Fluorescence in situ hybridization. Nucleic acid in situ hybridization The method is based on the Application of fluorescent in situ hybridization - FISH technology

Lecture 4.

Chromosome hybridization

Introduction

To determine the localization of individual genes on chromosomes (that is, gene mapping), a whole arsenal is used special methods. One of the main ones is molecular hybridization (formation of a hybrid) of a gene or its fragment with chromosome preparations fixed on a solid support, isolated from cells in pure form(this is called in situ hybridization). The essence of the in situ hybridization method is the interaction (hybridization) between denatured (unbraided) DNA strands in chromosomes and complementary nucleotide sequences added to the chromosome preparation of single-stranded DNA or RNA (they are called probes).

Fluorescent in situ hybridization (FISH)

This method made it possible to move from studying the morphology of chromosomes to analyzing the DNA sequences that make up them. The FISH method uses fluorescent molecules for intravital staining of genes or chromosomes. The method is used for gene mapping and identification of chromosomal aberrations.

The technique begins with the preparation of short DNA sequences, called probes, that are complementary to the DNA sequences that represent the target of interest. The probes hybridize (bind) to complementary regions of DNA and, due to the fact that they are labeled with a fluorescent label, make it possible to see the localization of the genes of interest within the DNA or chromosomes. Unlike other methods for studying chromosomes, which require active cell division, FISH can be performed on non-dividing cells, giving the method flexibility.

FISH can be used for a variety of purposes using three probes various types:

• locus-specific probes, binding to certain regions of chromosomes. These probes are used to identify the existing short sequence of isolated DNA, which is used to prepare a labeled probe and its subsequent hybridization with a set of chromosomes;
• alphoid or centromeric repeat probes are repeating sequences of the centromeric regions of chromosomes. With their help, each chromosome can be painted a different color, which allows you to quickly determine the number of chromosomes and deviations from their normal number;
• probes for the entire chromosome are a set of small probes complementary to individual sections of the chromosome, but generally covering its entire length. Using a library of such probes, it is possible to “color” the entire chromosome and obtain a differential spectral karyotype of an individual. This type of analysis is used to analyze chromosomal aberrations, such as translocations, when a piece of one chromosome is transferred to the arm of another.

The material for the study is blood, bone marrow, tumor biopsy, placenta, fetal tissue or amniotic fluid. Both metaphase and interphase cell preparations can be used. Specific DNA probes labeled with fluorescent labels hybridize with chromosomal DNA, and multiple probes for different loci can be used simultaneously.

FISH is a useful and sensitive cytogenetic analysis method for detecting quantitative and qualitative chromosomal aberrations, such as deletions (including microdeletions), translocations, duplications and aneuploidy. FISH on interphase chromosomes serves quick method prenatal diagnosis of trisomy 21, 18 or 13 chromosomes or sex chromosome aberrations. In oncology, FISH can detect a number of translocations associated with hematological malignancies. The method can also be used to monitor residual cancer after chemotherapy and bone marrow transplantation and identify enhanced oncogenes associated with poor prognosis in some tumors. FISH is also used to monitor the survival of bone marrow allograft obtained from an individual of the opposite sex. FISH is also used to detect and determine the location of specific mRNAs in a tissue sample. In the latter case, the FISH method makes it possible to establish the spatiotemporal features of gene expression in cells and tissues.

FISH is a sensitive method for the identification of chromosomal aberrations and the rapid analysis of large (>500) numbers of cells at one time. The method is highly accurate in identifying the nature of chromosomes and unknown fragments of chromosomal DNA.

Thus, general form The protocol for staging FISH can be presented as follows:

1) Preparation of histological or cytological specimen

The preparation of the histological specimen is carried out according to the standard procedure: cutting, marking, wiring, filling, microtomy, placing the section on a glass slide and dewaxing. When preparing a cytological preparation, special precipitating solutions and centrifugation are used, which makes it possible to obtain a concentrated cell suspension.

2) Pre-treatment (if necessary)

The drug is treated with proteases to eliminate the presence of proteins that impede hybridization.

3) Application of a DNA probe to the preparation and subsequent denaturation

In order to denature the probe and sample DNA, they are treated with formamide and heated to a temperature of about 85–90ºC.

