Fluorescence Microscope: Definition, Uses, Principle, Parts.

The concept of using fluorescence to study biological materials dates back to the late 19th century, but the first practical fluorescence microscope was not developed until the 1930s. In 1938, Robert Goldstein and Richard Manly, researchers at the University of California, Berkeley, published a paper describing the use of fluorescence to study cells and tissues in living animals. This marked the beginning of the modern era of fluorescence microscopy.

Since then, fluorescence microscopy has undergone significant technological advances. In the 1960s, the development of lasers allowed for the use of laser-excited fluorescence, which greatly increased the sensitivity and resolution of fluorescence microscopes. In the 1980s, the development of confocal laser scanning microscopy allowed for the creation of high-resolution, three-dimensional images of fluorescence samples. And in the 1990s and 2000s, the development of superresolution fluorescence microscopy techniques, such as stimulated emission depletion (STED) and photoactivated localization microscopy (PALM), allowed for the visualization of subcellular structures with resolutions much higher than was previously possible.

What is a Fluorescence Microscope?

A fluorescence microscope is a microscope that uses fluorescence and phosphorescence to generate an image. It works by illuminating a sample with a specific wavelength of light, which causes certain molecules in the sample to emit light at a different, longer wavelength. This emitted light, called fluorescence, is then captured by a detector and used to create an image of the sample. Fluorescence microscopes are used in a variety of fields, including biology, chemistry, and materials science, to study the structure and function of cells and other biological materials, and to analyze chemical compounds and other materials at the microscopic level. They are also used in research to visualize fluorescently labeled proteins and other biomolecules, which can help researchers understand how cells function and how diseases develop.

Working Principle of Fluorescence Microscope

The specimen is irradiated with light of a certain wavelength (or wavelengths), which causes the fluorophores to emit light of longer wavelengths (i.e., of a different colour than the absorbed light). Using a spectral emission filter, the illuminating light is separated from the significantly weaker fluorescence. A light source (xenon arc lamp or mercury-vapor lamp are popular; more sophisticated types are high-power LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beamsplitter), and the emission filter are typical components of a fluorescence microscope (see figure below). The filters and dichroic beamsplitter are chosen to correspond with the excitation and emission spectrum properties of the fluorophore employed to label the specimen. This method images the distribution of a single fluorophore (colour) at a time. Multiple single-color photos of various types of fluorophores must be combined to create multicolor images.

The majority of in-use fluorescence microscopes are epifluorescence microscopes, in which excitation of the fluorophore and detection of the fluorescence occur via the same light channel (i.e. through the objective). The confocal microscope and the total internal reflection fluorescent microscope are based on this architecture (TIRF).

The majority of fluorescence microscopes, particularly those used for life sciences, are of the epifluorescence design depicted in the diagram. Through the objective lens, excitation wavelength light illuminates the specimen. The fluorescence emitted by the specimen is focused onto the detector by the same objective used for excitation, which requires objective lenses with a higher numerical aperture for higher resolution. Since the majority of excitation light is transmitted through the specimen, only reflected excitation light and emitted light reach the objective, resulting in a high signal-to-noise ratio for the epifluorescence technique. The dichroic beamsplitter functions as a wavelength-specific filter, allowing fluoresced light to pass through to the eyepiece or detector while reflecting any residual excitation light back towards its source.

Sample Preparation for Fluorescence Microscope

There are present different types of fluorescent staining techniques such as;

1. Biological fluorescent stains

• DAPI, Hoechst, DRAQ5, and DRAQ7  stains are used for the staining of nucleic acids.
•  Phalloidin used to stain actin fibers in mammalian cells.

2. Immunofluorescence

• This method uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample.

