Fluorescence is the physical effect caused when a substance emits light energy on being irradiated with light or other forms of electromagnetic radiation. A fluorescence microscope is an optical microscope that makes use of fluorescence and phosphorescence in place or in combination with reflection and absorption to study the optical properties of organic and inorganic objects. A fluorescence microscope helps in visualising the sub-cellular and internal structures of an object with better clarity. It was discovered during the early twentieth century by August Köhler, Carl Reichert, and Heinrich Lehmann.
Working Principle of Fluorescence Microscope
A fluorescence microscope typically works on the principle of staining the components and structures of the sample with dyes. This is because most of the cellular components are colourless in nature. Hence, they can not be easily distinguished from each other. The fluorophore or the fluorescent molecules irradiated with short-wavelength light or electromagnetic rays tend to emit light. The emitted light has a longer wavelength as compared to the incident light. Typically, the time duration between the irradiation and emission is in nanoseconds and can be neglected easily. The emitted light is separated from the incident light, which tells the exact location of the fluorophores. The image of the sample is produced on the basis of the secondary light rather than on the basis of the primary incident light.
Working of Fluorescence Microscope
A fluorescence microscope works in a similar manner as an optical microscope. It consists of a strong light source, special bandpass filters, and fluorescent labelling of a sample. The sample to be studied is placed under the microscope. A light ray that has a wavelength short enough to excite the fluorophore is made to fall on the sample. This ray of light is focused on the specimen with the help of an objective lens. The excited ray emitted by the specimen is then focused on the detector via the objective lens. A major part of the exciting light ray gets transmitted through the sample object, while some part of it gets reflected back and reach the detector.
Components of Fluorescence Microscope
The fluorophore is a chemical compound that has the ability to emit light on being excited by an electromagnetic or light wave. It is also known as a fluorescent dye. Fluorophores basically consist of a combination of aromatic groups or plane/cyclic molecules with various π bonds.
2. Light Source
A light source is one of the most essential components of a fluorescence microscope. Here, the most common light sources used for excitation include lasers, high power LEDs, xenon arc lamps, mercury vapour lamps, etc.
3. Excitation Filter
An excitation filter used in a fluorescence microscope is typically a bandpass filter that allows the signal that falls into a certain bandwidth while rejecting all the others. The wavelengths absorbed by the fluorophore gets easily passed, while the reflected light radiations get blocked.
4. Dichroic Mirror
A dichroic mirror acts as a thin-film filter. The main use of a dichroic mirror is to selectively pass light of a small range of colours while reflecting all the others. It is also known as an accurate colour filter.
5. Emission Filter
The emission filter is a type of bandpass filter. The working of the emission filter is similar to that of an excitation filter. It passes the light radiations emitted by the fluorophore while blocking the excitation light.
Advantages of Fluorescence Microscope
1. The sensitivity of a fluorescence microscope is significantly high, i.e., approximately equal to 50 molecules per cubic micrometres.
2. With the help of a fluorescence microscope, different molecules can be stained with different colours, thereby allowing the user to picture multiple molecules of a substance at the same time.
3. Fluorescent microscope is comparatively more efficient than traditional optical microscopes.
4. It can be used for both in vivo and in vitro imaging.
5. A fluorescence microscope has the ability to isolate individual proteins with a high degree of precision.
6. Fluorescence microscopy is best suited for imaging and studying the dynamic behaviour exhibited by living cells.
7. A fluorescent microscope is able to produce images with high resolution and clarity, thereby enhancing the research.
8. It is a combination of the magnifying properties of an optical microscope and the visualization of fluorescence.
9. A fluorescent microscope can be used to generate a three-dimensional magnified image of the sample that is being studied.
Disadvantages of Fluorescence Microscope
1. The fluorescent chemical compound or the fluorophore present in the fluorescent microscopes tend to lose its ability to emit light over time when excited due to the process called photobleaching. Photobleaching generally occurs when the fluorophore molecules undergo chemical damage due to electron excitation during Fluorescence.
2. It only allows observation and study of certain structures of the sample that have already been highlighted and labelled for fluorescence.
3. When a fluorescent material is illuminated with light rays, the fluorophore molecules tend to generate chemical species, which further leads to phototoxicity. This phototoxicity effect disrupts the original nature of the sample under observation. The phototoxicity is comparatively high for short-wavelength light radiations.
4. A fluorescence microscope is unable to provide information regarding the lipid bilayer, especially for the SUVs.
5. A fluorescence microscope can not generate any conclusive observations related to the bilayer characteristics or lamellarity of the vesicles.
6. The addition of probes and dyes tend to interfere with the properties of the lipid vesicles, which can inaccurate data interpretation, thereby disrupting the authenticity of the experiment.
7. Photo-induced lipid peroxidation may lead to domain formation.
Uses of Fluorescence Microscope
1. Fluorescence microscopes are widely used to study inner structures of a sample at the micro-level with maximum quality.
2. Fluorescence microscope is best suited to study live biological samples.
3. Such microscopes are generally preferred in labelling the innermost structures of a cell or a microorganism.
4. It is helpful in the measurement of the physiological state of a cell or the sample under observation.