(E-190) ISOTROPIC CELLULAR RESOLUTION ACROSS CENTIMETER FIELD OF VIEW USING SUBVOXEL AXIALLY SWEEPING LIGHT SHEET MICROSCOPY (SV-ASLSM)

Introduction::
Light sheet microscopy (LSM) has emerged a prominent optical sectioning modality, due to millimeter penetration depth and ability to tune optical section thickness from single to several microns( >20um). However, LSM resolution and imaging Field-of-View (FOV) is dependent on the numerical aperture (NA) of the objective lens (OL) used for producing the light section[1]. Axially sweeping light sheet microscopy (AS-LSM), provides promising avenues for decoupling dependance of NA on FOV[1]. Original AS-SLM methods sweep the focus spot longitudinally across the sample, by beam refocusing via mechanical actuators and a rolling shutter (RS) camera pixel strategy for capturing extended FOV. However, a puzzling conundrum of choosing an appropriate NA without sacrificing resolution is still encountered. To address the resolution constraint, we propose a new generation of AS-LSM utilizing maximum likelihood estimation (MLE) based LSM[1], by off-axis sample detection with respect to beam propagation axis to improve voxel density and enable in silico resolution enhancement.




Materials and Methods:: A Voice Coil Actuator (VCA) was used to offset the optical section at detection objective lens [2], and a cylindrical lens was used to focus 1D light sheet into a high working distance (WD) objective to produce hyperbolic planar light sheet at focus spot. Furthermore, we have designed a tilted scan stage for SV-LSM. The off axis scanned raw volume is split into multiple sub-stacks, that are treated as low-resolution (LR) volumes containing redundant information due to significant image overlap produced by oblique scan. Using MLE, LR stacks are modelled as probability distributions with subtle spatial correlations and furthermore, a cost function based on image degradation operators is minimized to produce a high-resolution estimate from LR image volumes[1].

Results, Conclusions, and Discussions::
Using AS-LSM, we extended the imaging FOV from 700 um (Figure A, scale bar=650 um) to 1400 um by axially sweeping the light sheet beam waist using a remote refocusing actuator (Figure B, scale bar-650 um), enabling arbitrary FOV tuning (micron-centimeter) independent of NA. Furthermore, we quantified 5 um fluorescent bead resolution (Figures C, D, scale bars = 20 um) as 16 um laterally (Figure G) and 19 um axially (Figure H) using 1x air detection lens. Using SV-LSM (Figures E, F, scale bars = 10 um), we improved lateral resolution 1.45x to 11 um (Figure I) and axial resolution 1.65x to 11.5 um (Figure J) in silico, using 1x detection and 4x excitation OL’s. Consequently, MLE-based SV-LSM intrinsically improves 3D voxel density due to oblique sample scan, regardless of low lens NA. We hypothesize integration of SV-LSM and AS-LSM can be used fully decouple dependance of resolution and confocal region on NA, and thereby enable isotropic cellular resolution using centimeter WD OL’s.




Acknowledgements (Optional): :

References (Optional): :
[1] T. Teranikar, J. Lim, T. Ijaseun, and J. Lee, “Development of Planar Illumination Strategies for Solving Mysteries in the Sub-Cellular Realm,” International Journal of Molecular Sciences, vol. 23, no. 3, 2022, doi: 10.3390/ijms23031643.




[2] T. Chakraborty et al., “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nature Methods, vol. 16, no. 11, pp. 1109–1113, Nov. 2019, doi: 10.1038/s41592-019-0615-4.