Prediction of single-cell RNA expression profiles in live cells by Raman microscopy with Raman2RNA.

Single-cell RNA sequencing and other profiling assays have helped interrogate cells at unprecedented resolution and scale, but are inherently destructive. Raman microscopy reports on the vibrational energy levels of proteins and metabolites in a label-free and nondestructive manner at subcellular spatial resolution, but it lacks genetic and molecular interpretability. Here we present Raman2RNA (R2R), a method to infer single-cell expression profiles in live cells through label-free hyperspectral Raman microscopy images and domain translation. We predict single-cell RNA sequencing profiles nondestructively from Raman images using either anchor-based integration with single molecule fluorescence in situ hybridization, or anchor-free generation with adversarial autoencoders. R2R outperformed inference from brightfield images (cosine similarities: R2R >0.85 and brightfield <0.15). In reprogramming of mouse fibroblasts into induced pluripotent stem cells, R2R inferred the expression profiles of various cell states. With live-cell tracking of mouse embryonic stem cell differentiation, R2R traced the early emergence of lineage divergence and differentiation trajectories, overcoming discontinuities in expression space. R2R lays a foundation for future exploration of live genomic dynamics.

Structures and topological defects in pressure-driven lyotropic chromonic liquid crystals.

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-Shot Quantitative Polarization Imaging of Complex Birefringent Structure Dynamics.

Polarization light microscopes are powerful tools for probing molecular order and orientation in birefringent materials. While a number of polarization microscopy techniques are available to access steady-state 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 performance of PSIM are validated by imaging a birefringent resolution target 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 µm2 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.

Regularized pseudo-phase imaging for inspecting and sensing nanoscale features.

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 d6/λ4, 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 noise-polluted nanostructure profiles with the pseudo-phase framework even when using a low-cost bright-field microscope.

Single-Shot Optical Anisotropy Imaging with Quantitative Polarization Interference Microscopy.

Optical anisotropy measurement is essential for material characterization and biological imaging. In order to achieve single-shot mapping of the birefringence parameters of anisotropic samples, a novel polarized light imaging concept is proposed, namely quantitative polarization interference microscopy (QPIM). QPIM can be realized through designing a compact polarization-resolved interference microscopy system that captures interferograms bearing sample's linear birefringence information. To extract the retardance and the orientation angle maps from a single-shot measurement, a mathematical model for QPIM is further developed. The QPIM system is validated by measuring a calibrated quarter-wave plate, whose fast-axis orientation angle and retardance are determined with great accuracies. The single-shot nature of QPIM further allows to measure the transient dynamics of birefringence changes in material containing anisotropic structures. This application is demonstrated by capturing transient retardance changes in a custom-designed parallel-aligned nematic liquid crystal-based device.

Three-dimensional resolution and contrast-enhanced confocal microscopy with array detection.

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.

Fluorescence emission difference with defocused surface plasmon-coupled emission microscopy.

A novel fluorescence emission difference method is proposed to improve the lateral resolution of SPCEM without increasing instrument complexity. We discovered the profile of transverse PSF in SPCEM will dramatically change from a hollow spot to a solid spot, when the axial position of sample varies within one wavelength in the vicinity of the focal plane. The subtraction of an image whose PSF is hollow spot and an image with solid PSF will greatly enhance the resolution and contrast of SPCEM images. The mechanism of the distinctive PSF is demonstrated through basic optics theories, and the improvement of lateral resolution is verified by theoretical simulations and experimental results. It is believed that our method will stand out for its pleasant resolution enhancement and its instruments' simplicity to facilitate many biological cellular observations.

Virtual fluorescence emission difference microscopy based on photon reassignment.

A method for high-resolution imaging that we call virtual fluorescence emission difference microscopy (vFED) is presented. In vFED the analyzed samples are scanned only by a doughnut-shaped pattern and imaged by a detector array, which is very different from the previous FED system. By using photon reassignment, we can obtain imaging results with matched solid and hollow point spread functions, and the difference between them is used to estimate the spatial distribution of the analyzed sample. This method results in greatly simplified equipment in the configuration and enhanced imaging speed. Results show that the resolution can be enhanced by at least 27% compared with that in confocal microscopy with a point detector, or is 1.8-2-fold higher than that in wide-field microscopy. Plus, negative intensities can be avoided by using vFED during the subtraction process, leading to the elimination of the deformation in reconstructed images.

Aspheric subaperture stitching based on system modeling.

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.

Resolution-enhanced surface plasmon-coupled emission microscopy.

A novel fluorescence emission difference technique is proposed for further enhancements of the lateral resolution in surface plasmon-coupled emission microscopy (SPCEM). In the proposed method, the difference between the image with phase modulation by using a 0-2π vortex phase plate (VPP) along with a diaphragm and the original image obtained from SPCEM is used to estimate the spatial distribution of the analyzed sample. By optimizing the size of the diaphragm and the subtractive factor, the lateral resolution can be enhanced by about 20% and 33%, compared with that in SPCEM with a single 0-2π VPP and conventional wide-field fluorescence microscopy, respectively. Related simulation results are presented to verify the capability of the proposed method for improving lateral resolution and reducing imaging distortion. It is believed that the proposed method has potentials to improve the performance of SPCEM, thus facilitating biological observation and research.