4) Hybridization

After denaturation, the drug is cooled to a certain temperature (37ºС in the case of clinical trials) and incubated in a humid chamber for several hours (the duration of incubation is specified in each specific protocol). Currently, automatic hybridizers are used for denaturation and hybridization.

5) Flushing.

After hybridization is complete, it is necessary to wash away unbound probes, which would otherwise create a background that would make it difficult to evaluate the results of the FISH analysis. A solution containing sodium citrate and chloride (SSC) is usually used for rinsing.

6) Counterpainting

Using fluorescent dyes (DAPI - 4,6-diamidin-2-phenylindole; propidium iodide), all nuclear DNA is stained.

7) Analysis of results using a fluorescence microscope

Particularly important for studying the human genome in the early stages of its research was a method called hybridization of somatic cells. When human somatic (non-reproductive) cells are mixed with cells of other animal species (most often, mice or Chinese hamster cells were used for this purpose), fusion of their nuclei (hybridization) can occur in the presence of certain agents. When such hybrid cells reproduce, some chromosomes are lost. By a happy accident for the experimenters, in human-mouse hybrid cells, most of the human chromosomes are lost. Next, hybrids are selected in which only one human chromosome remains. Studies of such hybrids have made it possible to associate some biochemical characteristics, characteristic of cells human, with certain human chromosomes. Gradually, through the use of selective media, they learned to achieve the preservation or loss of individual human chromosomes carrying certain genes.

To facilitate cell fusion different types, Sendai virus, inactivated by ultraviolet irradiation, or polyethylene glycol is added to the culture medium. To select fused cells from the original human and mouse cells, the cells are grown in a special selective medium that allows only hybrid cells to proliferate.

To date chromogenic in situ hybridization is a more accessible method than fluorescent. When it comes to fluorescent in situ hybridization, the DNA probe is conjugated to a fluorescent label. The results of such a study are evaluated under a fluorescent microscope. In the case of chromogenic in situ hybridization, the DNA probe is conjugated with peroxidase or something else and stained with the chromogen. In this case, the results are assessed under a conventional light microscope.

ü Advantages of the FISH method

As main advantages FISH can be distinguished as follows:

1) the possibility of studying genetic material in interphase nuclei;

2) obtaining objective results on a “yes/no” basis is a quantitative method;

3) relatively simple interpretation of results;

4) high resolution.

ü Disadvantages of the FISH method

1) fluorescent dyes quickly “fade”;

2) A high-quality fluorescence microscope is required to analyze the results.

FISH (fluorescent in situ hybridization) is an indispensable method in the diagnosis of cancer. Using specific fluorescent probes, this method makes it possible to identify the presence of genomic rearrangements, that is, to clarify the diagnosis, clarify the prognosis and select adequate therapy, depending on the specific case. First of all, this approach is used for oncohematological diseases. Previously, conventional karyotyping was used for these purposes, but if the patient’s cells do not show significant growth in culture, this seriously complicates diagnosis using this method. In these cases, the use of FISH significantly expands the capabilities of laboratory diagnostics. In addition, complex chromosomal rearrangements are easier to interpret using FISH.

The laboratory uses probes to centromeres, specific regions of chromosomes, and genes. Two-color probes are selected to search for translocations in such a way that if fragments of two genes, which are normally located in different parts of the genome, are nearby, two signals of different colors - one from each probe - merge into one, different in light from the original ones. For example, BCR-ABL translocations, common among leukemias of various types, are identified. Genes such as MLL, TEL and RARα can rearrange themselves to form chimeric genes with different sequences. In this case, two probes are selected to different edges of the gene. If the gene is intact, there will be one dot in each nucleus on the specimen; if it is broken, there will be two dots of different colors. Due to this, FISH is a more flexible technique for detecting chromosomal translocations than PCR. The probe will sit on the chromosome regardless of how exactly the break and joining of a fragment of another chromosome occurred within any specific region, unlike oligonucleotides used in PCR, which recognize specific, albeit common, rearrangements.

A panel of markers detected by FISH allows one to assess the prognosis of chronic lymphoid leukemia. Deletions of 11q and 17p are associated with an unfavorable prognosis, while deletions of 13q, like a normal karyotype, are associated with a favorable one. With trisomy 12, the case can be classified as an intermediate risk group.

The risk category for myeloma is also associated with a combination of deletions and translocations; translocations t(4;14), t(14;16) and 17p deletion are associated with an unfavorable prognosis. Such diagnostic studies are performed on red bone marrow biopsies after enrichment. A small inversion, accompanied by the formation of the chimeric EML4-ALK gene, is characteristic of non-small cell lung cancer. The chimeric gene product is a target for targeted therapy. Such rearrangements can also be detected using the FISH method. HER2 amplification is often assessed indirectly by increasing expression levels using immunohistochemistry, but in some countries it is recommended to use FISH for this purpose, or at least use this method for confirmation.

Fluorescence in situ hybridization

Fluorescence hybridization in situ , or FISH method (eng. Fluorescence in situ hybridization - FISH ) - a cytogenetic method that is used to detect and determine the position of a specific DNA sequence on metaphase chromosomes or in interphase nuclei in situ. Additionally, FISH is used to detect specific mRNAs in a tissue sample. In the latter case, the FISH method makes it possible to establish the spatiotemporal features of gene expression in cells and tissues.

Probes

With fluorescent hybridization in situ use DNA probes (DNA probes) that bind to complementary targets in the sample. DNA probes contain nucleosides labeled with fluorophores (direct labeling) or conjugates such as biotin or digoxigenin (indirect labeling). With direct labeling, the DNA probe bound to the target can be observed using a fluorescence microscope immediately after hybridization is completed. In the case of indirect labeling, an additional staining procedure is required, during which biotin is detected using fluorescently labeled avidin or steptavidin, and digoxigenin is detected using fluorescently labeled antibodies. Although the indirect version of DNA probe labeling requires additional reagents and time, this method usually allows one to achieve a higher signal level due to the presence of 3-4 fluorochrome molecules on the antibody or avidin molecule. In addition, in the case of indirect labeling, cascading signal amplification is possible.

To create DNA samples, cloned DNA sequences, genomic DNA, PCR reaction products, labeled oligonucleotides, and DNA obtained by microdissection are used.

Labeling of the probe can be done in different ways, for example, by nick translation or by PCR with labeled nucleotides.

Hybridization procedure

Scheme of the fluorescent hybridization experiment in situ to localize the position of a gene in the nucleus

The first stage involves the construction of probes. The size of the probe should be large enough for hybridization to occur at a specific site, but not too large (no more than 1 thousand bp) so as not to interfere with the hybridization process. When identifying specific loci or when staining entire chromosomes, it is necessary to block the hybridization of DNA probes with non-unique repeated DNA sequences by adding unlabeled DNA repeats to the hybridization mixture (for example, Cot-1 DNA). If the DNA probe is double-stranded DNA, it must be denatured before hybridization.

At the next stage, preparations of interphase nuclei or metaphase chromosomes are prepared. Cells are fixed on a substrate, usually on a glass slide, and then the DNA is denatured. To preserve the morphology of chromosomes or nuclei, denaturation is carried out in the presence of formamide, which allows the denaturation temperature to be reduced to 70°.

Visualization of bound DNA probes is carried out using a fluorescence microscope. The intensity of the fluorescent signal depends on many factors - the efficiency of labeling with the probe, the type of probe and the type of fluorescent dye.

Literature

• Rubtsov N.B. Methods of working with mammalian chromosomes: Textbook. allowance / Novosibirsk. state univ. Novosibirsk, 2006. 152 p.

• Rubtsov N.B. Nucleic acid hybridization in situ in the analysis of chromosomal abnormalities. Chapter in the book “Introduction to Molecular Diagnostics” Vol. 2. “Molecular genetic methods in the diagnosis of hereditary and oncological diseases” / Ed. M.A. Paltseva, D.V. Zaletaeva. Educational literature for medical students. M.: Medicine, 2011. T. 2. P. 100–136.

Notes

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See what “Fluorescent in situ hybridization” is in other dictionaries:

This term has other meanings, see hybridization. DNA hybridization, nucleic acid hybridization, in vitro combination of complementary single-stranded nucleic acids into one molecule. With complete complementarity... ... Wikipedia

In situ hybridization capabilities can be greatly enhanced by using multiple fluorescent colors simultaneously. Multicolor fluorescence in situ hybridization (FISH), in its simplest form, can be used to label (stain) many characteristics because different fluorophores are used in hybridization. By using combinations of colors rather than single colors, digital imaging microscopes can simultaneously detect many more of the characteristics highlighted by the dyes in individual cells.

