This PDF file contains the front matter associated with SPIE Proceedings Volume 11972, including the Title Page, Copyright information, and Table of Contents.

Photodynamic therapy (PDT) is an established treatment which uses a photosensitiser drug and light source to destroy superficial lesions. This therapy is not applicable to deep-seated tumours due to limited light penetration. Recently, it has been found that replacing light in PDT with X-rays (thus named radiodynamic therapy (RDT)) can stimulate nano formulated photosensitiser drugs including Verteporfin and generate reactive oxygen species to kill the cancer cells. Herein, we investigated aspects of cellular metabolic processes after RDT in comparison with PDT and radiotherapy using label-free hyperspectral autofluorescence microscopy and image analysis. Biochemical signatures of metabolically relevant fluorophores (NAD(P)H, flavins, and optical-redox-ratio) were identified by developing a semi-unsupervised unmixing method combining supervised and unsupervised unmixing in a novel way.

Oblique back-illumination capillaroscopy (OBC) has recently demonstrated high resolution, label-free images of human blood cells in vivo. This technology shows promise for a new chapter in blood analysis, where blood cell counts, morphology, and dynamics can be probed non-invasively. OBC provides high quality blood cell images when applied to the ventral tongue, where capillaries are superficial and melanin is minimal. However, the anatomy of this location has a unique and challenging constraints due to the highly muscular and mobile nature of the tongue, and its presence within the oral cavity. This manuscript presents a portable and ergonomic dual- channel OBC system that is optimized for imaging the ventral tongue. The portable OBC system uses pneumatic stabilization to reduce capillary motion and is built upon an ophthalmic slit lamp housing to allow comfortable stabilization of the head and fine, 3-axis translation of the imaging probe. The signal from two diametrically opposed LEDs (530nm and 650nm) are imaged onto two time-synchronized CMOS sensors, providing combined phase-weighted and absorption-weighted contrast of blood cells at 200 Hz with a 165 x 220μm field-of-view. This functional implementation of OBC technology will enable high resolution blood cell imaging of patients with hematologic disease.

Globally, colorectal cancer was the second leading cause of cancer death in 2020. Research suggests that collagen, a major structural protein, plays a pivotal role in cancer development and metastasis, and by extension, subject prognosis. Collagen surrounding tumor cells undergoes structural changes that can be quantitatively studied with second harmonic generation (SHG), a subset of multiphoton microscopy (MPM). MPM as an imaging modality is difficult to implement in an endoscope because of the complex and expensive miniaturized scanning components required. Endoscope complexity can be greatly reduced by implementing a simpler, non-synchronized scanning mechanism. This study investigates whether non-imaging, randomly sampled SHG intensity measurements are sufficient to distinguish normal tissue from tumor/tumor-adjacent tissue. Unstained tumor, normal, and adjacent formalin-fixed, paraffin-embedded thin sections from 12 colorectal cancer subjects were imaged using a multiphoton microscope with 850nm excitation and 400-430nm emission band, constant power, and consisting of 1024x1024 pixels over 425x425μm. SHG signal from collagen fibers was isolated by grayscale thresholding, and the grayscale mean of the thresholded image was calculated. Then, random supra-threshold pixels in the image were selected. The mean SHG signal from normal samples was significantly greater than adjacent samples (p = 0.014) and cancer samples (p = 0.007). For both tumor and adjacent comparisons to normal tissue, p value becomes reliable after randomly sampling only 1000 pixels. This study suggests that reliable diagnostic information may be obtained through simple non-imaging, random-sampling SHG intensity measurements. A simple endoscope with this capability could help identify suspicious masses or optimum surgical margins.

