Light Sheet Theta Microscopy for Uniform, High-Resolution Imaging of Large Intact Tissues

Migliori B, Datta MS, Dupre C, Apak MC, Asano S, Gao R, Boyden ES, Hermanson O, Yuste R, Tomer R. Light sheet theta microscopy for rapid high-resolution imaging of large biological samples. BMC Biol 2018;16(1):57. doi: 10.1186/s12915-018-0521-8.

Background: Light sheet fluorescence microscopy (LSFM) has advanced large-scale volumetric imaging in biology, particularly in cleared tissue samples. However, conventional LSFM configurations often face trade-offs between axial resolution, field of view and working distance, especially when imaging large intact organs like mouse brains.

Hypothesis: This study tested the hypothesis that reconfiguring the light sheet illumination geometry can overcome resolution and coverage limitations in conventional LSFM, enabling uniform high-resolution imaging across large, intact biological samples.

Methods: The authors developed Light Sheet Theta Microscopy (LSTM), a novel LSFM architecture in which two obliquely oriented illumination arms deliver light sheets at a non-orthogonal (theta) angle to the detection objective. Unlike conventional orthogonal LSFM, LSTM allows the detection arm to be configured like in standard upright or inverted light microscopy, while removing lateral sample size constraints and extending the accessible imaging depth within a specimen. A simultaneous two-axis scan mode was implemented, enabling synchronous translation of the light sheet and detection focal plane, and facilitating uniform illumination across large tissue volumes.

Results: LSTM enables high-resolution imaging over large fields of view with minimal photo-bleaching and phototoxicity. The system produces near-isotropic resolution and demonstrated superior imaging performance in large cleared mouse brains, human brain slices and densely labeled Drosophila larvae compared to standard LSFM approaches. LSTM is particularly effective at minimizing artifacts due to tissue curvature and scattering.

Conclusions: LSTM offers a powerful solution to the scalability and resolution challenges of conventional LSFM. Its non-orthogonal geometry enables deep, uniform, high-resolution imaging of large intact tissues, making it especially well-suited for neuroscience and developmental biology applications.