Rice. 1. Multicolor FISH

For a detailed overview of fluorescence microscopes from the world's leading manufacturers of optical systems and related equipment, visit our catalog or contact our specialists and receive full professional advice on any questions you have.

Figure 1 shows a typical multicolor fluorescence hybridization - FISH pattern. Normal male lymphocytes were hybridized with FITC-biotin stained with Chr2l and ChrY probes and CY3-digoxigenin stained with Chrl3 and ChrY probes. Top left is a photograph of DNA nuclei stained with DAPI, obtained using a DAPI filter. Top right is a snapshot of Chr2l and ChrY stained with FITC, obtained using a FITC filter. Bottom left is a snapshot of Chrl3 and ChrY stained with CY3, obtained using the CY3 filter. The bottom right image is a composite image showing all target chromosomes in color. This sample was provided by Dr. Tim Houseil, Integrated Genetics, Framingham, MA.

Multicolor FISH techniques combined with digital imaging techniques now offer unparalleled non-isotopic detection capabilities of multiple nucleic acid sequences for the analysis of cellular components, chromosomes and genes.

Fluorescence - a phenomenon in which a chemical compound is excited at one wavelength of light and emits at another, usually longer - is used throughout the biological sciences to study a variety of structures and intracellular processes. Technological advances in the development of dyes and microscopes have led to rapid development of fluorescence techniques over the past decade.

This review article will outline the fundamentals of the FISH method, the limitations that researchers have encountered over the years using FISH, recent developments in hardware, software, dyes and reagents that have influenced the development of this method, and current directions in the field. The latest advances in this method, which have led to its use not only in research laboratories, but in clinical diagnostics, will also be discussed in this article.

FISH Method Overview

The applications of FISH are rapidly growing in genomics, cytogenetics, prenatal research, neoplastic biology, radiolabeling, gene mapping, gene amplification, and basic biomedical research. In principle, this method is quite simple.

By hybridization, target genomic sequences are identified, or tagged, so that their location and size can be observed. DNA or RNA sequences from suitable probes, depending on the chromosome, are first labeled with reporter groups, which are later identified using fluorescence microscopy. The labeled DNA or RNA probe is then hybridized to metaphase chromosomes or resting nuclei on a glass slide. After washing and signal amplification, the sample is examined for reporter groups using fluorescence microscopy.

FISH allows one to achieve very high spatial resolution of morphological and genomic structures. This method is quite fast, easy to implement and is characterized by high dye stability. Depending on the probe used, the genome of an individual, entire chromosomes, sections of chromosomes, and sequences of unique copies can be determined.

Previous restrictions

Until recently, FISH was limited by hardware, software, reagents, dye production technology, and high execution costs. Commercially available microscope hardware optimized for multicolor FISH was not available until the mid-1990s. Previously, microscopes had to be specially configured for FISH applications. Most microscopic optics were not designed to detect the low-level light signals characteristic of FISH. As the resolution of genomes has significantly improved using this method, the requirements for microscopic optics have also increased. Chromatic aberrations at many wavelengths were also a problem. For multicolor analysis in particular, all lenses, including the converging lens, had to be corrected for chromatic aberration. In addition, it was very difficult to adjust epifluorescent light sources to obtain uniform illumination.

Analyzing multicolor FISH images requires separating the different signals either by (a) separate filter cubes or (b) using excitation filter packs with broadband dichroic and threshold filters. Advances in filter technology have corrected some of the earlier problems caused by optical misalignment and misalignment caused by mechanical switching between individual filter cubes. Replaceable excitation light filters used with multiband dichroic and threshold filters can effectively handle three colors by using separate excitation filters for each color without shift detection. But when working with more than three colors, you still need to use single-band filters.

High-speed color film or CCD (charge-coupled device) were used to collect data, and both had problems with color accuracy. In addition, there were problems with overlapping images of different colors taken from the same sample using different dye probes.

Software for quantitative analysis of samples prepared with fluorescent reagents also left much to be desired, as existing image analysis systems were not optimized to handle fluorescent sampling samples. Visual analysis is a labor-intensive and often subjective procedure, so without the use of advanced fluorescence imaging, analysis of fluorescence samples was difficult and subject to ambiguity. Researchers typically had to employ their own programmers to develop their own image analysis programs.

