Abstract
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.
Highlights
Multifocal microscopy is a useful method that allows simultaneous imaging of multiple object planes to realize high-speed 3D imaging
Sample images resulting from Pixelflipper, Iterative Fourier Transform Algorithm (IFTA) and in-situ iterative methods are displayed in Fig 4(b), 4(c) and 4(d), respectively
We used our in situ iteratively optimized Spatial Light Modulator (SLM) phase patterns to acquire 3D images of GFPlabeled tubulin in MeOH-fixed TPX2 Hela Kyoto cells [12]
Summary
Multifocal microscopy is a useful method that allows simultaneous imaging of multiple object planes to realize high-speed 3D imaging. The Pixelflipper algorithm was designed to generate uniformlyilluminated subimages in existing multifocal microscope systems [3] This algorithm uses a software-based method that finds the phase pattern of a grating unit cell of size Pu × Pu pixels that gives the highest uniformity among the diffraction orders in the computed Fourier plane. In Uniform intensity in multifocal microscopy addition to other system aberrations, SLMs suffer from pixel-to-pixel crosstalk effects [8,9] that further alter the resultant diffraction pattern This issue is worsened when only few SLM pixels form the repeated grating pattern.
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