Development of High-Resolution High-Throughput Open-Top Light Sheet Fluorescence Microscopy

Abstract

Light sheet fluorescence microscopy (LSFM) is a high-speed 3D imaging technique that employs selective light sheet illumination and planar imaging. By offering much higher imaging speed with minimal photo-damage than conventional point scanning confocal microscopy, LSFM proved particularly valuable for conducting 3D live imaging of small animals such as embryos. However, conventional LSFMs had sample size constraints and open-top LSFM (OT-LSFM) techniques were developed for large tissue specimens by having all the optics below the sample stage. OT-LSFMs have two different configurations of dual objective (DO) and single objective (SO) types. DO types had orthogonally arranged illumination and imaging arms with separate objective lenses. Both the illumination and imaging objective lenses were angled with respect to the sample for open-top configuration. DO-OT-LSFMs had additional interfacing devices, such as the liquid prism, to minimize optical aberrations. DO-OT- LSFMs had advantages of utilizing full numerical aperture (NA) of the imaging objective lens for high resolution, while having the disadvantages of limited working distance, high sensitivity to aberration, and high complexity. SO type OT-LSFMs have both oblique light sheet illumination and planar imaging implemented in single objective lenses. An oblique illumination light sheet was generated by utilizing an offset aperture of the objective lens. Emission light from the sample was collected by the same objective lens and then relayed to the camera through a remote focusing. SO-OT- LSFMs had advantages of low sensitivity to optical aberration and the utilization of full working distance of the objective lens. However, SO types had relatively low image resolutions compared to DO types due to signal loss in the relaying of the oblique image plane and the trade-off of the cone angles of illumination and emission light within the limited NA of the single objective lens and their small crossing angle of less than 90°. In this research, I have developed three high-performance OT-LSFMs to advance OT- LSFM technology: (1) high-resolution axially swept OT-LSFM for the high-resolution imaging of optically cleared biological specimens in all dimensions. (2) dual-scale SO- OT-LSFM for optical biopsy application by enabling the switching between rapid macro-scale imaging and detail micro-scale imaging of biopsied specimens, and (3) axially swept dual-scale SO-OT-LSFM for the dual-scale imaging of both optically cleared and biopsied tissue specimens. First, I developed a high-resolution open-top axially swept light sheet microscopy (HR-OTAS-LSFM) for the sub-micron resolution imaging of optically cleared biological tissue in all dimensions. HR-OTAS-LSFM was a DO type system with a liquid prism interface. The high lateral resolution was realized by using a high-NA immersion imaging objective lens. The high axial resolution was achieved by axially sweeping an aberration corrected tightly focused excitation light sheet. Both the aberration correction and axial sweeping was achieved by using a deformable mirror (DM) in the illumination arm. After design, implementation, and characterization, the proposed system was applied to optically cleared mouse brain and transparent mouse retina specimens for performance verification. HR-OTAS-LSFM visualized 3D cell structures of optically cleared biological specimens in high resolution. In addition to the 3D cell network visualization, another important application of OT-LSFM is slide-free optical biopsy overcoming the limitations of conventional slide based pathological examination. HR-OTAS-LSFM was not appropriate for optical biopsy application. The fixed high-magnification objective lens of HR-OTAS-LSFM was inadequate for rapid cellular examination, and the liquid prism interface made sample mounting and aberration free imaging difficult. Instead of DO types, SO types were more appropriate for optical biopsy application. Therefore, I developed an SO-OT-LSFM called dual-scale and dual-channel oblique plane microscopy (D2OPM) for optical biopsy application. D2OPM used two switchable sample objective lenses with different magnifications for switching between rapid meso-scale imaging and detail micro-scale imaging. D2OPM was designed to do meso-scale imaging for the visualization of the gross tissue structure and the selection of regions of interest (ROIs), and then to do micro-scale imaging of the selected ROIs for detailed visualization. D2OPM incorporated two excitation laser sources in the illumination path and a custom image splitter in the detection path for dual-color imaging and the simultaneous visualization of cells labeled with nuclear and cytoplasmic fluorophores. The captured dual-channel volumetric images were processed to generate enface surface pseudo hematoxylin and eosin (H&E) images. After development and characterization, D2OPM was applied to various tissues including human cancer specimens in colon, stomach, and pancreas. The performance was evaluated in comparison with conventional slide-based H&E histology. D2OPM enabled both rapid meso-scale and detail micro-scale imaging for optical biopsy application. However, the current D2OPM had a limited depth of field (DOF) of 20 µm in micro-scale imaging mode by using a tightly focused light sheet, and its application to both optically cleared tissue specimens and tissue specimens with rough surface profiles was difficult. To extend the imaging DOF, I developed dual-scale and dual-channel axial swept oblique plane microscopy (D2-AS-OPM) by adapting the axial sweeping to D2OPM. A DM was used for the axial sweeping of tightly focused excitation light sheets in micro-scale imaging. D2-AS-OPM had an extended DOF of 200 µm. After design, implementation, and characterization, the performance of D2-AS-OPM was verified in the imaging of human pancreas specimens. Although the specimens had high surface roughness, all in focus high-resolution surface images were obtained with the extended DOF. Currently, preliminary results are presented. More experiments are underway for full verification. In conclusion, I developed three high-performance open-top light sheet microscopies for the rapid and high-resolution imaging of large sized tissue specimens. These imaging systems are anticipated to pave the way for 3D cellular network study of biological tissue and intraoperative optical biopsy application.