Chemotherapy is one of the most common anti-cancer treatments, that targets rapidly dividing cells by inducing DNA damage and inhibition of mitosis. Most chemotherapeutic drugs are known to induce apoptosis, which is programmed cell death. Therefore, monitoring cell death mechanism, in addition to its viability, is important for understanding the efficacy of treatment, and is particularly important during drug screening. Here we present an automated label-free method of testing the efficacy of chemotherapeutic drugs by identification of apoptotic cell death, based on the scattering signature of cancer cells, using dark field microscopy. Breast cancer cells (BT-20) were treated with different chemo-drugs and simultaneously imaged during the drug incubation step. A neural network was trained to identify the cells that remain alive, as well as distinguish between the cells undergoing apoptosis and necrosis, the two most common cell death mechanisms. The network identifies the cell death mechanism, based on the temporal changes in morphological properties of the cells, e.g. volume shrinkage, blebbing, membrane damage etc. Our results show that the network trained using a specific chemo-drug can then be used for identifying the cell death processes induced by other types of chemo-drugs, with over <95% accuracy, confirmed using western blot assay. This automated technique which can predict the cell death mechanism and viability in real time, during drug incubation, eliminates the additional steps, such as staining or adding conjugates, required for fluorescence imaging and western blot respectively, thereby making it user friendly, cost-effective and high throughput.

Glycolysis, glutaminolysis, and oxidative phosphorylation (OXPHOS) are the main cellular metabolic pathways used to generate energy. Reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) are coenzymes in these metabolism pathways, and detection of their endogenous fluorescence lifetime offers a label-free and quantitative method to study redox state and model cellular metabolism. Many cancer cells depend on glycolysis instead of oxidative phosphorylation to produce energy even in an aerobic environment, which is known as the Warburg effect. Here, autofluorescence lifetime images of NADH and FAD were obtained from MCF-7 breast cancer cells using multiphoton fluorescence lifetime microscopy. Cells were cultured in the Dulbecco’s Modified Eagle’s Medium (DMEM), and glycolysis, glutaminolysis, and OXPHOS were inhibited using 2-DG (2-Dexoy-D-glucose), sodium cyanide, and BPTES (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide) respectively. The fraction of free NADH decreases when glycolysis is inhibited and increases when OXPHOS is inhibited. The mean NADH lifetime is increased when glycolysis is inhibited and is reduced when OXPHOS is inhibited. NADH and FAD fluorescence lifetime features vary when inhibiting glutaminolysis. Altogether, this investigation offers a non-invasive method to image key metabolic pathways at a cellular level, which improves the identification of different metabolic states and drug responses of cancer cells.

Gastrointestinal neuroendocrine tumors (GI-NETs) including gastric carcinoids and duodenal neuroendocrine tumors (DNETs), represent a growing class of cancer.1 There is a strong need for intraoperative localization to facilitate diagnosis and treatment of DNETs, particularly those related to the hereditary MEN1 syndrome. However, these demands are precluded by a lack of in vivo model systems that accurately recapitulate disease heterogeneity and progression. Optical imaging markers are commonly used diagnostically to probe early tissue changes that occur with the onset of cancer. Promising techniques include autofluorescence imaging (AFI), which probes intrinsic biochemistry and metabolic markers, and optical coherence tomography (OCT), which provides a robust microstructural reference. Both have demonstrated widespread promise for non-invasive disease screening, making them potential candidates for localization of DNETs. Here we apply AFI and OCT to a mouse model of human MEN1 syndrome to identify unique optical markers associated with neuroendocrine cell reprogramming. Using Cre-lox technology, we generated a glial cell-directed Men1 knockout mouse model that exhibits enhanced neuroendocrine cell differentiation in the stomach and duodenum. We measured variations in optical imaging markers using AFI and OCT images of transgenic and wild type mice. The transgenic lines exhibit significant fluctuations in optical imaging markers compared to wild type mice, both in the scope of AFI and OCT (p<0.01). These results suggest that AFI and OCT may further inform biological and metabolic changes associated with initial neuroendocrine cell reprogramming prefacing tumor formation. Further studies are needed to fully elucidate the significance of these optical markers in GI-NET pathogenesis.

