As mentioned in 'Introduction to fluorescence' there are four key components in a fluorescence detection system: 1) The emission light source 2) The fluorophore 3) A filter 4) The detector
There are lots of different systems used to detect fluorescent signals in biomedical research including fluorescence microscopes, spectrophotometers, scanners and flow cytometers but in this section we focus on fluorescent microscopy.
Fluorescent microscopy allows us to visualise flurophores on a microscopic scale in 2D or 3D. While some flurophores occur naturally, in biomedical research we typically introduce these fluorphores into cells and tissues to tag proteins of interest or report on cellular processes such as calcium dynamics.
Some example uses of fluorescent microscopy include: - Determining where the PSD95 protein is located in neurons by tagging the protein with antibodies coupled to fluorphores. - Using a fluorphore-coupled antibody to GFAP to determine whether a cell in culture is likely to be an astrocyte. - Using DAPI dye to count of the number of cells in a slice of tissue. - Testing whether transfection of a plasmid containing a fluorescent reporter has been successful. - Using a proximity ligation assay to determine if two proteins exist in close proximity to each other. - Measuring voltage changes in neurons using a voltage-dye.
There are lots of different possible light sources including: - Incandescent bulbs - Arc lamps - LEDs - Lasers