Fluorescence microscopy is a light microscope that works on the principle of fluorescence. A substance is said to be fluorescent when it absorbs the energy of invisible shorter wavelength radiation (such as UV light) and emits longer wavelength radiation of visible light (such as green or red light). This phenomenon, also called fluorescence, is widely used in clinical and diagnostic settings to detect microorganisms, antibodies, and many other substances rapidly.
Some cells fluoresce naturally under ultraviolet light because they contain fluorescent substances such as chlorophyll. If the specimen to be viewed does not naturally fluoresce, it can be stained with fluorescent dyes called fluorochromes. Commonly used fluorescent dyes are; DAPI (49,6-diamidino-2-phenylindole), acridine orange, auramine-rhodamine, Alexa Fluors, or DyLight 488.
When fluorescence microscopy is used for the detection of antigen-antibody reaction, it is known as immunofluorescence.
Components of a Fluorescence Microscope
The fluorescence microscope has a
- Light source: Xenon arc lamps or mercury-vapor lamps are a common source of ultraviolet light; power LED and lasers are used in more advanced forms.
- A set of optical filters: Optical filters include a set of a compatible excitation filter, emission filter, and dichroic beam splitter;
- An excitation filter selects the wavelengths to excite a particular dye within the specimen.
- A dichroic beam splitter/ dichroic mirror reflects light in the excitation band and transmits light in the emission band, enabling the classic epifluorescence incident light illumination.
- An emission filter provides quality control by letting only the wavelengths of interest emitted by the fluorophore pass through.
- Darkfield condenser: It provides a black background against which the fluorescent objects glow.
The filters are often plugged together in a filter cube (compound microscopes) or a flat holder (mainly stereo microscopes).
To observe the sample through a fluorescence microscope, it should first be labeled with fluorescent dyes/substances known as fluorophores.
Higher energy shorter wavelength lights (UV rays or blue light) generated from mercury vapor arc lamp pass through the excitation filter. The excitation filter allows only the short wavelength of light to pass through and removes all other non-specific wavelengths of light. The filtered light is reflected by the dichroic filter and falls on the fluorophore-labeled sample. The fluorochrome absorbs shorter wavelength rays and emits rays of longer wavelength (lower energy) that pass through the emission filter. The emission filter blocks (suppresses) any residual excitation light and passes the desired longer emission wavelengths to the detector. Thus the microscope forms glowing images of the fluorochrome-labeled microorganisms against a dark background.
To the observer, the background is dark, as there is no visible light and only the labeled specimen (cells, microorganisms, etc.) appear bright (fluoresce).
Types of Fluorescence Microscopes
There are various types of fluorescence microscopes. Some of the common types are:
It is the most common type of fluorescence microscope. In this microscope, excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e., through the objective).
In this type of fluorescence microscope, high‐resolution imaging of thick specimens (without physical sectioning) can be analyzed using fluorescent-labeled dye.
In this type of microscope, multiphoton fluorescence excitation captures high-resolution three-dimensional images of specimens tagged with highly specific fluorophores.
Total internal reflection fluorescence (TIRF) microscope
Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices.
Applications of Fluorescence Microscope
Fluorescence microscopy is widely used in diagnostic microbiology and microbial ecology (for enumerating bacteria in natural environments). Some organisms, such as Pseudomonas, fluoresce naturally when irradiated with ultraviolet light. Other organisms, such as Mycobacterium tuberculosis and Treponema pallidum, are treated with fluorochrome.
- Acid-fast bacilli (AFB) in sputum or CSF are detected when stained with auramine fluorescent dye.
- Detection of Trichomonas vaginalis, intracellular gonococci, and other parasites when stained by acridine orange.
- In immunodiagnosis of infectious diseases, using both direct and indirect antibody techniques. Immunofluorescence is especially useful in diagnosing syphilis and rabies.
Limitations of Fluorescence Microscope
- Fluorophores gradually lose their ability to fluoresce as they are illuminated in photobleaching. Photobleaching can severely limit the time a sample can be observed by fluorescence microscopy. However, several techniques exist to reduce photobleaching, such as using more robust fluorophores, minimizing illumination, or using photoreactive scavenger chemicals.
- Fluorescence microscopy has enabled the analysis of live cells, but fluorescent molecules generate reactive chemical species under illumination that enhances the phototoxic effect, to which live cells are susceptible.
- Fluorescence microscopy only allows observation of the specific structures labeled for fluorescence. For example, observing a tissue sample prepared with a fluorescent DNA stain by fluorescence microscopy only reveals the organization of the DNA within the cells and reveals nothing else about the cell morphologies.
References and Further Readings
- Sanderson, M. J., Smith, I., Parker, I., & Bootman, M. D. (2014). Fluorescence Microscopy. Cold Spring Harbor Protocols, 2014(10), pdb.top071795. https://doi.org/10.1101/pdb.top071795
- Fluorescent Microscopy. Microscopy. Retrieved May 18, 2020, from https://serc.carleton.edu/microbelife/research_methods/microscopy/fluromic.html
- Fish, K. N. (2009). Total Internal Reflection Fluorescence (TIRF) Microscopy. Current Protocols in Cytometry / Editorial Board, J. Paul Robinson, Managing Editor … [et Al.], 0 12, Unit12.18. https://doi.org/10.1002/0471142956.cy1218s50