We demonstrate label-free imaging of renal function with a unilateral ureteral obstruction (UUO) kidney mouse model. The imaging was performed by optical coherence microscopy which is capable of measuring tissue dynamics. Two different studies comprising of 1-week and 2-week UUO models were performed. A circular ring-shape high dynamics appearance at the periphery of the tissue surface was found in the 1-week UUO model for both obstructed and contralateral non-obstructed kidneys. In the 2-week UUO model, several vertical high dynamics regions were observed in cross-sectional dynamics images for both obstructed and non-obstructed kidneys. The results were validated by histological analysis.

Nanoparticles are currently the subject of numerous research activities due to their unique physical and chemical properties, such as for industrial applications, but also in studies on environmental toxicity and human health effects. Since the toxicity of nanoparticles is mainly based on their interaction and uptake by cells, the aim of this study was to quantify their cellular internalization. Quantitative phase imaging (QPI) has proven to be a versatile method for minimally invasive label-free imaging of biological specimens, whereas laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows the label-free identification and quantification of nanomaterials in biological tissues. However, the determination of nanoparticle concentrations within single cells in vitro is challenging for LA-ICP-MS as precise information about the cell volume is required. We thus combined the LA-ICP-MS analysis with digital holographic microscopy (DHM), a QPI technology, which allows single cell volume determination prior to measurements by LA-ICP-MS. The implementation of this method at the single cell level in vitro was investigated by determining the concentration of Ce in RLE-6TN lung epithelial cells after exposition to CeO2 nanomaterial. Results from correlative LA-ICP-MS and DHM QPI investigations on nanoparticle-loaded cells show significant effects on intracellular Ce-levels for RLE-6TN cells treated with different CeO2 nanoparticle concentrations and therefore demonstrate the feasibility of this new concept.

Cell death pathways in multicellular organisms play an important role in maintaining homeostasis and any irregularities can cause diseases like cancer. Studying cell death mechanism will aid in understanding its role in different diseases and their translational implications. It is particularly important for screening new drugs. Here we present a high-throughput and label-free method of identifying the two most common forms of cells death, programmed (apoptosis) and unprogrammed (necrosis), using lensless holographic microscopy. Breast cancer cells BT-20 were treated with a series of drugs, that induce different types of cell death, and imaged continuously during the incubation step. The cells were imaged by illuminating them with partially coherent light and the in-line holograms of the cells, resulting from the interference between the transmitted wave and scattered wave, were recorded in a CMOS imaging chip. The holograms were digitally backpropagated to reconstruct the phase and amplitude images of the cells. The absence of lenses enables imaging at unit magnification over an area <10 mm², which includes over a thousand mammalian cells. The temporal changes in cell morphology, such as membrane blebbing, shrinking, swelling, and membrane rupture, which are reflected in the phase image was used to identify the cell death mechanism. This process was further automated using deep learning, which enabled the classification of the cell death process with <93% accuracy. This label-free approach of identifying cell death mechanisms enables high-throughput toxicity studies unlike the conventional biochemical assays, e.g., western blot, and can be useful for a variety of biomedical applications.

Transport of intensity (TIE) and digital holographic microscopy (DHM) are imaging techniques capable of real-time high resolution phase reconstructions. DHM is a widely used technique that provides phase maps through numerical reconstruction of light propagation of captured hologram intensities generated by interference between an object and a reference beam. TIE is a bright-field compatible technique that yields phase reconstructions through intensity measurements of a single object beam at symmetric planes about the focal plane. A TIE setup is simpler than DHM due to its non-interferometric nature and may yield a higher resolution reconstruction than DHM. Since TIE is a somewhat less-mature technique, we have developed a setup capable of both TIE and DHM measurements and simultaneously measured the volume changes of biological cells using both techniques. The setup is based on a modified bright-field microscope, with the addition of laser illumination for the DHM measurements. Live C6 glial cells were monitored as a hydrogen peroxide solution was introduced to the sample media to produce a visible and measurable decrease in cell volume through apoptosis. This decrease in volume was simultaneously measured by TIE and DHM, and the results were directly compared. Additionally, volume changes in C6 glial cells undergoing methamphetamine-induced apoptosis were tracked and compared.