3. Fluorescent proteins

• In this method, different Fluorescent proteins are used such as, Green fluorescent protein (GFP), Yellow fluorescent protein (YFP), Red fluorescent protein (RFP)

Light Path In Fluorescence Microscopy

1. Fluorescence microscope uses a high-intensity Mercury lamp as a Source of light.  This lamp emits white light..
2. The exciter filter transmits only blue lights to the specimen and blocks out all other colors.
3. The blue light is reflected downward to the specimen by a dichroic mirror, which reflects the lights of certain colors but transmits light of other colors.
4. The specimen is stained with a fluorescent dye certain portions of the specimen retains the dye others do not.
5. The stained portion absorbs blue light and emits green light,  which passes upward penetrates the dichroic mirror and reaches the barrier filter.
6. This filter allows the green light to pass to the eye; however, it blocks out any residual blue lights from the specimen which may not have been completely deflected by the dichroic mirror.
7. Thus the eye perceives the stained portion of the specimen as glowing green against a jet black background whereas the unstained portion of the specimen is invisible.

Fluorescence Microscope Parts

1. A powerful light source (xenon or mercury arch lamp)

• The light emitted by mercury arc lamps is ten to one hundred times brighter than that of typical incandescent lamps, and it emits a broad spectrum of wavelengths, from ultraviolet to infrared.

2. Fluorescent dyes or Fluorochromes

• Fluorescent dyes are a type of chemical compound that emits light rays when they are excited with UV, blue rays.
• It consists of aromatic groups, or plane or cyclic molecules which contain several π bonds.

3. Excitation filter

• It is a type of bandpass filter, which passes the wavelengths absorbed by the fluorophore.
• It minimizes the excitation of other sources of fluorescence.
• Block the excitation light in the fluorescence emission band. 
• The excitation filter is designed to filter out all wavelengths of the light source except for the excitation range of the inspected fluorophore. Minimum transmission % of the filter determines the brightness and brilliance of images. The optimal transmission rate is greater than 85 percent.

4. Emission filter

• It is also a type of bandpass filter.
• It only passes only those wavelengths are emitted from a fluorophore.
• It blocks all unwanted wavelengths outside this band – especially the excitation light. As a result of this action, it creates a dark background.
• In the image path of a fluorescence microscope, the emission filter is situated. Its function is to filter out the whole excitation range while transmitting the emission range of the inspected fluorophore.

5. The Dichroic mirror (beamsplitter)

• It is a type of accurate color filter.
• It only passes small range of colors and reflects other colors.
• Between the excitation filter and emission filter, the Dichroic mirror or beamsplitter is positioned at a 45° angle.
• A dichroic filter is designed to reflect the excitation signal towards the fluorophore while transmitting the emission signal towards the detector.

6. Objective lens

• The objective lens transmits light to the sample in order to generate an image. Before reaching the objective lens, light travels downwards through a dichroic mirror.

7. Camera system

• A camera system helps to capture high-resolution photos of the specimen. In the system, CCD (Charge Coupled Device) cameras are frequently employed.
• These electron multiplying cameras are able to image single-photon events without sacrificing sensitivity. They do not require an image intensifier and can take photos quickly.

Types of Fluorescence Microscopes

There are several types of fluorescence microscopes, which differ based on the specific technology used to excite and detect the fluorescence in the sample. Some common types of fluorescence microscopes include:

1. Epifluorescence microscopes: These are the most common type of fluorescence microscopes. They use a light source, such as a mercury arc lamp or a laser, to excite the fluorescence in the sample. The emitted fluorescence is then passed through a series of filters to select the specific wavelength of light and to remove any excess light. The fluorescence is then detected by a photodetector, such as a charge-coupled device (CCD) camera or photomultiplier tube (PMT), and used to create an image.
2. Confocal laser scanning microscopes: These microscopes use a laser as the light source and a series of lenses to focus the laser beam onto the sample. The laser excites the fluorescence in a small, confined area of the sample, and the emitted fluorescence is detected by a photodetector. The laser is then moved to a different location and the process is repeated, creating a series of images that can be combined to create a high-resolution, three-dimensional image of the sample.
3. Two-photon microscopes: These microscopes use a laser that generates two photons of light simultaneously, which can excite the fluorescence in the sample. Two-photon microscopes are particularly useful for studying living tissues because the laser used in these microscopes has a longer wavelength and is less damaging to the sample than the lasers used in other types of fluorescence microscopes.
4. Superresolution microscopes: These microscopes use techniques such as STED and PALM to achieve resolutions much higher than is possible with traditional fluorescence microscopes. They are able to visualize subcellular structures and are used to study the fine details of cells and other biological materials.
5. Multiphoton microscopes: These microscopes use lasers to excite multiple photons of light simultaneously, which can be used to study the fluorescence of samples in three dimensions. They are similar to two-photon microscopes, but have higher sensitivity and can be used to study thicker samples.
6. Total internal reflection fluorescence (TIRF) microscope: Total internal reflection fluorescence microscopy (TIRFM) takes advantage of the unique qualities of a produced evanescent wave or field in a confined specimen region next to the interface of two mediums with differing refractive indices.

