Comparison of different Light Sources in Fluorophore
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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 life (~200 hours). |
| Xenon Arc Lamp (XBO) | Relatively continuous spectrum in the visible range. Much greater output in the Near-IR (NIR). | Excitation filters require excellent blocking in the NIR (>800 nm). Filters must withstand higher thermal loads. | Preferred for quantitative analysis due to continuous spectrum. | Not as bright as mercury lamps of equivalent wattage. Larger arc size limits focusable light. Short rated life (~400 hours). |
| Metal-Halide Arc Lamp | Spectrum generally similar to mercury arc lamps, but with greater relative intensity between emission lines. High output in the IR. | Requires handling of high IR output. | Long useful life (up to 2000 hours). Easy and stable alignment due to integral elliptical reflector. | Average brightness is similar to xenon arc lamps due to larger arc size (~1.2 mm). |
| Light-Emitting Diode (LED) | Light emitted in a relatively narrow spectral bandwidth (10 to 40 nm FWHM). Limited number of spectral bands available. Peak wavelength variability (up to 20 nm) between batches. | Simplified excitation filter design; narrow spectral band eliminates the need for extended blocking ranges. | Long lifetimes (10,000+ hours). Output can be controlled very rapidly (millisecond order). Nearly as bright as arc lamps within their narrow band. | Limited spectral bands available (lack a strong green source). Batch variability and output sensitivity to temperature/current (up to 5 nm shift). |
| Lasers (Solid State/DPSS) | Extremely high brightness over narrow (monochromatic) spectral bands. Often polarized. | Excitation filters must block the entire output spectrum (e.g., NIR output at 1064 nm from DPSS lasers). Filters should attenuate light primarily by reflection to reduce thermal load. Beamsplitters must be designed considering polarization conditions. | Ubiquitous in Confocal Microscopy due to high brightness. | Requires stricter optical quality (TWD, wedge) in all filter components. Excitation filters must be highly reflective. |