Migrasome is a type of recently discovered organelle that plays a vital role in the release of cytosolic contents, regulation of zebrafish embryo formation, mitochondria quality control process, etc. Fluorescence microscopy is widely used to investigate biological specimens, including migrasomes. However, the labelling of fluorescence probes not only requires additional preparation steps, but also may interfere with cellular functions and potentially result in phototoxicity, while only a limited number of labelled structures can be observed at one time. Optical diffraction tomography, as a label-free imaging technique complementary to fluorescence imaging tools, is able to characterize the biophysical properties of organelles. Here we propose to apply optical diffraction tomography for three-dimensional (3D) imaging of migrasome and monitoring its dynamics in living cells.

Functionalized Single-walled carbon nanotubes (SWCNTs) are used for a variety of near-infrared (nIR) imaging and sensing applications, including in vivo imaging within Caenorhabditis elegans (C. elegans) nematodes, and sensing proteins, small molecules, and enzymatic activity. The optical properties of SWCNT open numerous possibilities for tailored molecular recognition and monitoring active processes in real-time, with optical signal transduction.

Label-Free detection of cardiac biomarkers has become an area of great interest with respect to point of care (POC) analysis of acute myocardial infarction and drug cardiotoxicity assays. DNA aptamers have become a potential replacement to traditional antibody detection of antigens in bioassays. In comparison to antibodies, DNA aptamers provide the advantages of lower cost, high flexibility, high batch-to-batch uniformity, stability at 37°C when immobilized on the sensor surface, and reusability with a regeneration solution. However, aptamer usage requires novel binding pathways that must be explored to ensure efficiency and consistency. Herein, a direct approach for Cardiac Troponin I (cTnI) detection was tested utilizing UV immobilization of Amino- and PolyT-modified aptamers on APTES or MPTMS modified and unmodified sensor surfaces composed of SiO₂ for a Photonic Crystal-Total Internal Reflection biosensor (PC-TIR). The detection of aptamer functionalization, and ultimately antigen detection, were monitored with a label-free bioassay system enabled by a PC-TIR sensor. Results from this study indicated that the binding pathways with the highest aptamer immobilization were: Amino modified aptamer on an APTES modified surface and PolyT modified aptamer on an MPTMS surface. Detection of the antigen was dependent on both aptamer secondary structure formation and aptamer immobilization following UV exposure.

Developing megapixel large-area CCD and CMOS sensor arrays in the 2000-s stimulated ideas about developing microscope systems operating without heavy and bulky microscope stands and objectives by using microoptics approach in combination with imaging by cellphone cameras. Due to limited magnification, however, the best resolution of such systems is currently limited by the finite size of the pixels at ~1.5 μm level. We propose a novel approach to designing such microscope systems based on using contact ball lenses with index of refraction close to 2, which are capable of imaging biomedical and nanoplasmonic objects with extraordinarily high magnification and resolution. By using ball lenses made from glass with index n = 2.02 at λ = 600 nm we build a cellphone camera-based microscope system with up to x50 magnification and resolution fundamentally limited at ~600 nm level due to diffraction of light. It is demonstrated that the operation of such system is a subject for strong dispersive effects in glass leading to a complicated tradeoff of magnification, resolution, and field-of-view (FOV) in the proximity to critical index of 2. Using this system, we performed imaging of melanoma samples which shows a potential of developing biopsy-free in vivo histology of skin using ball lensassisted smartphone microscopy.

Materials in thin-film structures for optical sensing applications must have multiple features: (i) enhancing the optical signal providing high optical sensitivity for the measurement of the interface processes, (ii) having appropriate chemical properties for supporting the adsorption of the molecules to be detected, (iii) having stability and selectivity. Development of materials that meet all these requirements is an ever lasting process with a lot of opportunities. In this work we propose porous silicon nanoparticles for the detection of biomolecules in plasmonic and Bragg multilayer enhanced Kretschmann-Raether ellipsometry configurations.