The reagents and dyes themselves were not sufficient for all applications. For example, the efficiency of determining the site of hybridization decreased with decreasing probe size, which imposed serious limitations on those samples that could be observed by fluorescence microscopy. The number of different fluorescent dyes was limited; In addition, they had low photostability. But developments in fluorescent dye technology and related technologies through the federally funded Human Genome Project are now bearing fruit. Probes already exist for all human chromosomes, and a growing number of gene probes are available. In situ hybridization kits and fluorescently labeled probes are now commercially available from several companies.

Cost was another major obstacle. Because there were no commercially available FISH systems on the market, researchers had to assemble custom systems, including reagents, probes, microscope, image processing hardware and software, data analysis, and reporting. Performing multicolor FISH with complex image analysis could cost a researcher more than $200,000, an amount difficult to afford for most clinical researchers. As a result, many researchers wishing to use FISH in their laboratories have not had the opportunity to do so.

FISH has become widely available

Many hardware and software manufacturers have developed affordable off-the-shelf systems as an alternative to custom systems. The collaborative atmosphere that has developed among many FISH firms and laboratories has led to new breakthroughs in the field. And the authors of published works consider their achievements precisely within the framework of such cooperation.

Rice. 2. MultiFluor operating screen

Operating in the United States and providing mid-range pricing for commercial FISH systems, the system combines components from many manufacturers and relies on the latest advances in imaging software, microscope hardware and other accessories. Clinical research laboratories find this system useful in many applications. Being integrated and automated, it allows clinical trials to be carried out in full.

The software system presented here is MultiFluor™, a multiparameter imaging system (Biological Structure Imaging Systems) developed on Microsoft® Windows (Microsoft Corporation, Bellevue, WA) for the detection, analysis and presentation of structural and molecular features derived from samples. multicolor FISH. Analysis time and accuracy of results are improved by correlating multiple features at different wavelengths in each sample. This system facilitates image acquisition, image storage, database management, automatic microscope control, and full-featured graphical data analysis.

Figure 2 shows the MultiFluor data overview screen. Users can view snapshots and their associated data, and compare multi-parameter data obtained in different colors (at different wavelengths) using a variety of plotting tools, including histograms, scatterplots, etc. Here, various plots are shown along with a set of multi-color cell snapshots (depicting DAPI nuclei, FITC-ChrX, CY3-ChrY and CY5-Chr2l), along with the original data presented in the table.

FISH researchers have the ability to automatically acquire images at multiple wavelengths at multiple focal planes, visualize multicolor FISH probes, annotate and print images, and store and retrieve large volumes of multicolor image data sets. Metaphase chromosomes can be analyzed by multicolor FISH by gene mapping, comparative genomic hybridization (CGH), and karyotype generation. User-selected areas of the sample can be scanned and analyzed. The program automatically focuses the system, acquires images at multiple wavelengths, remembers the position of cells on the slide, and measures various characteristics including probe counts, fluorescence intensity, and cell morphometry. Different characteristics at different wavelengths can be related to each other.

An additional characteristic of the system is the ability to work with personal computers (PCs) connected to a network. In a typical configuration, one computer is an online station connected to the camera and microscope hardware. This computer controls the acquisition of images and performs instant analysis. Other PCs are secondary information analysis stations, where data received from the first PC is processed, or where some special analysis is performed autonomously.

The program allows you to present all components in bright pseudo-colors for simultaneous multi-color visualization. For example, simultaneous images of a four-color experiment (using DAPI (blue), FITC (green), CY3 (red), and the FITC-CY3 combination (yellow)) can be presented separately or combined into a single image (as shown in Figure 1). . Each image can be interactively enhanced to highlight characteristics of interest. Using the program, it is easy to create histograms, scatterplots, tables, line graphs and other forms of presenting and evaluating data (Figure 2). In addition, the data is stored in easily accessible and popular formats such as TIFF, JPEG, GIF and others.

Oncological, prenatal and biological research

The FISH systems described are designed to be more accessible to researchers than previous custom-made systems. They are increasingly used in oncology, pathology studies, cytogenetics and developmental biology. Applications include spot count analysis of interphase cells observed with multicolor dyes, immunophenotyping, cell morphometry, and DNA composition.

