On/Off and intensity controls on Nikon Ti microscope base
The PEKA light engine®, first introduced in 2014, provides a powerful alternative to conventional tungsten-halogen sources for transmitted light microscopy. Unlike tungsten-halogen lamps, the color temperature of the PEKA light engine’s white light output does not change when the intensity is turned up or down. Consequently, the PEKA light engine is able to deliver consistent color rendition while dispensing with the need to use neutral density filters for light attenuation. Furthermore, unlike phosphorcoated LED sources, the PEKA light engine does not generate background emission in the epifluorescence light path. The newest addition to the PEKA light engine product line (90-10184) is configured for fully integrated operation on Nikon Ti microscopes. Light on/off and intensity adjustments are directly controlled by the push button and rotary dial located next to the focus knob on the Nikon Ti base.
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It’s a well-known, yet often overlooked, fact. No component makes a bigger contribution to optical throughput in epi-fluorescence microscopy than the objective. To a large extent, this is because the optical path passes through the objective twice — once for excitation and again for emission detection. Doubling the numerical aperture (NA) of the objective with the same magnification increases the detected fluorescence intensity by a factor of 16.
Although optimizing the optical throughput of a fluorescence microscope requires attention to many factors including light source output, light source coupling to the microscope, optical filtering and camera quantum efficiency, objective selection remains most important of all.
For example, here is fluorescence image of a brine shrimp imaged with a 10X 0.5 NA objective. The two plots below the image are digital representations of the image data, showing the effect of switching to a 10X 0.3 NA objective with all other image acquisition parameters unchanged.
10X 0.5 NA OBJECTIVE
Specimen: Eosin-stained brine shrimp
SPECTRA X light engine®, single band exciter 470/24 (CWL/FWHM), 10% maximum output intensity. Semrock FF506-Di03 dichroic and FF01-545/55 emitter.
Nikon Ti microscope, 10X, 0.5 NA objective OR 10X 0.3 NA objective, Hamamatsu ORCA-Flash4.0, 50 ms exposure.