Question

COMPARE FLUORESCENT MICROSCOPY WITH DARK FIELD MICROSCOPY . WHAT SIMILAR ADVANTAGE DO THEY PROVIDE? AND HOW DO THEY DIFFER?

Answer 1

Similar advantages between fluorescence and dark field microscopy (Comparison):

1. Both microscopes provide images wherein the background is dark and the cells/specimen are lighted bright. Hence, it is easier to visualize the specimen clearly. Both microscopes produce images in visible light wavelength, making it easier to visualize the specimen.

2. Live specimens can be visualized by both techniques. Live cells can be imaged with fluorescence imaging where the cells can be modified to express the fluorescent protein before imaging.

3. High contrast images are created by both microscopes which have higher resolution. This is because of the dark background providing better contrast and not obscuring the specimen.

4. Both techniques can be used to identify infectious microorganism from the sample. Treponema pallidum is identified by dark field microscopy. Based on protein expression, fluorescence microscopy can be used to detect the pathogen based on expression of specific proteins. Legionella infections are identified by fluorescein antibody test.

Differences between fluorescence and dark field microscopy:

Fluorescence microscopy	Dark field microscopy
1. Fluorescence dye are used which absorb light in UV range and emit light of different colors in visible range. A filter will remove all other light and only allows detection of the emitted wavelength of light.	Dark field microscopy is used to visualize unstained objects. A hollow cone of light is focused on the specimen by placing a small opaque disk between the illuminator source and the condenser. The specimen will then refract the light and is illuminated as white light. The background remains dark.
2. Ultraviolet light, which has a shorter wavelength is used to illuminate the specimen. Light emitted by the specimen is of a longer wavelength (visible range) and is visualized by the color emitted. UV light is absorbed by the specimen. Electrons get excited and then emit light.	Visible light is used to illuminate the specimen. Visible light of same wavelength is scattered or refracted by the specimen. The light refracted is white light. There is only scattering of light, and the specimen does not absorb this light.
3. Internal structure, proteins and other components can be visualized when the fluorescent dye is conjugated to an antibody specific to that protein. This technique is known as immunofluorescence.	Internal structures/proteins cannot be visualized by this technique. Specimen is observed as whole entity.
4. Multiple proteins/structures can be visualized at the same time by conjugating different fluorophore dyes that emit light of different colors. Further, live cell imaging is possible with fluorescence microscopy (via confocal imaging).	Multiple proteins cannot be visualized by this technique.
5. The fluorescence can be damaged by photo bleaching. Hence, exposure to visible light will decrease the intensity of fluorescence emitted. Phototoxicity is also observed if the specimen is exposed for linger duration. Autofluorescence is emitted by lipids in the cells. which may affect the analysis.	Photo bleaching and phototoxicity does not occur as visible light is used to illuminate the object. Autofluorescence does not affect the results.
6. Both fixed and unfixed sample are used for this method. In fixation method, the fixation agents such as paraformaldehyde, methanol, acetone etc may create artifacts in the specimen.	Unstained specimens are only visualized. Hence, there is no artifact created in the sample as no fixation by chemicals is performed.