Using these systems, abnormalities in the number of chromosome copies, correlated with the overall DNA composition, are analyzed, which are associated with the formation of tumors Bladder. In prenatal studies, these systems can be used to detect aneuploidies in quiescent nuclei associated with prenatal defects, including Down syndrome, Turner syndrome, Klinefelter syndrome, and others. In cell and developmental biology, this system can be used to map cell surface markers and their relative distribution, such as receptors, cytoplasmic markers including cytoskeletal proteins, messenger RNAs, and specific genes.

Diagnostic potential

Over the past decade and a half, it has become clear that FISH has enormous potential not only as a tool in research, but also in clinical diagnostics in areas such as prenatal diagnosis, cytogenetics and tumor development. The lack of high-quality, affordable systems has not only hindered the spread of FISH among researchers, but has made this method unattainable for diagnostic centers in many medical institutions

Results that previously could only be achieved with expensive, custom-made equipment can now be obtained with this system - and this is one of its most important advantages. The dream of applying FISH not only to a wider range of biomedical research, but also putting it directly at the service of patients may become a reality in the not-too-distant future.

Fluorescence stereomicroscopy

Epifluorescent lighting

Until recently, fluorescent illumination was only available on research microscopes equipped with special high-aperture lenses. The need for stereomicroscopy in this technique has increased with the advent of genetically encoded and biologically specific fluorescent proteins such as GFP (green fluorescent protein).

Rice. 1. Stereomicroscope with epifluorescent illuminator

The use of stereo microscopes for observing GFP is so common that stereo fluorescent illuminators are more commonly referred to as GFP illuminators, despite the fact that they can be used in many other applications, both in the life sciences and in the electronics industry. Large specimens such as larvae, nematodes, zebrafish, oocytes and mature insects are easy to observe (and manipulate) if they are stained with GFP and illuminated with fluorescence light. Fluorescent illumination reveals which organisms produce the fluorescent protein, and the stereoscopic viewing method, combined with a large field of view and long working distance, allows the observer to use tweezers, pipettes or micromanipulators during the experiment. Other, more typical specimens can also be easily examined using fluorescent-illuminated stereomicroscopes.

The epifluorescence illuminator on a stereomicroscope functions similarly to those found on more complex microscopes. Typically, the fluorescent illuminator is a xenon or mercury arc lamp placed in an external illuminator unit, which is connected to the microscope through an intermediate tube (or vertical illuminator, see Figures 1 and 2) located between the microscope zoom lens and the eyepiece tubes. To date, this type of illumination has been limited to applications using common main objective (CMO) stereo microscopes because off-the-shelf parts cannot be used to tune a Greenough stereo microscope or other converged stereo microscope to fluorescence light.

The light emitted by the arc lamp is directed through a customizable collecting lens to an excitation filter located in the combination filter bank (as shown in Figures 2 and 3). This filter transmits light only in a certain wavelength range (passband). The light passing through the filter is then deflected along the optical path of the microscope to its lower part (the zoom module and the lens in the stereo microscope) and is directed onto the sample by a dichroic mirror, which, depending on the setting, reflects, selectively filters and/or transmits light of certain lengths waves/spectrum regions. The term dichroic (or dichromatic) reflects the ability of a filter or mirror to “distinguish” the colors of incident light, reflecting light of a color that falls below a given wavelength limit, and transmitting colors above that limit.

Rice. 2. Beam path in a fluorescence stereomicroscope

The focused beam of excitation light passes through the zoom lens and lens, where it forms an inverted cone of light that irradiates the sample, causing the excitation of all fluorophores in the sample, whose absorption band matches the passband of the irradiating light. Secondary fluorescent light (usually at a wavelength longer than the excitation light) emitted from the sample is captured by the common main objective of the stereo microscope and directed back through the zoom lens to a threshold filter, which blocks light at the excitation wavelength and allows only light at the emission wavelength to pass through. The microscope tube in Figures 1 and 2 is designed in such a way that the longer wavelengths of fluorescent emission passing back through the left and right optical zoom channels are focused independently before they reach the epifluorescent illuminator. Light from the left channel passes directly to the threshold filter, after which it is directed to the eyepiece tubes or to the photo port. In contrast, light in the right channel is first directed back through the dichroic mirror and then to the threshold filter and eyepieces. This light does not exit into the camera port and can only be used to observe the sample.

