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Refractive index is an optical property explored in the light scattering measurement of micro- and nano-particles as well as in label-free imaging of cells and tissues. Because the refractive index value is a major input to the characterization and quantification of the analyzed specimens, various methods have been developed targeting at different sample types. In this paper, we demonstrate a technique for the refractive index measurement of homogeneous microspheres and liquids in the short-wave infrared (SWIR) range. We use synthetic phase microscopy (SPM), which records a scattering-corrected projection of the 3D refractive index distribution, in combination with a least-squares fitting to a theoretical model of a sphere. Using the method, we determine the refractive index dispersion of two polymer microspheres (polymethyl methacrylate and polystyrene), two glass microspheres (silica and soda lime), and three microscopy mounting media (glycerol, FluorSave, and Eukitt) in the SWIR range of 1100–1650 nm. 

We demonstrate hyperspectral confocal microscopy in the short-wave infrared (SWIR) range of 1100–1600 nm using a wavelength-scanning laser in tandem with laser scanning confocal microscopy. Confocal microscopy in the SWIR range allows for high-resolution inspection of an integrated circuit (IC) chip, while hyperspectral imaging, together with a chemometric analysis, enables us to identify functional circuit block groups in the acquired image. With the extended capability, the developed instrument can be potentially used for inline inspection and non-invasive failure analysis of IC chips.

 

Optical projection tomography (OPT) is a three-dimensional (3D) fluorescence imaging technique, in which projection images are acquired for varying orientations of a sample using a large depth of field. OPT is typically applied to a millimeter-sized specimen, because the rotation of a microscopic specimen is challenging and not compatible with live cell imaging. In this Letter, we demonstrate fluorescence optical tomography of a microscopic specimen by laterally translating the tube lens of a wide-field optical microscope, which allows for high-resolution OPT without rotating the sample. The cost is the reduction of the field of view to about halfway along the direction of the tube lens translation. Using bovine pulmonary artery endothelial cells and 0.1 µm beads, we compare the 3D imaging performance of the proposed method with that of the conventional objective-focus scan method.

 

Spectroscopic microtomography provides the ability to perform 4D (3D structural and 1D chemical) imaging of a thick microscopic specimen. Here, we demonstrate spectroscopic microtomography in the short-wave infrared (SWIR) wavelength using digital holographic tomography, which captures both the absorption coefficient and refractive index. A broadband laser in tandem with a tunable optical filter allows us to scan the wavelength from 1100 to 1650 nm. Using the developed system, we measure human hair and sea urchin embryo samples. The resolution estimated with gold nanoparticles is 1.51 μm (transverse) and 1.57 μm (axial) for the field of view of 307 × 246 μm2. The developed technique will enable accurate and efficient analyses of microscopic specimens that have a distinctive absorption or refractive index contrast in the SWIR range. 

Abstract Hyperspectral fluorescence imaging is widely used when multiple fluorescent probes with close emission peaks are required. In particular, Fourier transform imaging spectroscopy (FTIS) provides unrivaled spectral resolution; however, the imaging throughput is very low due to the amount of interferogram sampling required. In this work, we apply deep learning to FTIS and show that the interferogram sampling can be drastically reduced by an order of magnitude without noticeable degradation in the image quality. For the demonstration, we use bovine pulmonary artery endothelial cells stained with three fluorescent dyes and 10 types of fluorescent beads with close emission peaks. Further, we show that the deep learning approach is more robust to the translation stage error and environmental vibrations. Thereby, the He-Ne correction, which is typically required for FTIS, can be bypassed, thus reducing the cost, size, and complexity of the FTIS system. Finally, we construct neural network models using Hyperband, an automatic hyperparameter selection algorithm, and compare the performance with our manually-optimized model. 

Hyperspectral three-dimensional (3D) imaging can provide both 3D structural and functional information of a specimen. The imaging throughput is typically very low due to the requirement of scanning mechanisms for different depths and wavelengths. Here we demonstrate hyperspectral 3D imaging using Snapshot projection optical tomography (SPOT) and Fourier-transform spectroscopy (FTS). SPOT allows us to instantaneously acquire the projection images corresponding to different viewing angles, while FTS allows us to perform hyperspectral imaging at high spectral resolution. Using fluorescent beads and sunflower pollens, we demonstrate the imaging performance of the developed system.