Propidium iodide binds to DNA in the nucleus and fluoresces red under ultraviolet light. You can see the dead cells in the same specimen if you mix in another dye called propidium iodide, which only penetrates the dead cells. Therefore, living cells will fluoresce green, and dead cells will not fluoresce. Living cells have an enzyme that removes the AM portion, traps the calcein within the cell and allows the calcein to bind calcium so that it fluoresces green under ultraviolet light. When you mix the calcein/AM with the solution bathing the cells, the dye crosses into the cell. The AM portion of the molecule hides a portion of the calcein molecule that binds calcium, which is fluorescent. For example, you can stain cells with a dye called calcein/AM. The fluorescent molecules within the specimen can either occur naturally or be introduced. This fluorescent light passes through the dichroic mirror and a barrier filter (that eliminates wavelengths other than fluorescent), making it to the eyepiece to form the image. The objective lens collects the fluorescent-wavelength light produced. ![]() The ultraviolet light excites fluorescence within molecules in the specimen. ![]() The dichroic mirror reflects the ultraviolet light up to the specimen. The light comes into the microscope and hits a dichroic mirror - a mirror that reflects one range of wavelengths and allows another range to pass through. Light path of an epifluorescence microscopeĪ fluorescence microscope uses a mercury or xenon lamp to produce ultraviolet light.
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