The design details of the fluorescence combination filter bank are shown in Figures 2 and 3. Each bank contains a single-band excitation light filter, two threshold filters, and a dichroic mirror. Light from a mercury arc lamp enters the filter block through the excitation filter, and is reflected from the surface of the dichroic mirror, as discussed above, and as shown in Figure 3. Secondary fluorescent radiation passes through threshold filters. The excitation filter, dichroic mirror and threshold filter of the left channel are glued into the system, and the threshold filter of the right channel is fixed in a small frame, which can be removed from the unit by loosening its mounting screws. By removing the threshold filter, you can access the dichroic mirror located inside the filter block. When installing replacement filters, be careful not to get any adhesive on the surface of the filters, and be sure to wear gloves before handling the filters and dichroic mirror to avoid getting fingerprints on the surface.

The fluorescent vertical illuminator can accommodate three filter banks and an empty dia filter bank (no filters) for routine brightfield observation. Filter units are mounted on guides and installed in the optical path using a handle used to control the position of the guides. Each unit is supplied with a corresponding identification plate. The labels are inserted into a slot in the illuminator housing in a sequential order so that the operator can easily select the desired filter bank for fluorescence observation.

Rice. 3. Combinations of fluorescence filters in a stereomicroscope

The set of filter combinations shown in Table 1. These filters cover a wide range of fluorescent excitation and emission and should be useful in many biological studies where conventional fluorescent dyes are used. These filter combinations are also suitable for industrial applications, such as the analysis of IC semiconductor wafers for contamination by fluorescent photoresist polymers. By selecting an appropriate combination of excitation/emission filters, fluorescent probes with excitation wavelengths ranging from 380 to 510 nanometers can be used (see Table 1). These filter combinations are also very useful in studies with various green fluorescent protein mutants, including the cyan and blue variants.

In live cell cultures with fluorescently tagged proteins, signal intensity can be significantly increased if filter combinations precisely match the excitation and emission profiles of the fluorophores. For example, in the case of DS-red signals, visual observation and recording of red fluorescence images by photodetectors can be greatly improved by shifting the red signal toward the emission of more orange light. In addition, combinations of filters selected for plant specimen studies with intense background chlorophyll autofluorescence are often more effective when the appropriate filter specifications and emission signal are precisely selected. Many of these criteria are taken into account by microscope design engineers to optimize the bandwidth of various stereomicroscope filter combinations.

Table 1. Combinations of fluorescent filters for stereomicroscopes

Filter kit	Excitation wavelength range	Dichroic mirror	Emission wavelength range
Blue GFP/DAPI
Blue (EGFP) GFP
GFP Bandwidth
Enhanced GFP Bandwidth
TRITC (Ds-red)
Yellow GFP Bandwidth

Like all sensitive interference filters, combination block filters fail over time due to intense light and ultraviolet radiation. Characteristics such as bandwidth and transmittance also change when filters are used and stored in a humid environment. To increase their service life, such filters should be stored in a drying cabinet or hermetically sealed container with a desiccant. When not observing, the illuminator shutter should be kept closed to reduce the amount of light passing through the filters. The filter can only be cleaned with dry air from a can, a soft camel hair brush or oil-free gas from a gas cylinder. To avoid scratches and abrasions, never wipe soft interference coated filters with a lens cloth.

Focusing and alignment of arc lamps

Gain hands-on experience in aligning and focusing arc lamps with this interactive tutorial, Mercury or Xenon Burner, which simulates all lamp alignment processes in a fluorescence microscope.

Stereo microscopes can be used to observe various samples under fluorescence light. With an objective magnification of 0.5x to 1.6x and a zoom range of up to 15x, these stereo microscopes can provide a total system magnification of 4x to 540x, comparable to the viewing range of classic compound microscopes. A wide range of magnifications allows microscopists to observe both large living specimens and fine details in thin sections stained with fluorochromes mounted on a glass slide. An example of a high magnification fluorescence observation is shown in Figure 4. It shows a thick sample of a mouse kidney stained with three markers. The sample was stained with DAPI, Alexa Fluor 488 WGA and Alexa Fluor 568 (using Alexa Fluor probes and a sample from Molecular Probes and observed using the three filter combinations from Table 1: Blue GFP/DAPI, Endow GFP Bandpass, TRITC DsRed. This image clearly demonstrates the possibilities of fluorescence stereomicroscopy at high magnifications when observing samples prepared for complex microscopes.

