Annular oblique epi-illumination design for ultra-large-scale and high- resolution fluorescence microscopy

摘要

Biological processes are inherently multi-scale, and supramolecular complexes at the cellular scale. However, in their current form these modalities are limited by a highly constrained field of view (FOV) and field-dependent imaging resolution. New-generation photographic instruments with ultra-large-scale and high-resolution imaging capabilities are strongly demanded in life science and medicine research, ranging from neuroscience to oncology. In practice, the size and quality of images from above instruments are severely limited by several factors related to the illumination. In this work, we present an efficient illumination design for ultra-large-scale and high-resolution fluorescence microscopy to be used on samples of any thickness, yielding strikingly clearer images. Compared with traditional epi-illumination device of fluorescence microscopy, the annular light of our design whose radius is large enough is incident on the sample directly from outside of the objective, rather than through the objective lens, which effectively improves the utility of off-axis illumination. Our design simplifies the overall system and reduces aberrations induced from dichroic mirror because the dichroic mirror is not required any more to separate the illumination path and imaging path. Meanwhile, more uniform illumination can be provided due to the symmetry of annular illumination. No need to consider the lighting angle, the proposed design is especially suitable for fluorescence microscopy because the direction of emitting light is spherical, regardless of the direction of excitation light. The apparatus is simple, robust and inexpensive, making it broadly useful to biological and clinical researchers alike.