For example, the detection of fluorescence not only depends on the absolute amount of targeted molecules, but also the local structural density which can limit the diffusion and binding of the antibodies. This missing information can confuse and sometimes mislead our interpretation. However, this molecular specificity comes with a dilemma that the structural context and other unspecified molecules would not appear in the images, i.e. One critical aspect of optical imaging is the 3D localization of molecular composites in tissues or cells, typically by using specific antibodies with fluorescent reporters. Volumetric optical imaging with cellular and sub-cellular resolution is essential for our fundamental understanding of biological systems. We also demonstrate potential applications in imaging human intestinal organoids (HIOs), colon mucosa, and retina. We demonstrate several mechanisms for molecular imaging, including intrinsically expressed GFP fluorescence, autofluorescence from Flavin proteins, and exogenous antibody-conjugated dyes. The current setup provides ~3.6 × 4.2 × 6.5 μm resolution in fluorescence imaging, ~7 × 7 × 3.5 μm in OCT in three dimensions, and the current speed of imaging is up to 100 frames per second (fps) over a volume about 0.8 × 1 × 0.5 mm 3. Here, the use of an obliquely scanning laser eliminates the z-stacking process, then allows highly efficient 3D OCT and fluorescence imaging by using only one raster scan. Conventional 3D laser scanning fluorescence microscopy requires repeated optical sectioning to create z-stacks in depth.

In this paper, we present a method termed oblique scanning laser microscopy (OSLM) to combine optical coherence tomography (OCT), for simultaneously volumetric structural and molecular imaging with cellular resolution in all three dimensions. Multi-modal three dimensional (3D) optical imaging combining both structural sensitivity and molecular specificity is highly desirable in biomedical research.

Conical scan raster scan