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This PDF file contains the front matter associated with SPIE Proceedings Volume 12835, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.
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By combining high beam quality picosecond pulsed optical parametric amplifiers at 2.94 μm with Rapid Evaportive Ionisation Mass Spectrometry (REIMS), we have demonstrated record spatial pixel resolutions for ambient mass spectrometry (MS) imaging of < 10 μm. In this contribution, we introduce our work in this area, demonstrating the platform workflow and highlighting recent results of metabolic imaging at the single cell resolution level.
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The polarization (Q = co-polarized - cross-polarized) of reflectance from a tissue of incident linearly polarized light serves as a gate that isolates superficially scattered light, rejecting multiply scattered diffuse light. Pathology often arises in superficial tissue layers. But to interpret an observed change in Q in the field of view, one must decide: ”Is the change due to an altered size of scatterers or to an altered concentration of scatterers?” This study uses the GPU-accelerated MCX version of Monte Carlo polarized light transport, based on light-scattering Mie spheres of varying size, to answer this question.
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In this study, we developed an imaging system that can acquire and produce high-resolution hyperspectral images of the retina. Our system combines the view from a high-resolution RGB camera and a snapshot hyperspectral camera together. The method is fast and can be constructed into a compact imaging device. We tested our system by imaging a calibrated color chart, biological tissues ex vivo, and a phantom of the human retina. By using image pansharpening methods, we were able to produce a high-resolution hyperspectral image. The images from the hyperspectral camera alone have a spatial resolution of 0.2 mm/pixel, whereas the pansharpened images have a spatial resolution of 0.1 mm/pixel, a 2x increase in spatial resolution. Our method has the potential to capture images of the retina rapidly. Our method preserves both the spatial and spectral fidelity, as shown by comparing the original hyperspectral images with the pansharpened images. The high-resolution hyperspectral imaging device can have a variety of applications in retina examinations.
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We report wide-field time-domain fluorescence lifetime imaging (TD-FLIM) using a high-photosensitivity 648x484- pixel time-resolved CMOS image sensor with four simultaneous time gates. The advantages of this image sensor are high spatial resolution, high quantum efficiency and high photon rate. In this report, we verified the applicability of a 648x484-pixel range imaging sensor developed in our laboratory for FLIM. In the measurements, four time-resolved images are obtained simultaneously. To improve the temporal sampling resolution, sub sampling is performed. The performance was compared with that of our previously developed 128x128-pixel TD-FLIM CMOS image sensor. The data was analyzed by the phasor method at 20MHz. The measured average fluorescence lifetimes for the new and previous sensors were 8.52 ns and 8.26 ns, and the standard deviations were 0.74 ns and 0.56 ns, respectively. We have achieved higher signal-to-noise ratios as well as high spatial resolution. Finally, this image sensor was used to perform two-dimensional imaging of the fluorescence lifetime of fluorescent acrylic plates.
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Non-obstructive azoospermia (NOA) is a severe form of male infertility characterized by impaired or absent sperm production in the testes. Microsurgical testicular sperm extraction (Micro TESE) is the primary treatment for NOA, but it faces challenges in differentiating between normal and abnormal seminiferous tubules based solely on morphology. To address this, our study employed stimulated Raman scattering (SRS) and second harmonic generation (SHG) microscopy to identify diagnostic features in human testicular tissues. Additionally, a deep learning-assisted diagnostic algorithm using multimodal imaging datasets demonstrated excellent performance in azoospermia diagnosis. Utilizing a weakly supervised Multiple Instance Learning-Convolutional Neuron Network (MIL-CNN) model framework, we achieved a 96% classification accuracy, surpassing the supervised CNN model. Gradient-weighted class activation mapping (Grad CAM) visualization confirmed the model’s focus on the spermatogenic region, demonstrating the potential of SRS/SHG microscopy coupled with deep learning to accurately classify normal and abnormal spermatogenic tubules, enhancing the efficiency and accuracy of pathological diagnosis.
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Currently, liquid biopsy method is mainly used for tumor detection based on genomic molecular alterations in vitro. Liquid biopsy is superior to traditional tissue biopsy techniques and its diagnosis time of disease and repeated diagnosis of liquid biopsy are new breakthroughs in clinical application. Liquid biopsy method can be used to detect most human disease based on genetic biomarkers from body fluids, among which, special biomarkers in blood and cerebrospinal fluid (CSF) samples are the main research objects, and have made good achievements in preliminary clinical applications. The application of optical spectroscopy in the field of liquid biopsy has aroused great interest among researchers and demonstrated the potential of its clinical application for oncology. The aim of this study is to reveal the optical spectroscopic characteristics of the main biochemical components of CSF of brain tumor using visible resonance Raman (VRR) spectroscopy ex vivo. Tumor-associated proteins, glucose, lactate and other metabolites released to CSF can be used as markers for liquid biopsy. We studied the VRR spectra of CSF samples from 7 types of brain tumor patients. The characteristic VRR modes that were found and may be used as a combination of multiple analyte biomarkers include amyloid-β and tau protein, excess neurotransmitters such as glutamic acid derived from the exchange with interstitial fluid (ISF), DNA, glucose, lactate, etc. for optical liquid biopsy analyses. Another interesting finding was that CSF of different types of tumors showed different images similar to the crystallization of water under the optical microscope. Considering our previous study, the current study on CSF provides another proof that the VRR system can provide a complete scan region of 200 - 4000cm-1 as a clinical tool for non-invasive diagnosis of brain disease.