Fluorescence microscope light source

The light source of a fluorescence microscope is an important component that is responsible for providing the excitation light that is used to excite the fluorophores in the sample.

There are several types of light sources that are commonly used in fluorescence microscopy, including:

1. Mercury arc lamps: Mercury arc lamps are one of the most commonly used light sources in fluorescence microscopy. They produce a broad spectrum of light that can be used to excite a wide range of fluorophores. However, they may produce unwanted emissions, which can interfere with fluorescence imaging.
2. Metal halide lamps: Metal halide lamps are a newer type of light source that produce a more focused spectrum of light than mercury arc lamps. They are often used to excite specific fluorophores and can provide higher intensity and more stable light output than mercury arc lamps.
3. LED light sources: LED (light-emitting diode) light sources are becoming increasingly popular in fluorescence microscopy due to their long lifetimes and low power consumption. They produce a narrow spectrum of light that can be used to excite specific fluorophores and can be easily controlled and modulated for advanced imaging applications.
4. Laser light sources: Laser light sources produce a highly focused and coherent beam of light that can be used to excite specific fluorophores. They are often used in advanced imaging techniques, such as superresolution microscopy, that require high-intensity, highly focused light.

Overall, the choice of light source for a fluorescence microscope depends on the specific requirements of the research application and the type of fluorophores being imaged.

Fluorescence microscope camera

A camera is an important component of a fluorescence microscope that is used to capture and record images of the sample. Fluorescence microscopes typically use specialized cameras that are sensitive to the wavelengths of light emitted by the fluorophores in the sample.

There are several types of cameras that are commonly used in fluorescence microscopy, including:

1. CCD cameras: CCD (charge-coupled device) cameras are widely used in fluorescence microscopy due to their high sensitivity and low noise. They are based on a linear array of pixels that convert light into an electrical signal, which can be processed and stored as an image.
2. CMOS cameras: CMOS (complementary metal-oxide-semiconductor) cameras are an alternative to CCD cameras that are based on a different type of pixel technology. They offer similar performance to CCD cameras and are often used in fluorescence microscopy due to their lower power consumption and smaller size.
3. Scientific-grade cameras: Scientific-grade cameras are specialized cameras that are designed for use in scientific research applications. They offer higher sensitivity and resolution than consumer-grade cameras and are often used in advanced imaging techniques, such as superresolution microscopy.

Overall, the choice of camera for a fluorescence microscope depends on the specific requirements of the research application and the type of images being captured. It is important to choose a camera that is compatible with the microscope and has the necessary sensitivity and resolution for the research application.

Application of Fluorescence Microscope

1. The Fluorescence microscope has become an essential tool in medical microbiology and microbial ecology.
2. Bacterial pathogens can be identified after staining them with fluorescent or specifically labeling them with fluorescent antibodies using immunofluorescence produce.
3. In ecological studies, the Fluorescence microscope is used to observe microorganisms stained with Fluorochrome-label probes or Fluorochromes that bind specific call constitutes.
4. Another Important use of the fluorescence microscope is the localization of specific proteins within the cell.
5. Fluorescence microscopy has applications in the biological, biomedical, and material sciences. The special capability of fluorescence microscopes allows for the accurate and detailed identification of cells and sub-microscopic cellular components.
6. Fluorescence microscopy is commonly utilised in the study of histochemistry to detect invisible particles such as neurotransmitter amines.
7. In food chemistry, it is used to evaluate the existence, structural structure, and spatial distribution of specific food components.
8. Fluorescence Speckle Microscopy is a further use of fluorescence imaging. It is a technique that employs fluorescence-labeled macromolecular assemblies, such as cytoskeletal protein, to investigate rates of mobility and turnover.
9. Fluorescence microscopy staining is also useful for applications in the science of mineralogy. It is commonly employed to analyse minerals including coal, graphene oxide, and others.
10. It is also commonly utilised in the textile sector for fibre dimension analysis. Epifluorescence microscopy aids in the investigation of fiber-based materials, such as paper and textiles.
11. Utilizing a fluorescent dye, fluorescence microscopy is appropriate for the study of porosity in ceramics. It is also applicable to semiconductor research.

