Video-ratein vivofluorescence imaging with a line-scanned dual-axis confocal microscope

Abstract

Abstract. Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of cancer-positive surgical margins using structured illumination microscopy

“…Techniques applied to date have included depth-sensitive tomography techniques such as optical coherence tomography1011121314 and photoacoustic tomography15, as well as optical sectioning techniques such as reflectance and fluorescence scanning confocal microscopy and label-free nonlinear microscopies1617181920212223242526272829. These techniques have shown promising results for imaging of smaller specimens such as skin cancer resections1721, gross pathology sections of larger resection specimens16172527, and core needle biopsies30.…”

Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology

“…A major goal of this study was to demonstrate the ability to miniaturize a LS-DAC microscope without sacrificing performance compared to a table-top LS-DAC prototype that was recently developed for high-speed optical sectioning [33,39]. The miniature system described here exhibits a lateral and axial resolution of 1 -2 μm, which is slightly superior to that of our previous tabletop systems.…”

“…In addition, while the low-NA collection of light would be expected to limit the collection efficiency and signal-to-noise ratio (SNR) of DAC microscopes, the relatively isotropic resolution of the DAC microscope enables the collection of light from a larger focal volume compared to a SAC microscope with an equivalent optical-sectioning thickness. Coupled with the increased pixel dwell times afforded by line scanning, the LS-DAC architecture is capable of sensitive optical-sectioning microscopy of fresh tissues (ex vivo and in vivo) at high frame rates (>16 frames/sec), as exhibited here as well as in a previous study with a large tabletop LS-DAC prototype [34,35,39].…”

“…Compared to a previous tabletop LS-DAC system [39], the only major differences for this miniature system are the incorporation of a miniature MEMS scanner (Mirrorcle Technologies Inc.), rather than a large galvanometric scanner, as well as the design and utilization of a custom 3x relay objective (Fig. 1).…”

“…For preliminary assessment of the optical design and to evaluate the performance of the microscope, a sCMOS camera (Hamamatsu Orca Flash 4.0) with a two-dimensional (2D) detector was used to mimic the one-dimensional (1D) linear detector that will be incorporated in the final design. A thin rectangular region of interest within the sCMOS array serves as a digital slit, as described previously for a large-scale tabletop LS-DAC microscope system [34,35,39]. In short, the sCMOS camera allows rapid acquisition of a 2048 × 8 pixel region at the center of the camera.…”