Multitrack Compressed Sensing for Faster Hyperspectral Imaging.

Hyperspectral imaging (HSI) provides additional information compared to regular color imaging, making it valuable in areas such as biomedicine, materials inspection and food safety. However, HSI is challenging because of the large amount of data and long measurement times involved. Compressed sensing (CS) approaches to HSI address this, albeit subject to tradeoffs between image reconstruction accuracy, time and generalizability to different types of scenes. Here, we develop improved CS approaches for HSI, based on parallelized multitrack acquisition of multiple spectra per shot. The multitrack architecture can be paired up with either of the two compatible CS algorithms developed here: (1) a sparse recovery algorithm based on block compressed sensing and (2) an adaptive CS algorithm based on sampling in the wavelet domain. As a result, the measurement speed can be drastically increased while maintaining reconstruction speed and accuracy. The methods were validated computationally both in noiseless as well as noisy simulated measurements. Multitrack adaptive CS has a ∼10 times shorter measurement plus reconstruction time as compared to full sampling HSI without compromising reconstruction accuracy across the sample images tested. Multitrack non-adaptive CS (sparse recovery) is most robust against Poisson noise at the expense of longer reconstruction times.

Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods.

Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture's viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical [Formula: see text]-galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-[Formula: see text]-galactosidase activity using the fluorogenic substrate, C[Formula: see text]FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in [Formula: see text]-galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p [Formula: see text] 0.001 for the fluorogenic C[Formula: see text]FDG method, and r = 0.72, p [Formula: see text] 0.05 for the forward scatter method), and good fold difference ranges (1.120-4.436 for total autofluorescence mean and 1.082-6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Bioinspired cell-in-shell systems in biomedical engineering and beyond: Comparative overview and prospects.

With the development in tissue engineering, cell transplantation, and genetic technologies, living cells have become an important therapeutic tool in clinical medical care. For various cell-based technologies including cell therapy and cell-based sensors in addition to fundamental studies on single-cell biology, the cytoprotection of individual living cells is a prerequisite to extend cell storage life or deliver cells from one place to another, resisting various external stresses. Nature has evolved a biological defense mechanism to preserve their species under unfavorable conditions by forming a hard and protective armor. Particularly, plant seeds covered with seed coat turn into a dormant state against stressful environments, due to mechanical and water/gas constraints imposed by hard seed coat. However, when the environmental conditions become hospitable to seeds, seed coat is ruptured, initiating seed germination. This seed dormancy and germination mechanism has inspired various approaches that artificially induce cell sporulation via chemically encapsulating individual living cells within a thin but tough shell forming a 3D "cell-in-shell" structure. Herein, the recent advance of cell encapsulation strategies along with the potential advantages of the 3D "cell-in-shell" system is reviewed. Diverse coating materials including polymeric shells and hybrid shells on different types of cells ranging from microbes to mammalian cells will be discussed in terms of enhanced cytoprotective ability, control of division, chemical functionalization, and on-demand shell degradation. Finally, current and potential applications of "cell-in-shell" systems for cell-based technologies with remaining challenges will be explored.

Autofluorescence spectroscopy in redox monitoring across cell confluencies.

Patient-specific therapies require that cells be manufactured in multiple batches of small volumes, making it a challenge for conventional modes of quality control. The added complexity of inherent variability (even within batches) necessitates constant monitoring to ensure comparable end products. Hence, it is critical that new non-destructive modalities of cell monitoring be developed. Here, we study, for the first time, the use of optical spectroscopy in the determination of cellular redox across cell confluencies by exploiting the autofluorescence properties of molecules found natively within cells. This was achieved through a simple retrofitting of a standard inverted fluorescence microscope with a spectrometer output and an appropriate fluorescence filter cube. Through spectral decomposition on the acquired autofluorescence spectra, we are able to further discern the relative contributions of the different molecules, namely flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH). This is then quantifiable as redox ratios (RR) that represent the extent of oxidation to reduction based upon the optically measured quantities of FAD and NADH. Results show that RR decreases with increasing cell confluency, which we attribute to several inter-related cellular processes. We validated the relationship between RR, metabolism and cell confluency through bio-chemical and viability assays. Live-dead and DNA damage studies were further conducted to substantiate that our measurement process had negligible effects on the cells. In this study, we demonstrate that autofluorescence spectroscopy-derived RR can serve as a rapid, non-destructive and label-free surrogate to cell metabolism measurements. This was further used to establish a relationship between cell metabolism and cellular redox across cell confluencies, and could potentially be employed as an indicator of quality in cell therapy manufacturing.

Identification of senescent cells in multipotent mesenchymal stromal cell cultures: Current methods and future directions.

