  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, 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 fabricated gratings in MFM, as they are
relatively simple and could find broad applications in the wider research
community.
