Optical filters must be precisely designed to match the specific spectral output of the light source used in fluorescence microscopy. Filter requirements vary dramatically depending on the illumination source. Light Source Output Spectrum Characteristics Filter Design Implications Advantages Disadvantages Mercury Arc Lamp (HBO) Non-continuous spectrum concentrated in narrow spectral lines (~10 nm wide). High brightness (luminance/radiance) in UV and visible. Excitation filters must transmit one or more specific mercury lines. Wide-band filters might be used but risk increasing autofluorescence noise. Most common historical source; high brightness/luminance. Non-continuous output. Short rated...
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The Stokes shift is simply defined as the difference in wavelength between two critical points for a fluorochrome:
1. The peak excitation intensity (the maximum wavelength of light absorbed).
2. The peak emission intensity (the maximum wavelength of light radiated as fluorescence).
Since fluorescence involves absorbing light and then radiating light of a lower energy, the emitted light always has a longer wavelength than the excitation light.
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