SignificanceThe ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition.AimHere, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells.ApproachMie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers.ResultsThe polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography.ConclusionsThe combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.
We present a polarized light-sheet microscopy and tomography system, PLμTo, for versatile multi-scale and multi-modal volumetric optical imaging. The system can utilize fluorescence and elastic scattering contrast for visualization of biochemical and structural information. PLμTo is compatible with traditional slide and well-plate sample mounting, as well as microfluidics for high-throughput volumetric imaging flow cytometry of mesenchymal stem cells cultured on spherical microcarriers in a bioreactor culture. Additionally, the system is capable of rapidly screening millimeter-sized cleared tissues with cellular level resolution in a few minutes. This system can be utilized to study biological processes at varying spatial and temporal scales.
Visualization-based monitoring provides an empirical and minimally-invasive means for evaluating monolayer cell cultures, but does not translate to three-dimensional microcarrier-based cultures. In toto visualization of cell density and morphology is imperative for producing high quality, high yield cell cultures. Here, the optical properties of commercial polystyrene and custom-fabricated hydrogel microcarriers are compared for compatibility with light-sheet imaging for visualization and enumeration of adherent cells. Additionally, Mie scattering simulations were performed to describe the angular scattering intensity distributions. This study shows the custom hydrogel microcarrier is compatible with in toto non-destructive and non-invasive visualization and monitoring of 3D adherent cell cultures.
Compact multispectral imaging systems can provide fast analysis of tissue properties, such as perfusion, that are useful parameters for clinicians to inform individualized care. Some sedative drugs used in both human and veterinary medicine have been found to induce changes in perfusion in small animals such as cats. The use of these drugs on veterinary patients must be carefully considered in the case of wound care or procedures such as axial pattern flaps as inhibited wound perfusion may inhibit successful treatment. In this preliminary study, we built a multispectral illumination module that consisted of a white light source and filter wheel to supplement a commercial laser speckle contrast system. A color camera of the laser speckle system was used for multispectral image acquisition. The system was used to obtain visible-light reflectance measurements of the skin of anesthesized healthy dogs undergoing routine dental cleaning. The goal of this study was to determine if multispectral data detects changes in skin reflectance as the dog was placed under anaesthesia. The changes observed may be related to changes in superficial skin perfusion as the patient receives sedation.
We present our 2nd generation handheld simultaneous multispectral frequency-domain FLIM endoscopic system for label-free metabolic imaging of oral cancer, with enhanced optical performance and system usability. Our custom-designed and 3D-printed handheld endoscope consists of an enclosure (6 x 3 x 3 cm3) with a rigid probe (1 cm diameter, 9 cm length) that weighs less than 125 g with all the system components, which, compared to our previous system, is significantly smaller and lighter, and has improved ergonomics and usability. The enclosure has mounts for a dual axes bi-directional MEMS scanner and a dichroic mirror, and plug-and-play ports for excitation, emission collection and rigid probe optics. The rigid probe used for oral mucosa imaging contains a three-lens imaging system that, compared to our previous system, has: an increased field of view (FOV) (6 x 6 mm2 vs. 16 x 16 mm2), improved lateral resolution (36 μm in the center and 65 μm at the edge, diffraction-limited performance across a central ± 5.5 mm field), and an extended working distance (10 mm vs. 40 mm). A 375 nm CW laser is used as the excitation source, and fluorescence emission is spectrally divided into three emission bands (405±10 nm, 440±20 nm, and 525±25 nm) targeting collagen, NADH, and FAD, which are relevant for early detection of oral cancer. Fluorescence emission is then detected by three APDs and further processed onboard an FPGA. Our clinically compatible handheld endoscope allows for noninvasive and fast in situ clinical metabolic imaging of the oral mucosa.
Rodent models are robust tools for understanding human retinal disease and function because of their similarities with human physiology and anatomy and availability of genetic mutants. Optical coherence tomography (OCT) has been well-established for ophthalmic imaging in rodents and enables depth-resolved visualization of structures and image-based surrogate biomarkers of disease. Similarly, fluorescence confocal scanning laser ophthalmoscopy (cSLO) has demonstrated utility for imaging endogenous and exogenous fluorescence and scattering contrast in the mouse retina. Complementary volumetric scattering and en face fluorescence contrast from OCT and cSLO, respectively, enables cellular-resolution longitudinal imaging of changes in ophthalmic structure and function. We present a non-contact multimodal OCT+cSLO small animal imaging system with extended working distance to the pupil, which enables imaging during and after intraocular injection. While injections are routinely performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the location and volume delivered is not precisely controlled and difficult to reproduce. Animals were imaged using a custom-built OCT engine and scan-head combined with a modified commercial cSLO scan-head. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. When combined with imagesegmentation, we believe OCT can be used to precisely identify injection locations and quantify injection volumes. Fluorescence cSLO can provide complementary contrast for either fluorescently labeled compounds or transgenic cells for improved specificity. Our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections, which may be used for real-time image-guided injections.
Intraocular injections are routinely performed for delivery of anti-VEGF and anti-inflammatory therapies in humans. While these injections are also performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the injection location and volume are not well-controlled and reproducible. We overcome limitations of conventional injections methods by developing a multimodality, long working distance, non-contact optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (cSLO) system for retinal imaging before and after injections. Our OCT+cSLO system combines a custom-built spectraldomain OCT engine (875±85 nm) with 125 kHz line-rate with a modified commercial cSLO with a maximum frame-rate of 30 fps (512 x 512 pix.). The system was designed for an overlapping OCT+cSLO field-of-view of 1.1 mm with a 7.76 mm working distance to the pupil. cSLO excitation light sources and filters were optimized for simultaneous GFP and tdTomato imaging. Lateral resolution was 3.02 µm for OCT and 2.74 μm for cSLO. Intravitreal injections of 5%, 10%, and 20% intralipid with Alex Fluor 488 were manually injected intraocularly in C57BL/6 mice. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. OCT enables quantitative analysis of injection location and volumes whereas complementary cSLO improves specificity for identifying fluorescently labeled injected compounds and transgenic cells. The long working distance of our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections and may be applied for imaging of ophthalmic surgical dynamics and real-time image-guided injections.
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