This PDF file contains the front matter associated with SPIE Proceedings Volume 12385, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

Time-resolved multispectral fluorescence microscopy provides a 4D hypercube dataset with high specificity for cellular examination. However, this is generally obtained by significantly increasing the measurement time, which is quite limiting for in vivo measurement or with photosensitive sample. It is possible to reduce the measurement effort with a novel microscopy framework exploiting compressive sensing based on single-pixel camera. In this work, we present a compressive sensing system and validate it with a cellular sample. Data fusion with a high-resolution camera image allows us to tackle the well-known problem of low-resolution in single-pixel imaging.

We propose a new single pixel microscope with optical sectioning properties by using structured illumination techniques. The method uses single-pixel imaging (SPI) techniques by interrogating the sample with a series of spatially resolved patterns and measuring the output intensities with a non-spatial resolution detector. Moreover, optical sectioning is obtained by adding a grating to the system and employing Structured Illumination Microscopy (SIM). The system allows us to perform 3D bright-field and fluorescence microscopy. We apply compressive sensing techniques to decrease the acquisition time.

High-speed imaging with light-sheet fluorescence microscopy poses several challenges throughout the whole pipeline, from data acquisition in the lab to image stitching and post-processing. Here we present our custom hardware and software solutions that allow us to map large biological samples at the cellular level, e.g. large portions of human brain cortex. Our custom optical setup—a dual-view, inverted, light-sheet microscope—is capable of simultaneous two-color acquisition at a data rate of 1 GB/s. Our open source tools include the instrument’s data acquisition and control software and also cover volumetric image stitching and post-processing.

Expansion microscopy (ExM) is a powerful imaging strategy that offers a low-cost solution for interrogating biological systems at the nanoscale using conventional optical microscopes. It achieves this by physically and isotropically magnifying preserved biological specimens embedded in a cross-linked water-swellable hydrogel. However, most reported techniques are unable to preserve endogenous epitopes due to strong protease digestion used to expand samples. In addition, these protocols rely on mechanically fragile hydrogels that only expand by at most 4.5× linearly. We present a new ExM framework, Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence MicroscopY (MAGNIFY), that exhibits a broad retention of nucleic acids, proteins, and lipids without the need for a separate anchoring step. By using a mechanically sturdy hydrogel, MAGNIFY is capable of expanding biological specimens up to 11×. This facilitates nanoscale imaging (~25-nm effective resolution) using an ∼280-nm diffraction-limited objective lens on a conventional optical microscope and can be furthered to ~15 nm effective resolution if combined with computational methods such as Super-resolution Optical Fluctuation Imaging (SOFI). Here, we demonstrate that MAGNIFY provides a generalized solution for imaging nanoscale subcellular features of a broad range of biological specimens. We also show that MAGNIFY provides a novel, accessible tool for improving the precision, utility, and generality of nanoscopy.

In this paper, numerical and semi-analytical methods are used for studying how well a fluorescent bead with a given radius in a polarizer-supplemented microscopy setup, can emulate a single fluorescent molecule.

Digital Holographic Microscopy (DHM) is an emerging label-free modality for quantitative imaging of semi-transparent biological specimens. DHM provides Quantitative Phase Imaging (QPI) of the light transmitted or scattered by an object, which allows to extract information about morphology and dynamics of investigated specimens. DHM can be implemented with other optical microscopy techniques like Bright Field (BF) or fluorescence, for example, to provide complementary information about cellular processes. Multimodal microscopy is often achieved by sequential acquisition of information from different imaging modalities or simultaneously by capturing images with different acquisition devices. However, such arrangements can be cost-intensive and/or limited in speed for imaging of dynamic processes. Here, we investigate a single-snapshot multimodal approach that enables simultaneous acquisition of DHM-based QPI and BF images and is compatible with common optical microscopes for the analysis of living cells. The technique is based on spatially multiplexed interferometric microscopy (SMIM), where off-axis holograms are generated by spatial multiplexing of the illuminated sample plane utilizing a diffraction grating, which is extended by additional superposition of complementary white-light image information. Image separation is achieved by Fourier-transformation-based numerical demultiplexing procedures. The technique is firstly characterized by investigations on microspheres. Then, the application on living adherent and suspended cells is illustrated.

Endoscopy through coherent fiber bundles plays a significant role in industrial and medical 2D imaging. By replacing the lens on the distal side with a diffuser, the 3D information of the measurement volume is encoded as 2D speckle patterns on the camera. Neural networks can then be employed to reconstruct the 3D object. Therefore, minimally invasive single-shot 3D imaging through a flexible low-cost endoscope with a diameter of less than 1 mm is enabled. However, the number of fiber cores is limiting the transferable information and reduces the reconstruction quality. In this paper, separate reconstruction for the diffuser and the coherent fiber bundle for different core numbers is explored. This approach enables biomedical applications for in vivo diagnostics, e.g. with fluorescence imaging in 3D.

Since additive manufacturing has become increasingly popular in prototyping, printed optics are also beginning to enter the market. Novel characterization methods for printed optics are needed because traditional, destructive methods often do not work on these optics. The scope of investigation is also different for additively manufactured optics. Homogeneity of subtractive manufactured optics such as glass lenses is usually granted but for printed optics the interfaces in-between layers can cause absorption, scattering or refraction. Functionalized optics can also have characteristics such as fluorescence that cannot be tested with traditional methods. The presented work tries to fill the void for this particular challenge by studying two non-destructive methods for optical characterization of such components and expanding their use by clever combination. In Scanning Laser Optical Tomography (SLOT), a needle-like beam is formed and focused into the sample. The sample is scanned to form projection images and rotated to allow for reconstruction, which yields volumetric data about scattering, transmission and fluorescence of sample structures. Simulated SLOT measurements with imperfect Refractive Index (RI) matching of sample and medium are presented. A method to correct distorted measurements is presented and evaluated. The simulations imply that a measurement with a RI mismatch of up to 0.1 can still yield reasonable results.

The advantages of confocal microscopy over widefield microscopy are their ability to produce optically sectioned images and their ability to produce multi-color imaging in which different organelles within the biological specimen are stained using multiple dyes, enabling colocalization studies. These features make confocal microscopy a widely used tool to provide valuable morphological and functional information within cells and tissues. One of the major drawbacks of confocal microscopy is its limited spatial resolution. Here, we train two generative adversarial networks using paired and unpaired data of low- and high-resolution images to improve the spatial resolution in confocal microscopy.

We present a novel way of designing multi-immersion microscope objectives inspired by both the mirror-based eyes of scallops and the Schmidt telescope, a wide-field telescope invented in the 1930s. Despite containing only two optical elements and without a correction collar, our design achieves diffraction-limited imaging performance over mm-scale field-of-views in air and in any homogeneous liquid medium. Based on our concept, we built a prototype two-photon microscope objective and demonstrated its versatility by imaging Xenopus tadpoles cleared with BABB (n=1.56) and by imaging the larval Zebrafish brain in vivo in water.

The point-spread function (PSF) plays a fundamental role in deciding the resolution of an optical microscope. One way to find the experimental PSF is to image sub-resolution nanobead. However, the PSF can also be approximated numerically by knowing the optical arrangement of the imaging system. In this paper, we estimate the experimental three-dimensional PSF of a widefield microscope by taking several z-stacks of an arbitrary target, followed by application of our 2D PSF estimation scheme to each z slice. Here we use a standard resolution test target as the arbitrary target whose geometrical-optics predicted image can be easily constructed.

Industrial inspection is critical for 3D semiconductor manufacturing process and automated optical inspection for 3D ICs has attracted a lot of attention in these years. Plenoptic imaging systems, based on a micro-lens array, acquire light field from parallax and computes 3D information with lower costs. To reduce aberrations from the optical design with microlens array, especially for off-axial micro lenses, the design flow for plenoptic imaging systems is proposed. Based on the parameters designed from paraxial approximation, lateral image quality is optimized by a commercial optical design software, and then depth-related performances are estimated from the simulated images of the optical system. The experimental system for validation is tested quantitatively with modulation transfer function (MTF), by the slanted-edge method of ISO 12233. The difference of MTF between the paraxial and off-axial regions is approximately 0.02, which is within the repeatability error 0.03. Moreover, the synthesized images of a PCIe card refocused on the chip and the board clearly show the elements at the refocusing depth only. The depth map and the all-in-focus image are estimated to build a 3D model. However, significant artifacts appear on depth maps when lighting is not uniform. With combination of the ring light and coaxial light, the depth maps of objects with different surface properties can be estimated with less artifacts. Furthermore, accuracy and resolution can be enhanced by deep-learning technologies, which will be implemented in the future.

Based on a simple ratiometric intensity computation, dual-focus optics have enabled us to track the vesicle transport in a living cell directly from the fluorescence microscopy images. However, because the acquired dual images from this 3D microscopy can be suffered from optical distortion, the calculation result might not accurate enough for the nano-scale. In this paper, we suggest a linear transformation-based image processing method for the accurate detection and tracking of a vesicle in a living cell. In addition, we present a pipeline for reconstructing the 3D trajectory of the vesicle directly from the images.