No abstract

Recovering tiny nanoscale features using a general optical imaging system is challenging because of poor signal to noise ratio. Rayleigh scattering implies that the detectable signal of an object of size d illuminated by light of wavelength λ is proportional to d 6 /λ 4 , which may be several orders of magnitude weaker than that of additive and multiplicative perturbations in the background. In this article, we solve this fundamental issue by introducing the regularized pseudo-phase, an observation quantity for polychromatic visible light microscopy that seems to be more sensitive than conventional intensity images for characterizing nanoscale features. We achieve a significant improvement in signal to noise ratio without making any changes to the imaging hardware. In addition, this framework not only retains the advantages of conventional denoising techniques, but also endows this new measurand (i.e., the pseudo-phase) with an explicit physical meaning analogous to optical phase. Experiments on a NIST reference material 8820 sample demonstrate that we can measure nanoscale defects, minute amounts of tilt in patterned samples, and severely noisepolluted nanostructure profiles with the pseudo-phase framework even when using a low-cost bright-field microscope.

What we believe is a novel method for improving confocal microscopy's resolution and contrast in 3D space is proposed. Based on a conventional confocal microscopy setup, we use an array detector composed of 32 photomultiplier tubes (PMTs) to replace one point-detector, where the location offset of each PMT caused a different effective point spread function (PSF). By applying array detection and the fluorescence emission difference method of an image with a solid PSF and another with a donut-shaped PSF, we can enhance lateral resolution about 27% in real time with only one scan, and improve the axial resolving ability by about 22% simultaneously. Experimental results of both fluorescent beads and living cells are presented to verify the applicability and effectiveness of our method.

Lyotropic chromonic liquid crystals are water-based materials composed of self-assembled cylindrical aggregates. Their behavior under flow is poorly understood, and quantitatively resolving the optical retardance of the flowing liquid crystal has so far been limited by the imaging speed of current polarization-resolved imaging techniques. Here, we employ a single-shot quantitative polarization imaging method, termed polarized shearing interference microscopy, to quantify the spatial distribution and the dynamics of the structures emerging in nematic disodium cromoglycate solutions in a microfluidic channel. We show that pure-twist disclination loops nucleate in the bulk flow over a range of shear rates. These loops are elongated in the flow direction and exhibit a constant aspect ratio that is governed by the nonnegligible splay-bend anisotropy at the loop boundary. The size of the loops is set by the balance between nucleation forces and annihilation forces acting on the disclination. The fluctuations of the pure-twist disclination loops reflect the tumbling character of nematic disodium cromoglycate. Our study, including experiment, simulation, and scaling analysis, provides a comprehensive understanding of the structure and dynamics of pressure-driven lyotropic chromonic liquid crystals and might open new routes for using these materials to control assembly and flow of biological systems or particles in microfluidic devices.

Single cell RNA-Seq (scRNA-seq) and other profiling assays have opened new windows into understanding the properties, regulation, dynamics, and function of cells at unprecedented resolution and scale. However, these assays are inherently destructive, precluding us from tracking the temporal dynamics of live cells, in cell culture or whole organisms. Raman microscopy offers a unique opportunity to comprehensively report on the vibrational energy levels of molecules in a label-free and non-destructive manner at a subcellular spatial resolution, but it lacks in genetic and molecular interpretability. Here, we developed Raman2RNA (R2R), an experimental and computational framework to infer single-cell expression profiles in live cells through label-free hyperspectral Raman microscopy images and multi-modal data integration and domain translation. We used spatially resolved single-molecule RNA-FISH (smFISH) data as anchors to link scRNA-seq profiles to the paired spatial hyperspectral Raman images, and trained machine learning models to infer expression profiles from Raman spectra at the single-cell level. In reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs), R2R accurately (r>0.96) inferred from Raman images the expression profiles of various cell states and fates, including iPSCs, mesenchymal-epithelial transition (MET) cells, stromal cells, epithelial cells, and fibroblasts. R2R outperformed inference from brightfield images, showing the importance of spectroscopic content afforded by Raman microscopy. Raman2RNA lays a foundation for future investigations into exploring single-cell genome-wide molecular dynamics through imaging data, in vitro and in vivo.

No abstract

Polarization light microscopes are powerful tools for probing molecular order and orientation in birefringent materials. While a multitude of polarization light microscopy techniques are often used to access steadystate properties of birefringent samples, quantitative measurements of the molecular orientation dynamics on the millisecond time scale have remained a challenge. We propose polarized shearing interference microscopy (PSIM), a single-shot quantitative polarization imaging method, for extracting the retardance and orientation angle of the laser beam transmitting through optically anisotropic specimens with complex structures. The measurement accuracy and imaging performances of PSIM are validated by imaging a rotating wave plate and a bovine tendon specimen. We demonstrate that PSIM can quantify the dynamics of a flowing lyotropic chromonic liquid crystal in a microfluidic channel at an imaging speed of 506 frames per second (only limited by the camera frame rate), with a field-of-view of up to 350×350 μm 2 and a diffraction-limit spatial resolution of ∼2 μm. We envision that PSIM will find a broad range of applications in quantitative material characterization under dynamical conditions.

Instead of various mathematical stitching algorithms, an aspheric subaperture stitching interferometric method relying on modern computer modeling technique is presented. Based on our previously reported non-null annular subaperture stitching interferometry (NASSI), a simultaneous reverse optimizing reconstruction (SROR) method based on system modeling is proposed for full aperture figure error reconstruction. All the subaperture measurements are simulated simultaneously with a multi-configuration model in a ray tracing program. With the multi-configuration model, full aperture figure error would be extracted in form of Zernike polynomials from subapertures wavefront data by the SROR method. This method concurrently accomplishes subaperture retrace error and misalignment correction, requiring neither complex mathematical algorithms nor subaperture overlaps. Experiment results showing the validity of SROR method are presented.