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

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.

Near Zero Photon Bioimaging

“…Further, the CMOS camera was integrated with an intensifier (iCMOS) to enable single photon detection at the Poisson limit ( Methods ). As per previous photonsparse demonstrations 25, 26 , such semiclassical operation is largely independent of specimen and magnification, and we have observed both in fluorescence and Raman imaging. As such, we anticipate our findings described below to be valid not only for fluorescence imaging, but also Raman and other microscopy modalities.…”

Video-rate spontaneous Raman imaging

“…By considering the total illumination area afforded by the long propagation length of the Airy beam (~75,000 pixels and 30 μm light-sheet scanning range at 40× magnification), we achieved less than 400 nsec pixel dwell times, which is comparable to Coherent Antistokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) imaging. 1 Similar to conventional light-sheet microscopy, we used low-magnification objectives (20× and 40×) in our setup. This naturally led to a 2-fold lower imaging resolution compared to the Nyquist limit (for a 40× objective and 6.6 μm pixel size).…”

“…Importantly, this increased magnification relied solely on the particle nature of light, without any need for additional optical elements or any penalties to the field of view. 1…”

“…8,9 Overall, the enhanced performance reported in this investigation can be attributed predominantly to the utilization of light-sheet microscopy, in conjunction with the accelerating Airy beam and photon-sparse detection techniques. 1 Figure 2: Average photon number per pixel required for the Raman scattered signal of yeast cells (Yarrowia lipolytica, 1997-2380 cm -1 ), a PDMS polymer slab (1405-1420 cm -1 ), and polystyrene (PS) particles (990.5-1005.5 cm -1 ) to converge to brightness levels with less than 5% error with respect to the ground truth. Both conventional (red) and photon-sparse (blue) microscopy results are shown.…”

三维拉曼显微成像技术研究进展（特邀）

“…2 Lightsheet Raman microscopic system and its application results. (a) System structure diagram of the lightsheet Raman microscopic system [12] ; (b) system diagram of Airylightsheet Raman microscopic imaging [15] ; (c) 3D imaging of polystyrene microspheres [15] ; (d) sparse photon image of a single liposolysaccharide Y. cell, and its corresponding 3D enlarged image and the time trajectory of the corresponding pixel [16] ; (e) 3D imaging results of c lipid metabolism in cells under different photon numbers [16] 0618010 -4 3 Raman projection tomographic system and its application results. (a) Structure diagram of dualmode optical -Raman projection tomographic system [25] ; (b) optical -Raman 3D fusion imaging of Arabidopsis stems [25] ; (c) Raman tomographic image of dog bone tissue [23] 0618010 -5 4 Coherent Raman scattering microscope and its application results.…”