Advantages of Fluorescence Microscope

There are several advantages of using a fluorescence microscope:

1. High sensitivity: Fluorescence microscopy allows for the detection of low levels of fluorescence, making it a sensitive technique for detecting and visualizing specific molecules or structures within a sample. Due to the presence of higher sensitivity, it can detect the 50 molecules per cubic micrometer.
2. High resolution: Fluorescence microscopy can provide high-resolution images, allowing for the visualization of small structures and details within a sample.
3. Multiplexing: Fluorescence microscopy can be used to visualize multiple different molecules or structures within a single sample using different fluorophores, which can be excited and imaged separately. This allows for the simultaneous visualization of multiple processes or pathways within a sample.
4. Specificity: Fluorescence microscopy can be used to selectively visualize specific molecules or structures within a sample, making it a specific technique for studying specific processes or pathways.
5. Non-destructive: Fluorescence microscopy is a non-destructive technique, meaning that it does not damage the sample being imaged. This allows for the sample to be imaged multiple times or for other analyses to be performed on the same sample.
6. Live-cell imaging: It is used to study the dynamic behavior exhibited in live-cell imaging.
7. Protein Location: It can trace the location of a specific protein in the cell.
8. Multicolor: It allows multicolor staining of the specimen.
9. Use in different Fields: Fluorescence microscopy has applications in the biological, biomedical, and material sciences. The special capability of fluorescence microscopes allows for the accurate and detailed identification of cells and sub-microscopic cellular components.
10. Histochemistry: Fluorescence microscopy is commonly utilised in the study of histochemistry to detect invisible particles such as neurotransmitter amines.
11. Food chemistry: In food chemistry, it is used to evaluate the existence, structural structure, and spatial distribution of specific food components.
12. Fluorescence imaging: Fluorescence Speckle Microscopy is a further use of fluorescence imaging. It is a technique that employs fluorescence-labeled macromolecular assemblies, such as cytoskeletal protein, to investigate rates of mobility and turnover.
13. Mineralogy: Fluorescence microscopy staining is also useful for applications in the science of mineralogy. It is commonly employed to analyse minerals including coal, graphene oxide, and others.
14. Textile sector: It is also commonly utilised in the textile sector for fibre dimension analysis. Epifluorescence microscopy aids in the investigation of fiber-based materials, such as paper and textiles.
15. Ceramics: Utilizing a fluorescent dye, fluorescence microscopy is appropriate for the study of porosity in ceramics. It is also applicable to semiconductor research.

Disadvantages of Fluorescence Microscope

Fluorescence microscopes are powerful tools for studying cells and other biological materials, but they do have some limitations. Some of the main limitations of fluorescence microscopes include:

1. Photobleaching: Fluorescent dyes and proteins can become bleached or damaged when exposed to the light used to excite the fluorescence. This can cause the fluorescence signal to fade over time, limiting the amount of time that a sample can be studied.
2. Phototoxicity: The light used to excite the fluorescence in a sample can also be harmful to living cells, causing them to become damaged or die. This can make it difficult to study living cells or tissues over extended periods of time.
3. Excitation and emission overlap: The wavelengths of light used to excite the fluorescence in a sample and the wavelengths of light emitted by the fluorescence can overlap, making it difficult to distinguish between the two. This can lead to false or misleading results.
4. Limited sample thickness: Fluorescence microscopes are limited in their ability to study samples that are too thick or too dense, as the light used to excite the fluorescence may not be able to penetrate the sample.
5. Limited multiple labeling: Fluorescence microscopes are limited in their ability to simultaneously visualize multiple different fluorophores in a sample. This can make it difficult to study complex systems or processes that involve multiple different molecules or structures.
6. Resolution limits: While superresolution microscopes have significantly increased the resolution of fluorescence microscopes, there are still limits to the level of detail that can be visualized with these instruments.