Rice. 4. Section of a mouse kidney under fluorescence light

Other stereomicroscope manufacturers offer alternative illumination methods for fluorescence excitation and observation. The most popular configuration, shown in Figure 5, uses external circuitry for fluorescence excitation and does not use the microscope's imaging optical system. Light from the illuminator unit first passes through the excitation filter and then through a tube located at the back of the microscope body. At the bottom of the tube there is a system of lenses that direct the excitation light onto the sample. In this configuration, light is directed directly at the sample at any zoom level, thereby providing the same fluorescent illumination intensity and a uniformly dark background at any zoom level.

Secondary fluorescent radiation emitted by the stained sample is captured by the common main objective (Figure 5) and passes through the channels of the zoom unit to the threshold filters at the top of the microscope. Light is then directed either to eyepieces for direct observation or to a camera tube for digital imaging or microphotography. This configuration does not require dichroic mirrors and this is its main advantage, another advantage is the independence from pre-assembled filter banks, which gives the researcher greater freedom in filter selection. However, this configuration can lead to errors in the work of inexperienced operators caused by the incorrect selection of filter combinations.

Rice. 5. Microscope with a separate path of excitation light rays

The intense ultraviolet radiation from a mercury arc lamp used as an excitation light source in fluorescence microscopy can cause severe damage to the retina. To avoid this, many microscope manufacturers have protective devices on the body that filter out the ultraviolet light that irradiates the sample on the stage. Other precautions include ultraviolet threshold filters in the viewing path and stray light protection around the illuminator assembly. When not in use, fluorescent interference filters often have special filter-shaped plugs inserted into the guides and rotating frames of fluorescent interference filters.

Fluorescence stereo microscopes are often equipped with a special diaphragm located somewhere between the mercury illuminator assembly and the vertical illuminator to block dangerous ultraviolet radiation emanating from the lamp when the sample is not being observed. When observations are not being made, this diaphragm must be placed in the light path.

When observing samples with fast apochromatic objectives (1.6x to 2.0x) in fluorescence stereomicroscopy, reflections, or hot spots, may appear in the lower parts of the field of view. This artifact usually appears only at low zoom ratios, and disappears at higher magnifications. In most cases, reflections do not occur if the lens magnification is low (0.5x to 1.0x), regardless of optical correction, and this effect is usually absent with high numerical aperture, low correction lenses (achromats or planchromats).

As shown in Table 2, there are many applications in fluorescence microscopy where stereo microscopes are used. The number of samples that are convenient to observe in this mode is very large, and they belong to a wide range of disciplines from biology to industrial production.

Table 2. Applications of fluorescence stereomicroscopy

Region	Applications - Analysis
Biology	Gene expression, cell sorting, dissection, developmental processes, eye and muscle studies
Botany
Pharmacology	Capillary flow, drugs, environmental observations
Hydrology	Water quality, cellular structures and filter membrane analysis
Agronomy	Seed studies, gene expression and transgenetics
Electronics	Solder paste, epoxy resin analysis, coating testing, polymer selection for integrated circuits
Semiconductors	Photoresist contamination, presence of foreign particles, production control
Polymers	Presence of foreign particles, voids, granules, non-polymerized areas
Metalworking	Cracks, surface defects, contamination, welding, damage analysis
Materials	Cracks, welding, carbon joints, orientation studies
Paper production	Fibers, coatings and inclusions
Forensic examination	Textile fibers, body fluids, fingerprints, banknotes, counterfeits

Fluorescence stereomicroscopy has a unique three-dimensional observation property, compared to a classical compound microscope. In addition, stereo microscopes have longer working distances and depths of field, which provide a wider panoramic field of view and more intense fluorescence. These characteristics are of great importance for researchers who have to work with large biological samples and materials, and for specialists performing preparatory work, such as component assembly, electronics production control or cutting. As increasingly precise filter combinations become available for specialized applications, the use of fluorescence in stereomicroscopy continues to grow.