Regardless of their tissue of origin, multipotent mesenchymal stromal cells (MSCs) are commonly expanded in vitro for several population doublings to achieve a sufficient number of cells for therapy. Prolonged MSC expansion has been shown to result in phenotypical, morphological and gene expression changes in MSCs, which ultimately lead to the state of senescence. The presence of senescent cells in therapeutic MSC batches is undesirable because it reduces their viability, differentiation potential and trophic capabilities. Additionally, senescent cells acquire senescence-activated secretory phenotype, which may not only induce apoptosis in the neighboring host cells following MSC transplantation, but also trigger local inflammatory reactions. This review outlines the current and promising new methodologies for the identification of senescent cells in MSC cultures, with a particular emphasis on non-destructive and label-free methodologies. Technologies allowing identification of individual senescent cells, based on new surface markers, offer potential advantage for targeted senescent cell removal using new-generation senolytic agents, and subsequent production of therapeutic MSC batches fully devoid of senescent cells. Methods or a combination of methods that are non-destructive and label-free, for example, involving cell size and spectroscopic measurements, could be the best way forward because they do not modify the cells of interest, thus maximizing the final output of therapeutic-grade MSC cultures. The further incorporation of machine learning methods has also recently shown promise in facilitating, automating and enhancing the analysis of these measured data.

Lasing with cell-endogenous fluorophores: parameters and conditions.

The notion of lasing with biologics has recently been realized and has rapidly developed with the collective objective of creating lasers in vivo. One major limitation of achieving this is the requirement of exogenous dyes and fluorescent materials. We thus investigate for the first time the possibility of lasing unlabelled cells, using just cell-endogenous fluorophores - the source of cell autofluorescence. In this work, we theoretically studied the lasing potential and efficiency of flavins and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) using a dye lasing model based on coupled rate equations. Analytical solutions for one- and two-photon pumped system were used in multi-parameter studies. We found that at physiological conditions, the more abundant NAD(P)H can be lased with a cavity quality factor of 105. We then recommended the tuning of intersystem crossing to make the lasing of flavins feasible even at their low physiological concentrations. Under conditions of reduced intersystem crossing, we concluded that it is more practical to lase unlabelled cells using flavins, because lasing thresholds and cavity quality factors were both at least an order lower. We also note the higher threshold requirements and lower efficiencies of two-photon pumping, but recognize its potential for realizing lasing in vivo.

Activation and inactivation of Bacillus pumilus spores by kiloelectron volt X-ray irradiation.

In this study, we investigated the inactivation efficacy of endospore-forming bacteria, Bacillus pumilus, irradiated by low-energy X-rays of different beam qualities. The different low-energy X-rays studied had cut-off energies of 50, 100 and 150 keV. Bacillus pumilus spores (in biological indicator strips) were irradiated at step doses between 6.5 to 390 Gy. The resulting bacteria populations were then quantified by a pour plate method. Results showed that X-rays of lower energies were more effective in inactivating bacterial spores. In addition, an increment in bacterial population was observed at doses below 13Gy. We attributed this increase to a radiation-induced activation of bacterial spores. Four kinetic models were then evaluated for their prediction of bacterial spore behavior under irradiation. This included: (i) first-order kinetics model; (ii) Shull model; (iii) Sapru model; and (iv) probabilistic model. From R2 and AIC analyses, we noted that the probabilistic model performed the best, followed by the Sapru model. We highlighted that for simplicity in curve fitting the Sapru model should be used instead of the probabilistic model. A 12-log reduction in bacterial population (corresponding to a sterility assurance level of 10-6 as required in the sterilization of medical devices) was computed to be achievable at doses of 1000, 1600 and 2300 Gy for the three different X-ray cut-off energies respectively. These doses are an order in magnitude lesser than that required in gamma irradiation. This highlights the applicability of cheaper and safer table-top X-ray sources for sterilization application.

In-fiber photo-immobilization of a bioactive surface.

We demonstrate the first in-fiber light-induced bioactive biotin-functionalization via photobleaching fluorophore-conjugated biotin. Photobleaching the fluorophores generated free radicals that bind to the albumin-passivated inner surface of pure silica photonic crystal fiber. The subsequent attachment of dye-conjugated streptavidin to the bound biotin qualified the photo-immobilization process and demonstrated a potential for the construction of in-fiber macromolecular assemblies or multiplexes. Compared with other in-fiber bioactive coating methods, the proposed light-induced technique requires only a low-power light source, without the need for additional preactivation steps or toxic chemical reagents. This method, hence, enables a simple and compact implementation for potential biomedical applications.

In-fiber fluorospectroscopy based on a spectral decomposition method.

We report a simplified model for the computation of light-fluorescence interactions within photonic crystal fibers (PCFs). It involved the plotting of ray trajectories confined by total internal reflection within a geometrically simplified PCF core. This was followed by the calculation of absorption and fluorescence emission at each point of reflection, which were subsequently summed and averaged over all the launched rays. The computation of these components for two specified wavelengths (peak excitation and emission) produced a dimensionless ratiometric relationship for varying concentrations of fluorescence dye. This hence eliminated the need for optical filters and minimized the effects of intensity fluctuations. Modeled results were demonstrated to concur well with that obtained experimentally for two PCFs with different microstructured cores.