Photon-Sparse, Poisson Light-Sheet Microscopy

Abstract

Light-sheet microscopy has revolutionized bioimaging by enabling approximately an order of magnitude reduction in specimen irradiance compared to confocal imaging. Here, we introduce a light-sheet imaging system that enables an additional order of magnitude reduction in specimen irradiance by operating at the Poisson limit. To operate at this limit, we integrated classical illumination with single-photon detection and wavelet-based image reconstruction. This integration enabled brightness quantification and object recognition from fewer than one detected photon per image pixel, corresponding to more than 10-fold lower irradiance levels than modern systems. We demonstrate how such photon-sparse imaging can eradicate photobleaching and enable both dim and bright object imaging, thus, further enhancing the related gains of light-sheet microscopy.

Video-rate spontaneous Raman imaging

“…2, we estimated this reduction to be ~200-fold, when coupled to dedicated computer vision routines for reconstructing/denoising the photon-sparse images. 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.…”

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

“…Further, we projected the Raman scattered photons onto an intensified complementary metal oxide semiconductor camera (iCMOS) that, additionally, synchronously modulated the laser intensity to ensure coincident illumination and collection ( Methods ). Due to the inevitable pixel-to-pixel crosstalk from the optical amplification process, each detected photon formed a cloud that occupied more than 1 pixel ( 35 , 36 ). We reduced the size of these clouds to a single pixel by estimating their centroids using procedures similar to single-molecule localization ( Methods ).…”

Scattered‐light‐sheet microscopy with sub‐cellular resolving power

“…The light-sheet imaging setup deployed in this work is illustrated in Figure 1A and Figure S1, and previously reported by the authors [12,15,30]. This setup includes a long-working distance illumination objective (20Â/0.42, Mitutoyo), a detection objective (40Â/0.6 HCX PL FLUOTAR L, Leica), and a sCMOS (scientific CMOS, ORCA-Flash 4.0, Hamamatsu) camera.…”

“…As detailed in Figure S1, the illumination/detection objectives and camera were integrated with a standard inverted microscope (DMi8, Leica). Here, light-sheet illumination of the sample was enabled through a custom-made microscope stage comprising of two linear stages (LS-50, Applied Scientific Instrumentation) and a piezoelectric stage (IPZ-3150, Applied Scientific Instrumentation) [12,15,30]. For light-sheet illumination, we employed an ultra-narrow linewidth (2 MHz bandwidth) Titanium: Sapphire ring laser (Matisse CR, Spectra Physics) operating at λ = 720 nm and pumped by a 532 nm diode pumped solid state laser (Millenia ev, Spectra Physics).…”

“…Overall, this geometrical reconfiguration has been found to reduce the total sample irradiance levels by more than one order of magnitude with respect to confocal microscopy [5,11]. Further, LSI and SPIM have been also integrated with pseudo-diffraction-free illumination sheets, such as those enabled by Bessel and Airy beams, and optical lattices, as well as photon-sparse detection to accelerate imaging rates and further decrease the levels of irradiance [12][13][14][15].…”