Resolution of fluorescence microscope

The resolution of a fluorescence microscope refers to its ability to distinguish fine details within a sample. Resolution is typically measured in terms of the smallest distance between two points that can be distinguished as separate entities by the microscope.

In fluorescence microscopy, the resolution is often limited by the wavelength of the excitation light and the numerical aperture (NA) of the objective lens. The shorter the wavelength and the higher the NA, the higher the resolution of the microscope.

There are several factors that can affect the resolution of a fluorescence microscope, including the quality of the objective lens, the quality of the fluorophore, and the quality of the imaging system.

There are several techniques that can be used to improve the resolution of a fluorescence microscope, including superresolution microscopy techniques such as stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). These techniques use specialized optics and image processing algorithms to achieve resolution beyond the diffraction limit of conventional microscopes.

Overall, the resolution of a fluorescence microscope can range from several micrometers for low-resolution microscopes to a few nanometers for high-resolution microscopes using superresolution techniques.

Fluorescence microscope magnification

The magnification of a fluorescence microscope refers to the size of the image of a sample as it appears through the microscope compared to its actual size. Magnification is typically expressed as a ratio, such as 10x or 100x.

In fluorescence microscopy, the magnification of the microscope is determined by the combination of the objective lens and the eyepiece. The objective lens is responsible for collecting and focusing light from the sample, while the eyepiece magnifies the image produced by the objective lens.

The magnification of a fluorescence microscope can be adjusted by changing the objective lens or the eyepiece. Most fluorescence microscopes come with a range of objective lenses with different magnifications, such as 4x, 10x, 40x, and 100x, allowing the user to choose the appropriate magnification for their sample.

The magnification of a fluorescence microscope is an important consideration when choosing a microscope, as it determines the level of detail that can be resolved in the image of the sample. Higher magnification microscopes can provide more detailed images, but may have a smaller field of view and may be more susceptible to chromatic aberrations. It is important to choose a microscope with the appropriate magnification for your specific research needs.

Fluorescence Microscopy Images

Examples

Zeiss fluorescence microscope

Zeiss is a well-known manufacturer of high-quality optical instruments, including fluorescence microscopes. Zeiss fluorescence microscopes are widely used in many different fields, including biology, medicine, and materials science.

Zeiss fluorescence microscopes typically use epifluorescence or total internal reflection fluorescence (TIRF) techniques to visualize fluorescence in samples. They offer high sensitivity and resolution, and can be used to visualize a wide range of fluorophores, including dyes, proteins, and nanoparticles.

Zeiss fluorescence microscopes are also equipped with advanced features such as automated focusing, high-speed imaging, and multi-channel imaging capabilities, which allow for the simultaneous visualization of multiple fluorophores.

Zeiss fluorescence microscopes are available in a range of configurations, including upright, inverted, and superresolution models, to suit the needs of different research applications. They are also compatible with a wide range of imaging software and accessories, such as camera systems and stage-mounted incubators, which allow for the acquisition and analysis of high-quality fluorescence images.

Nikon fluorescence microscope

Nikon is a well-known manufacturer of high-quality optical instruments, including fluorescence microscopes. Nikon fluorescence microscopes are widely used in many different fields, including biology, medicine, and materials science.

Nikon fluorescence microscopes typically use epifluorescence or total internal reflection fluorescence (TIRF) techniques to visualize fluorescence in samples. They offer high sensitivity and resolution, and can be used to visualize a wide range of fluorophores, including dyes, proteins, and nanoparticles.

Nikon fluorescence microscopes are also equipped with advanced features such as automated focusing, high-speed imaging, and multi-channel imaging capabilities, which allow for the simultaneous visualization of multiple fluorophores.

