Histological classification of atherosclerotic arteries using high-speed confocal Raman microscopy with machine learning.

Confocal Raman microscopy is a useful tool to observe composition and constitution of label-free samples at high spatial resolution. However, accurate characterization of microstructure of tissue and its application in diagnostic imaging are challenging due to weak Raman scattering signal and complex chemical composition of tissue. We have developed a method to improve imaging speed, diffraction efficiency, and spectral resolution of confocal Raman microscopy. In addition to the novel imaging technique, the machine learning method enables confocal Raman microscopy to visualize accurate histology of tissue sections. Here, we have demonstrated the performance of the proposed method by measuring histological classification of atherosclerotic arteries and compared the histological confocal Raman images with the conventional staining method. Our new confocal Raman microscopy enables us to comprehend the structure and biochemical composition of tissue and diagnose the buildup of atherosclerotic plaques in the arterial wall without labeling.

Pupil function design for multifocal confocal, STED, and isoSTED microscopy.

Point scanning super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy are powerful tools to observe biological samples at sub-diffraction limited resolution in three dimensions. However, scanning the sample with only a single beam limits the imaging speed in these microscopes. Here, we propose a concept to increase this speed by introducing highly flexible multifocal illumination and detection. We introduce phase patterns in the objectives' pupil planes to create arrays of foci in the sample plane with negligible loss of laser power. High uniformity of these foci's intensities is achieved by iteratively applying a weighted Gerchberg-Saxton phase retrieval algorithm. We characterize the performance of this iterative approach numerically and present simulation results that demonstrate the high quality of the focus arrays for future implementations in laser-scanning STED and isoSTED microscopes. The same approach can also be applied in diffraction-limited confocal laser scanning microscopy.

DMA-tudor interaction modules control the specificity of in vivo condensates.

Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.

Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

Effect of partial coherence on dimensional measurement sensitivity for DUV scatterfield imaging microscopy.

Optical scatterfield imaging microscopy technique which has the capability of controlling scattered fields in the imaging mode is useful for quantitative nanoscale dimensional metrology that yields precise characterization of nanoscale features for semiconductor device manufacturing process control. To increase the sensitivity in the metrology using this method, it is required to optimize illumination and collection optics that enhance scatterfield signals from the nanoscale targets. Partial coherence of the optical imaging system is used not only for enhancing image quality in the traditional microscopy or lithography but also for increasing the sensitivity of the scatterfield imaging microscopy. This paper presents an empirical investigation of the effect of partial coherence on measurement sensitivity using a deep ultraviolet scatterfield imaging microscope platform that uses a 193 nm excimer laser as a source and a conjugate back focal plane as a unit for controlling partial coherence. Dimensional measurement sensitivity is assessed through analyzing scatterfield images measured at the edge area of periodic multiline structures with nominal linewidths ranging 44-80 nm on a Molybdenum Silicide (MoSi) photomask. Intensities scattered from the targets under the illuminations with various partial coherence factors and two orthogonal polarizations are assessed with respect to sensitivity coefficient. The optimization of partial coherence factor for the target dimension is discussed through the sensitivity coefficient maps.

Compact fiber optic dual-detection confocal displacement sensor.

We propose a dual-detection confocal displacement sensor (DDCDS) with a compact fiber-based optical probe. This all-fiber-optic sensor probe is simple and robust, since it only requires simple alignment of a gradient refractive index lens and a double-clad fiber (DCF). The DDCDS is composed of two point detectors, one coupled to a single mode fiber and the other coupled to a multimode fiber, which are used to measure the light intensity from a core and an inner clad of a DCF, respectively. The ratio of the axial response curves, measured by the two detectors, can be used to obtain a linear relationship between the axial position of the object plane and the ratio of the intensity signals. We demonstrate the performance of the proposed method by measuring micromovement and fast vibration.

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.