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®.
• 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)