Light Source Power Levels
Choosing the appropriate light source for investigations in optical microscopy is highly dependent upon the illumination strategy (transmitted or episcopic), specimen parameters, microscope configuration, and the detector sensitivity. In fluorescence microscopy, the class of fluorophore (synthetics, fluorescent proteins, quantum dots, etc.), filter set characteristics (bandwidth and profile), and detector acquisition speed are variables that must be also considered. As a general rule, synthetic fluorophores and quantum dots can be used with fixed cells with high illumination power, whereas in live-cell imaging, fluorescent proteins and other probes should be excited at much lower power levels. The tables presented below are compilations of the comparative output powers (measured at the objective focal plane) for the most popular non-coherent light sources used in epi-fluorescence optical microscopy.
Comparative Optical Power of Light Sources
1ZEISS Filters 2Semrock Filters 3LED Peak Wavelength 4Tungsten-Halogen Lamp Voltage = 12.2 V
The optical output power (presented in milliwatts/cm2) of the most commonly used light sources in widefield fluorescence microscopy are compared in Table 1. Power levels were measured at the focal plane of the microscope objective (40x fluorite dry, numerical aperture = 0.85) using a photodiode-based radiometer. Lamphouses were either coupled directly to the microscope epi-illumination optical system (xenon, mercury, tungsten-halogen, and LED sources), or directed through a liquid light guide and a collimating lens attached to the illuminator (metal halide source). Light from the source was directed through the microscope optical train and into the selected fluorescence filter sets listed in the first column of Table 1. Power measurements represent the amount of light that passes through the excitation filter and is reflected into the objective by the dichromatic mirror for each filter set. Note that the tungsten-halogen light source, which exhibits the lowest excitation power of any source listed in the table, was operated at the highest possible voltage for these measurements.
Comparative Optical Power of Metal Halide Light Sources
1ZEISS Filters 2Semrock Filters
Presented in Table 2 are the optical output power values of several commercial metal halide light sources after passing from a liquid light guide through the microscope optical train and selected fluorescence filter sets. Power (in milliwatts/cm2) was measured at the focal plane of the microscope objective (40x fluorite dry, numerical aperture = 0.85) using a photodiode-based radiometer. Either a mirror with greater than 95% reflectivity from 350 to 800 nanometers or a standard fluorescence filter set was used to project light through the objective and into the radiometer sensor. The light throughput loss in a microscope illumination system can vary between approximately 50 and 99 percent of the input power, depending upon light source coupling mechanism and the number of filters, mirrors, prisms, and lenses in the optical train. As an example, for a typical research-grade inverted microscope coupled to an external metal halide illumination source, less than 5 percent of the light exiting the liquid light guide at the entrance of the collimating lens system is available for excitation of fluorophores positioned at the objective focal plane. A similar range of light loss occurs with traditional xenon and mercury arc discharge lamps secured directly to the illuminator through a lamphouse.
Christopher S. Murphy and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.