Multifocal microscopy enables high-speed three-dimensional (3D) volume imaging by using a multifocal grating in the emission path. This grating is typically designed to afford a uniform illumination of multifocal subimages for a single emission wavelength. Using the same grating for multicolor imaging results in non-uniform subimage intensities in emission wavelengths for which the grating is not designed. This has restricted multifocal microscopy applications for samples having multicolored fluorophores. In this paper, we present a multicolor multifocal microscope implementation which uses a Spatial Light Modulator (SLM) as a single multifocal grating to realize near-uniform multifocal subimage intensities across multiple wavelength emission bands. Using real-time control of an in-situ-optimized SLM implemented as a multifocal grating, we demonstrate multicolor multifocal 3D imaging over three emission bands by imaging multicolored particles as well as Escherichia coli (E. coli) interacting with human liver cancer cells, at ∼ 2.5 multicolor 3D volumes per second acquisition speed. Our multicolor multifocal method is adaptable across SLM hardware, emission wavelength band locations and number of emission bands, making it particularly suited for researchers investigating fast processes occurring across a volume where multiple species are involved.
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