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

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.

Measuring extinction with digital holography: nonspherical particles and experimental validation.

Through simulations and experiment, this Letter shows how a particle's extinction cross section can be extracted from a digital hologram. Spherical and nonspherical particles are considered covering a range of cross-sectional values of nearly five orders of magnitude. The extracted cross sections are typically less than 10% in error from the true values. It is also shown that holograms encompassing a sufficiently large angular range of scattered light yield an estimate for the absorption cross section.

Using holography to measure extinction.

This work presents a new concept to measure the extinction cross section for a single particle in situ. The concept involves recording the hologram produced by the interference of a particle's forward-scattered light with the incident light. This interference pattern is fundamentally connected to the energy flow that gives rise to extinction, and, by integrating this measured pattern, one obtains an approximation for the cross section. Mie theory is used to show that this approximation can be as little as 1% in error of the true value for many cases of practical interest. Moreover, since an image of the particle can be computationally reconstructed from a measured hologram using the Fresnel-Kirchhoff diffraction theory, one can obtain the cross section simultaneously with the particle shape and size.

Backscatter digital holography of microparticles.

This work investigates a method for digital holographic imaging of microparticles. Traditional digital holographic techniques use a particle's forward scattered light to form the hologram, whereas here we use the backscattered light. Images of a particle are then computationally reconstructed from the backscatter hologram, and several examples of such reconstructions are presented. A potential advantage of this technique is that the backscatter holograms may be more sensitive to particle-surface details.

Scattered-light-sheet microscopy with sub-cellular resolving power.

Since its first demonstration over 100 years ago, scattering-based light-sheet microscopy has recently re-emerged as a key modality in label-free tissue imaging and cellular morphometry; however, scattering-based light-sheet imaging with subcellular resolution remains an unmet target. This is because related approaches inevitably superimpose speckle or granular intensity modulation on to the native subcellular features. Here, we addressed this challenge by deploying a time-averaged pseudo-thermalized light-sheet illumination. While this approach increased the lateral dimensions of the illumination sheet, we achieved subcellular resolving power after image deconvolution. We validated this approach by imaging cytosolic carbon depots in yeast and bacteria with increased specificity, no staining, and ultralow irradiance levels. Overall, we expect this scattering-based light-sheet microscopy approach will advance single, live cell imaging by conferring low-irradiance and label-free operation towards eradicating phototoxicity.