Multifocal microscopy (MFM) offers high-speed three-dimensional imaging through the simultaneous image capture from multiple focal planes. Conventional MFM systems use a fabricated grating in the emission path for a single emission wavelength band and one set of focal plane separations. While a Spatial Light Modulator (SLM) can add more flexibility as a replacement to the fabricated grating, the relatively small number of pixels in the SLM chip, cross-talk between the pixels, and aberrations in the imaging system can produce non-uniform intensity in the different axially separated image planes. We present an in situ iterative SLM calibration algorithm that overcomes these optical- and hardware-related limitations to deliver near-uniform intensity across all focal planes. Using immobilized gold nanoparticles under darkfield illumination, we demonstrate superior intensity evenness compared to current methods. We also demonstrate applicability across emission wavelengths, axial plane separations, imaging modalities, SLM settings, and different SLM manufacturers. Therefore, our microscope design and algorithms provide an alternative to the use of fabricated gratings in MFM, as they are relatively simple and could find broad applications in the wider research community.
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