A house-built MFLFM prototype: (a) a fluorescence microscope, (b) the MFG (multi-focus grating) optics, and (c) an MLA (micron-lens array) and EMCCD.
Abstract: Light field microscopy (LFM) is an emerging technology for high-speed wide-field 3D imaging by capturing 4D light field of 3D volumes. However, its 3D imaging capability comes at a cost of lateral resolution. In addition, the lateral resolution is not uniform across depth in the light field dconvolution reconstructions. To address these problems, here, we propose a snapshot multifocal light field microscopy (MFLFM) imaging method. The underlying concept of the MFLFM is to collect multiple focal shifted light fields simultaneously. We show that by focal stacking those focal shifted light fields, the depth-of-field (DOF) of the LFM can be further improved but without sacrificing the lateral resolution. Also, if all differently focused light fields are utilized together in the deconvolution, the MFLFM could achieve a high and uniform lateral resolution within a larger DOF. We present a house-built MFLFM system by placing a diffractive optical element at the Fourier plane of a conventional LFM. The optical performance of the MFLFM are analyzed and given. Both simulations and proof-of-principle experimental results are provided to demonstrate the effectiveness and benefits of the MFLFM.
Kuan He, Xiaolei Wang, Zihao W. Wang, Hannah Yi, Norbert F. Scherer, Aggelos K. Katsaggelos, and Oliver Cossairt, “Snapshot multifocal light field microscopy,” Opt. Express 28, 12108-12120 (2020).
Schematic diagrams comparison between (a) a conventional microscopy, (b) LFM and (c) proposed MFLFM.
The LFM (b) records light field of 3D volumes by placing a microlens array in front of a sensor. The MFLFM (c) simultaneously collects multiple light fields by placing a multifocal grating at the Fourier plane of the system. Each light field has its corresponding focal plane in the volume.
Schematic of single-shot 3D MFLFM.
An MFG is placed at the Fourier plane of the first relay lens system to separate and focus different depths of a 3D sample onto different diffraction orders, which later propagate through different sub-regions of the MLA and form differently focused light fields on the EMCCD sensor in a single shot. The insets show the patterns of the MFG and lenslet in the array.
Comparison of experimental PSFs of a conventional LFM (a) and the MFLFM (b) by imaging a sub-resolution fluorescent bead at different z-positions (columns).
The proposed MFLFM system is able to capture 9 differently focused light field in a single shot. The scale bar denotes the length in the object space.
The MFLFM proof-of-principle experimental results.
(a) A snapshot captured MFLFM light fields measurement. (b) The light field 3D reconstructions for the LFM (top row) and MFLFM (bottom row) using synthetic focusing algorithms. Each column shows reconstructions of the 3D volume as it was translated to the different z-heights denoted above each image.
Quantitative analysis of experimental MFLFM results.
(c) Wide-field microscopy images when three beads outlined in a red dash rectangle were in focus (first column) and out-of-focus (second to last columns). (d) The LFM and (e) MFLFM refocused 2D slice of the three beads. (f-h) Linecuts comparisons indicated by red solid lines in (c-e), respectively. The beads could not be resolved by the LFM when it refocused at z-position larger than 6 um, but could be well separated by the proposed MFLFM even it refocused at z = -10 um, demonstrating that the MFLFM could extended the DOF of the LFM without sacrificing its spatial lateral resolution.
MFLFM simulation setup.
(a) Ground truth image of a USAF 1951 resolution target, (b) simulated MFLFM PSF and (c) simulated MFLFM image under Poisson noise corruption.
MFLFM simulation reconstruction results.
Simulation results: deconvolution reconstructions for (a) a conventional wide-field microscope, (b) the LFM, and (c) proposed MFLFM as a USAF 1951 resolution test target was moved to different z-positions denoted above each image. (d-e) The line-cuts comparisons indicated by blue line in (b) and (c). The three lines with a spacing of about 0.68 um could not be distinguished by the LFM (d) except at z=1 um depth but could be super resolved within the whole DOF in the MFLFM.
PSNR (left) and SSIM (right) of the LFM (red) and MFLFM (blue) reconstructed images.
The low uniformity of reconstruction quality in the LFM was significantly improved by the proposed MFLFM method.
Impact of signal-to-noise ratio on the light field reconstructions for the LFM (dash lines) and MFLFM (solid lines).
The colors of lines denote different numbers of photon collected per pixel. Here, the format n1/n2 in the legend denotes n1 and n2 average photon per pixel for the MFLFM and LFM, respectively, to ensure the same total photon budget for both imaging systems.
Funding: National Science Foundation (NSF) CAREER grant IIS-1453192;
Biological Systems Science Division, Office of Biological and Environmental Research, Office of Science, U.S. Dept. of Energy, under Contract DE-AC02-06CH11357.