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.
Highlights
While microscopy of ex vivo tissues provides valuable information for biological investigations and clinical diagnoses, key insights into dynamic processes can only be obtained under in vivo settings
Our results indicate that video-rate line-scanned dual-axis confocal (LS-DAC) microscopy is capable of in vivo fluorescence imaging of blood cells trafficking within the capillaries of a mouse ear at a frame rate of 15 fps (FOV ∼500 × 500 μm) or 30 fps (FOV ∼250 × 500 μm)
Video-rate imaging at 30 fps has been achieved with an LS-DAC microscope with sufficient speed, contrast, and sensitivity to visualize in vivo blood cell dynamics
Summary
While microscopy of ex vivo tissues provides valuable information for biological investigations and clinical diagnoses, key insights into dynamic processes can only be obtained under in vivo settings. Considerable efforts have been undertaken to develop miniature optical-sectioning microscopes for applications such as in vivo microendoscopy and point-of-care pathology.[5,10,11,12,13,14,15,16,17,18,19,20,21,22] Regardless of the specific modality—e.g., confocal microscopy, optical coherence tomography, photoacoustic tomography, or multiphoton microscopy—a general goal has been to maximize the imaging depth, resolution, contrast, field of view (FOV), and speed of imaging. A number of miniature microscopes have been developed that utilize a point-scanned dual-axis confocal configuration (PS-DAC).[23,24,25] A DAC microscope is a unique confocal architecture that utilizes low-numerical aperture (NA) off-axis illumination and collection beams that intersect precisely at their foci. The DAC architecture decreases the undesired background from multiplying scattered and out-
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