The #fluorescence microscopes are an integral part of #cellbiology labs these days with their applications ranging from localization of #proteins to determination of intracellular ion uptake. Lets learn a little about the innerworkings.
Principle:
The first barrier filter or #excitation filter transmits only such UV or blue wavelengths that are near the absorption maximum of dyes used in staining the sample.
The #dichroic mirror is a beam splitter that transmits only selected long wavelengths in a single direction and reflects back other <500nm wavelength rays.
The second barrier filter or #emission filter absorbs remaining short wavelength, to transmit pure long wavelength rays as far as possible, and also darken the background field.
Merits:
It allows #specific and #sensitive #staining of a specimen to detect distribution of molecules of interest.
It allows analysis of #co-localization of #proteins by using different color filters.
Demerits:
#Hazing Effect: No sharp image because of rays coming from all planes of the specimen, out of focus rays.
Specimen fixation may cause artefacts and #overexpression may form from inclusion bodies.
#Fluorochromes lose their ability to fluoresce as they are illuminated, in a process known as photo-bleaching.
The live cells under analysis are susceptible to photo-toxicity, particularly with short wavelength rays.
Time consuming, and preparations are laborious.
The process is antibody-dependent, so any cross-reactivity may result in false report.
#Fluorescent molecules have a tendency to generate reactive oxygen species ( #ROS ) under illumination. This can hamper cells during live cell imaging.
Only 2-D image is generated.
Applications:
#Localization of unknown proteins inside cells: Cells can be transfected with a shuttle vector encoding the protein of interest tagged with #GFP. The cells or tissue is then fixed with a fixative like paraformaldehyde or ethanol and viewed under blue filter of fluorescence microscope to localize the protein inside cells.
#Determination of Ca2+ uptake inside cells: The role of Ca2+ as a second messenger is well established in the context of cell signaling. #Fura2 is a ca2+ sensitive fluorescent dye that has 5 #carboxylate groups and cannot penetrate cell membrane. With ethanol, #Fura2 is converted to #Fura2-ester outside cells and allowed to enter cells because of its lipophilic nature. Inside cells, F#ura2-ester is converted to #Fura-2 again by cytoplasmic #esterase and ester hydrolase. This #Fura-2 then binds to intracellular Ca2+ and cannot come out of cell because of its now non-lipophilic nature. Now when excited with a particular #wavelength under fluorescence microscope, we can measure Ca2+ uptake by cells.
More to munch on:
# Bradbury, S. and Evennett, P., Fluorescence microscopy, Contrast Techniques in Light Microscopy., BIOS Scientific Publishers, Ltd., Oxford, United Kingdom (1996).
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