Video-rate Raman-based metabolic imaging by Airy light-sheet illumination and photon-sparse detection

Author:

Dunn Lochlann1,Luo Haokun2,Subedi Nava R.1,Kasu Ramachandran1ORCID,McDonald Armando G.3ORCID,Christodoulides Demetrios N.2,Vasdekis Andreas E.1

Affiliation:

1. Department of Physics, University of Idaho, Moscow, ID 83844-0903

2. The College of Optics and Photonics, University of Central Florida, Orlando, FL 32816-2700

3. Department of Forest, Rangeland, and Fire Sciences, University of Idaho, Moscow, ID 83844-1132

Abstract

Despite its massive potential, Raman imaging represents just a modest fraction of all research and clinical microscopy to date. This is due to the ultralow Raman scattering cross-sections of most biomolecules that impose low-light or photon-sparse conditions. Bioimaging under such conditions is suboptimal, as it either results in ultralow frame rates or requires increased levels of irradiance. Here, we overcome this tradeoff by introducing Raman imaging that operates at both video rates and 1,000-fold lower irradiance than state-of-the-art methods. To accomplish this, we deployed a judicially designed Airy light-sheet microscope to efficiently image large specimen regions. Further, we implemented subphoton per pixel image acquisition and reconstruction to confront issues arising from photon sparsity at just millisecond integrations. We demonstrate the versatility of our approach by imaging a variety of samples, including the three-dimensional (3D) metabolic activity of single microbial cells and the underlying cell-to-cell variability. To image such small-scale targets, we again harnessed photon sparsity to increase magnification without a field-of-view penalty, thus, overcoming another key limitation in modern light-sheet microscopy.

Funder

U.S. Department of Energy

Publisher

Proceedings of the National Academy of Sciences

Subject

Multidisciplinary

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