Nikon fluorescence microscopes are available in a range of configurations, including upright, inverted, and superresolution models, to suit the needs of different research applications. They are also compatible with a wide range of imaging software and accessories, such as camera systems and stage-mounted incubators, which allow for the acquisition and analysis of high-quality fluorescence images.

Evos fluorescence microscope

The EVOS fluorescence microscope is a brand of microscope produced by Thermo Fisher Scientific, a leading manufacturer of scientific instrumentation. The EVOS fluorescence microscope is a versatile, high-performance microscope that is widely used in many different fields, including biology, medicine, and materials science.

The EVOS fluorescence microscope uses epifluorescence or total internal reflection fluorescence (TIRF) techniques to visualize fluorescence in samples. It offers high sensitivity and resolution, and can be used to visualize a wide range of fluorophores, including dyes, proteins, and nanoparticles.

The EVOS fluorescence microscope is equipped with advanced features such as automated focusing, high-speed imaging, and multi-channel imaging capabilities, which allow for the simultaneous visualization of multiple fluorophores. It is also compatible with a range of imaging software and accessories, such as camera systems and stage-mounted incubators, which allow for the acquisition and analysis of high-quality fluorescence images.

Overall, the EVOS fluorescence microscope is a powerful and versatile tool for studying the structure and function of cells, tissues, and molecules at the microscopic level.

FAQ

Q1. When do we use a fluorescence microscope?

Fluorescence microscopy is a widely used technique in many different fields, including biology, medicine, and materials science. Some common applications of fluorescence microscopy include:

1. Cell biology: Fluorescence microscopy is frequently used to study the structure and function of cells and their components, such as organelles, proteins, and nucleic acids.
2. Molecular biology: Fluorescence microscopy can be used to visualize and study specific molecules within a sample, such as DNA, RNA, or proteins.
3. Developmental biology: Fluorescence microscopy is commonly used to study the development and differentiation of cells and tissues in living organisms.
4. Neuroscience: Fluorescence microscopy is used to study the structure and function of neurons and the connections between them.
5. Drug discovery and development: Fluorescence microscopy can be used to study the mechanisms of action of drugs and to identify potential new drugs.
6. Materials science: Fluorescence microscopy is used to study the properties and behavior of materials at the microscale.
7. Environmental science: Fluorescence microscopy is used to study the presence and distribution of contaminants in water, soil, and air.

Overall, fluorescence microscopy is a powerful and versatile tool for studying the structure and function of cells, tissues, and molecules at the microscopic level.

Q2. Fluorescence microscope price?

The price of a fluorescence microscope can vary widely depending on the manufacturer, model, and features. Entry-level fluorescence microscopes may cost several thousand dollars, while more advanced models with additional features and capabilities can cost tens of thousands of dollars or more.

Some factors that can affect the price of a fluorescence microscope include:

1. Type of microscope: Upright microscopes are generally less expensive than inverted microscopes, which are typically more expensive due to their more complex design.
2. Magnification: Higher magnification microscopes are generally more expensive than those with lower magnification.
3. Resolution: Higher resolution microscopes are generally more expensive than those with lower resolution.
4. Fluorophores: Fluorescence microscopes that can visualize a wide range of fluorophores are generally more expensive than those that are limited to a specific range of fluorophores.
5. Additional features: Fluorescence microscopes with advanced features such as automated focusing, high-speed imaging, or multi-channel imaging capabilities may be more expensive than those without these features.

Overall, the price of a fluorescence microscope can range from several thousand dollars for an entry-level model to tens of thousands of dollars or more for a high-end model with advanced features. It is important to consider the specific requirements and budget of your research when choosing a fluorescence microscope.

Reference

• https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy
• https://www.semrock.com/introduction-to-fluorescence-filters.aspx
• https://www.slideshare.net/VIVEKKUMARSINGH109/fluorescence-microscopy-72205439
• https://www.slideshare.net/doctorrao/fluorescent-microscopy
• https://en.wikipedia.org/wiki/Fluorescence_microscope
• http://www.scholarpedia.org/article/Fluorescent_proteins
• https://pubmed.ncbi.nlm.nih.gov/18228363/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711767/