Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

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. Video-rate microscopy (defined here as ≥15  fps) has shown utility for visualizing cellular-level dynamics, such as blood cells trafficking within capillary networks.1–4 In addition, high-frame-rate microscopy is useful for mitigating motion artifacts due to respiration or mechanical jitter when utilizing an intravital, handheld, or endoscopic microscope.5–9 In recent years, considerable efforts have been undertaken to develop miniature optical-sectioning microscopes for applications such as in vivo microendoscopy and point-of-care pathology.5,10–22 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. In practice, various trade-offs are necessary to optimize a device for a particular application. Among various methods, a number of miniature microscopes have been developed that utilize a point-scanned dual-axis confocal configuration (PS-DAC).23–25 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-of-focus photons, which in turn improves imaging contrast and depth compared to a conventional single-axis confocal microscope.26 Point-scanned confocal microscopes achieve optical sectioning through spatial filtering with a pinhole detector, in which an image is obtained in a point-by-point fashion by mechanically scanning the sample or by scanning the focal volume within the sample. However, with frame rates that are typically lower than 10 Hz, the images obtained by a point-scanned confocal microscope are often susceptible to motion artifacts.27 In addition, such slow frame rates (<10  Hz) are lower than the frequency response of the human visual system (∼15  Hz) when perceiving moving objects.28 In order to address the drawback of limited imaging speed, modifications were made to a DAC microscope to accommodate line scanning instead of point scanning. Even though confocality is lost in one dimension for a line-scanned dual-axis confocal (LS-DAC) microscope, which limits its ability to image deeper within tissues, previous feasibility studies have shown that a slow stage-scanned LS-DAC microscope (with 2-fps imaging rate and 1-ms line-acquisition rate) can maintain the resolution and contrast of a PS-DAC microscope at moderate depths of up to several hundred microns.29 In addition, previous Monte-Carlo simulations suggest that a LS-DAC configuration provides superior contrast (signal to background ratio, SBR) in comparison to a line-scanned single-axis confocal microscope.30 Recently, we have also introduced a variation on the LS-DAC technique called sheet-scanned dual-axis confocal (SS-DAC) microscopy,31 in which a scientific complementary metal–oxide–semiconductor (sCMOS) camera enables an oblique light-sheet to be imaged at each scanned position. However, both the previous LS-DAC microscope and the recently developed SS-DAC microscope were slow stage-scanned prototypes (frame rate at 2 fps) that did not utilize a fast-scanning galvo (up to 30 Hz), high-speed camera acquisition (10-kHz line acquisition rate), and speed-optimized software. On the other hand, a major challenge for many high-frame-rate fluorescence optical-sectioning devices (with short pixel dwell times) is achieving adequate sensitivity. This is especially true for DAC microscopy, in which low-NA optics are utilized. Here, we demonstrate for the first time that an LS-DAC microscope is capable of achieving video-rate imaging of fluorescently labeled tissues with sufficient sensitivity for a number of potential biological and clinical applications. The results of this study demonstrate that video-rate LS-DAC microscopy is capable of sensitive in vivo video-rate imaging of blood cells trafficking within the capillaries of a mouse ear at 15 fps (FOV ∼500×500  μm) or 30 fps (FOV ∼250×500  μm). In addition, the 100-μs camera exposure time (line-acquisition rate) used for video-rate in vivo imaging experiments provides sufficient sensitivity to image excised mouse tongue tissues, topically stained with methylene blue, at subcellular resolution. To further demonstrate the versatility of video-rate LS-DAC microscopy for clinical applications, images are obtained from surgically excised human brain tumor specimens (glioma) expressing 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence.