Small-molecule Raman probes for cellular imaging have attracted great attention owing to their sharp peaks that are sensitive to environmental changes. The small cross section of molecular Raman scattering limits dynamic cellular Raman imaging to expensive and complex coherent approaches that acquire single-channel images and lose hyperspectral Raman information. We introduce a new method, dynamic azo-enhanced Raman imaging (DAERI), to couple the new class of azoenhanced Raman probes with a high-speed line-scan Raman imaging system. DAERI achieved high-resolution low-power imaging of fast cellular dynamics resolved at ∼270 nm along the confocal direction, 75 μW/μm 2 and 3.5 s/frame. Based on the azo-enhanced Raman probes with characteristic signals 10 2 −10 4 stronger than classic Raman labels, DAERI was not restricted to the cellular Raman-silent region as in prior work and enabled multiplex visualization of organelle motions and interactions. We anticipate DAERI to be a powerful tool for future studies in biophysics and cell biology.
Microscopic imaging and imaging flow cytometry have wide
potential
in point-of-care assays; however, their narrow depth of focus necessitates
precise mechanical or fluidic focus control of a sample in order to
acquire high-quality images that can be used for downstream analysis,
increasing the cost and complexity of the imaging system. This complexity
represents a barrier to miniaturization and translation of point-of-care
assays based on microscopic imaging or imaging flow cytometry. To
address this challenge, we present a simple drop-in phase mask with
a physics-informed, circularly symmetric asphere phase profile that
extends the depth of focus by >5-fold while largely preserving
the
image quality compared to other depth extending methods. We show that
such a focus-extended system overcomes manufacturing tolerances in
low-cost sample chambers, enlarges the useable field-of-view of low-cost
objectives, and permits increased throughput and precision in flow
imaging systems without the need for complex flow-focusing. As the
image quality is preserved without the need for postacquisition image
restoration, our solution is also highly appropriate for on-line applications
such as cell sorting.
Laser
tweezers Raman spectroscopy enables multiplexed, quantitative
chemical and morphological analysis of individual bionanoparticles
such as drug-loaded nanoliposomes, yet it requires minutes-scale acquisition
times per particle, leading to a lack of statistical power in typical
small-sized data sets. The long acquisition times present a bottleneck
not only in measurement time but also in the analytical throughput,
as particle concentration (and thus throughput) must be kept low enough
to avoid swarm measurement. The only effective way to improve this
situation is to reduce the exposure time, which comes at the expense
of increased noise. Here, we present a hybrid principal component
analysis (PCA) denoising method, where a small number (∼30
spectra) of high signal-to-noise ratio (SNR) training data construct
an effective principal component subspace into which low SNR test
data are projected. Simulations and experiments prove the method outperforms
traditional denoising methods such as the wavelet transform or traditional
PCA. On experimental liposome samples, denoising accelerated data
acquisition from 90 to 3 s, with an overall 4.5-fold improvement in
particle throughput. The denoised data retained the ability to accurately
determine complex morphochemical parameters such as lamellarity of
individual nanoliposomes, as confirmed by comparison with cryo-EM
imaging. We therefore show that hybrid PCA denoising is an efficient
and effective tool for denoising spectral data sets with limited chemical
variability and that the RR-NTA technique offers an ideal path for
studying the multidimensional heterogeneity of nanoliposomes and other
micro/nanoscale bioparticles.
Traditional line-scan Raman imaging features a rapid imaging speed while preserving complete spectral information, yet has diffraction-limited resolution. Sinusoidally structured line excitation can yield an improvement in the lateral resolution of the Raman image along the line’s direction. However, given the need for the line and spectrometer slit to be aligned, the resolution in the perpendicular direction remains diffraction limited. To overcome this, we present here a galvo-modulated structured line imaging system, where a system of three galvos can arbitrarily orient the structured line on the sample plane, while keeping the beam aligned to the spectrometer slit in the detection plane. Thus, a two-fold isotropic improvement in the lateral resolution fold is possible. We demonstrate the feasibility using mixtures of microspheres as chemical and size standards. The results prove an improvement in the lateral resolution of 1.8-fold (limited by line contrast at higher frequencies), while preserving complete spectral information of the sample.
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