmitted signals in red, green and blue (RGB) to create a true-color image, similar to what is done in some digital color cameras. Such a receiver is especially useful for pathologists, who routinely observe actual colors in tissue under transmitted light and overlay these images with fluorescent data for analysis.