Fluorescence Microscopy | Definition | Working | 3 Important Limitations

Fluorescence Microscopy

Contents

What is fluorescence microscopy?

Fluorescence microscopes a type of compound microscope that works with several variants of light sources having different wavelength ranges for fluorescing a specimen to produce images replacing the use of transmission, absorption, reflection, and scattering. These microscopic variants are mainly used for observing biological samples. Fluorescence microscopy can be achieved by using the simple design of an epi-fluorescence microscope or a complex con-focal microscope.

How does fluorescence microscopy work?

The sample is kept on the microscopic stage first. The sample is then illuminated depending on the amount of light needed. In fluorescence microscopy, the light of different wavelengths is used to illuminate the specimen and this light is then absorbed by the fluorophores present in the specimen that further emits a light beam of longer wavelengths or of a different color than that of the incoming light. A spectral emission filter is used for separating the weaker emitted light from the stronger illuminating light. A general fluorescence microscope has the following parts.

  1. An excitation filter.
  2. A dichroic mirror or beam-splitter.
  3. An emission filter.
  4. A light source (that can be a xenon arc lamp, mercury-vapor lamp, high-power LEDs, or lasers).
  5. A set of objective lenses.
  6. An ocular lens.
  7. A stage to hold the sample.
  8. A detector.

The dichroic beam splitter and the excitation and the emission filters are selected as such that the spectral excitation should be compatible with emission characteristics, of the fluorophore utilized for labeling samples and this is how the distribution of a single color or fluorophore is detected at that time. For observing multicolor images several single-color images need to be combined. Certain microscopic designs come with a CCD (charge-coupled device) in the microscope tube in place of an eyepiece lens. In a CCD-installed microscope, the image formation occurs on a monitor screen by placing the CCD on the intermediate image plane.

What is epi-fluorescence microscopy?

In Epi-fluorescence microscopy the excitation of the sample with fluorescence and the detection of emitted fluorescent light is done by following the same path of light i.e. through the objective lens. Such microscopes are specially used for observing living samples. For obtaining greater resolution of images, the numerical aperture of the objective lens has o be increased. Epi-fluorescence produces a high signal-to-noise ratio as some amount of reflected illuminating light combines with the emitted light from the sample. For this reason, the dichroic beamsplitter is used. This beamsplitter acts as a selective wavelength filter and transmits only the emitted fluorescence light to the eyepiece or ocular lens.

What are the demerits of fluorescent microscopy?

  1. With the illumination of the fluorophores over time as a result of the photobleaching process, the fluorophores may lose their ability to fluoresce. Photobleaching happens when the fluorescent molecules gradually suffer from the chemical damage due to the excitation of electrons during fluorescence. Photobleaching can severely affect the time period of observing a sample by fluorescence microscopy. 
  2. Fluorescence microscopy has allowed the examination of live cells. However, these live cells are prone to phototoxicity (mainly with short-wavelength light) and Fluorescent molecule might produce specific reactive agents when it is illuminated, this further increases the photo-toxic effect.
  3. Fluorescence microscopy can allow the imaging and observation of only certain structures that have been labeled for fluorescence. i.e., a tissue sample could be observed with the help of fluorescence microscopies if the samples are presented with a fluorescent DNA stain. This might show the DNA organization within the cells but is unable to reveal anything about the cell morphology.

How is a fluorescent sample prepared?

We know that, fluorescence microscopy allows the imaging and observation of only certain structures that have been labeled for fluorescence. Therefore, the sample to be observed is made to be fluorescent by various techniques such as:

  1. Biological fluorescent stains: Several types of fluorescent stains are made, that are compatible with a range of biological specimens. Some examples of such fluorescence stains are DRAQ7 and DRAQ5 (excited by red light), Hoechst and DAPI (excited by Ultraviolet wavelength light), phalloidin, Collagen Hybridizing Peptide, etc.
  2. Immunofluorescence: Immunofluorescence is used for binding an antibody.
  3. Fluorescent proteins: Fluorescent proteins are used for modifying DNA such that the molecules demonstrate fluorescence.
A sample of herring sperm stained with SYBR green in a cuvette illuminated by blue light. image source: Zephyris – Own work
CC BY-SA 3.0
File:FluorescenceMicroscopeSample HerringSpermSYBRGreen.jpg
A fluorescent stain. Image source: Swastipandey – Own work
CC BY-SA 4.0
File:Fluorescent dye.jpg

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About Sanchari Chakraborty

I am an eager learner, currently invested in the field of Applied Optics and Photonics. I am also an active member of SPIE (International society for optics and photonics) and OSI(Optical Society of India). My articles are aimed towards bringing quality science research topics to light in a simple yet informative way. Science has been evolving since time immemorial. So, I try my bit to tap into the evolution and present it to the readers.

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