Light Engine Color Channel Selection Facilitates Multicolor Fluorescence Imaging

Light Engine Color Channel Selection Facilitates Multicolor Fluorescence Imaging

The capacity to electronically switch light output from one color channel to another is advantageous in sequential acquisition multicolor fluorescence imaging. Typically, SPECTRA light engine®, SPECTRA X light engine® or AURA light engines® with internal excitation filters matched to the reflection bands of polychroic beamsplitters and emission filters are used for this purpose. Channel switching can be controlled by serial commands (~10 ms) or TTL signals (~10 μs) depending on the speed of sequential acquisition required. Acquiring similar data using a white light source requires switching excitation filters using an accessory filter wheel, which is both slower (~50 ms) and susceptible to motion-related color registration artifacts. Alternatively, a multiband excitation filter + white light source allows simultaneous excitation of multiple fluorophores but requires a color camera (or human vision) to render a color separated image (see Figure A). The MIRA light engine®, with manual or serial command controlled color channel selection, can be used to generate a color separated image using a multiband excitation filter and a monochrome camera (see Figure B). The principle is the same as that applied to obtain color images in transmitted light microscopy using a monochrome camera and the LIDA light engine®

Technical Details
MIRA light engine, dual band exciter 390/22 + 475/33. Semrock FF409/493/573/652-Di01 polychroic and FF01 432/515/595/730 quad
• Nikon Ti microscope, 40X objective, Andor Zyla 5.5 camera, 100 ms exposures
• Sample: BPAEC cells labeled with DAPI (nucleus) and Alexa Fluor 488 phalloidin (cytoskeletal actin)

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