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It is challenging to assess the spectral absorption of sub-surface medium using non-contact diffuse reflectance spectroscopy (DRS). A demanding application of such is assessing myoglobin oxygenation at a depth of >1mm to inform beef discoloration. Common to the broad-band DRS and especially challenging to non-contact DRS conducted in continuous-wave, probing the sub-surface medium needs to deal with several geometry-dictated limitations in the measurements. For example, photons of longer pathlength are to be acquired to probe the deeper medium. However, the information associated with the photons with paths specific to only the sub-surface layer of the deep-probing path cannot be easily isolated in the measurement when additional information specific to only the shallower layer is absent. Another challenge to instrumentation relates to the substantially different scales of the magnitudes of the shallower probing and deeper probing DRS signals when originating from the same source of illumination at a similar timeframe. We demonstrate a novel dual-channel non-contact DRS for the assessment of spectral absorption at a depth of below 1mm. The dual-channel non-contact DRS combines a center-illuminated-central- acquired (CIC-A) geometry and a center-illuminated-central-blocked (CIC-B) geometry that are co-centric with respect to the same point-of-illumination (POI). The CIC-A geometry enables probing the surface layer of less than 1mm deep. And the CIC-B geometry acquires deep-probing photons while rejecting the short-path-shallow-probing- only components that would confound otherwise. The combination of CIC-A and CIC-B geometries allows assessing the below-surface spectral absorption. The principle is demonstrated by measurements from phantoms and tissues conforming to a two-layer geometry.
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We aim to realize non-invasive blood glucose measurements in daily life. The human body emits light with an intensity that depends on the body temperature (approximately 300 K). Mid-infrared passive spectroscopic imaging obtains component information from this radiated light. Using radiated light measurements of the arm from a distance of 600 mm, we identified the specific emission spectral peaks of glucose components at wavelengths of 9.25 μm and 9.65 μm. In addition, we determined the correlation between the intensity of radiated light at the peak wavelength and blood glucose level. From these results, we previously reported the possibility of non-invasive blood glucose measurements from a distance using mid-infrared passive spectroscopic imaging. Therefore, we developed a passive one-shot Fourier spectrometer to apply this method to wearable devices. The apparatus was designed with a numerical aperture (N.A.) of 0.77 for passive spectroscopy of the living body. The field curvature due to the increased N.A. was mitigated by combining an imaging lens with a phase shifter. Additionally, the apparatus was configured with two lenses to enhance transmission. Owing to the small object lens (diameter of 6 mm) and short optical path (axis length of approximately 14 mm), the apparatus was mountable on wearable devices. Moreover, the apparatus was equipped with a multi-slit to prevent loss of interference sharpness. The multi-slit was designed with five lines comprising aperture patterns that enabled the detection of glucose. As a result, we succeeded in detecting spectral characteristics of polypropylene using a blackbody as the background light source.
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Diabetes is a major global health issue. Prolonged exposure to high blood glucose results in the formation of intermediate glycation products such as HbA1c and eventually leads to the formation of advanced glycation end products (AGEs) which contribute to diabetic pathogenesis. Since some AGEs are fluorescent, they may be used as long-term glycemic markers. In this study, we investigate the spectral properties of fluorescent AGEs from monosaccharides that are encountered in human physiology. Both the absorption and fluorescent properties are characterized in the UV-visible range. The spectral information may be of value in developing assays using fAGEs as biomarkers.
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The decay-kinetics of slow delayed luminescence from organic materials informs conditions such as the oxidative stress of the organism. And delayed luminescence can be induced by external stimulations not limited to photo-irradiation. Acquiring the decay-kinetics of delayed luminescence thus renders studying the response of the organism to external stress. At the absence of external stress causing delayed luminescence, however, organic materials may produce spontaneous ultraweak photon emission due to the residual oxygen demand. The value of steady-state ultraweak photon emission to studying the residual state of organism has been under debate. To better understand the dynamic and residual oxidative state of organism, it will be desirable to acquire both the stress-induced delayed luminescence and the steady-state photon emission. This, however, is challenging due to the significantly different scales of photon counts in the two types of photon-emission. The decay of delayed luminescence from an organism will reach a steady noisy baseline, which however may not be a random noise. Instead, the noisy baseline could be the spontaneous ultraweak photon-emission, which is challenging to measure by time-gated configuration for acquiring delayed luminescence, due to the weak signal overlaying with the noise. We demonstrate an irradiation-acquisition interleaved time-integrated imaging to acquire both the slow-decay kinetics of delayed luminescence and the steady-state ultraweak photon emission. The integration-mode acquisition enabled differentiating the steady-state ultraweak photon emission from the random baseline noise. And repetitive irradiation interleaved with integration at various durations of acquisition allowed the extraction of the decay-kinetics. Results from yeasts demonstrate the approach.
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Diffuse reflectance spectroscopy relies upon the spectral responsivity of the photon remission to the medium’s property for application. Practically, it is imperative to assess the conditions at which the photon remission becomes illy responsive or unresponsive to the change of an otherwise sensible property of the medium. Such limiting cases may be particularly relevant to single-fiber reflectance (SfR) for minimally invasive sensing and center-illuminatedcentral- acquired (CIC-A) geometry for non-contact sensing. The steady-state photon remission of SfR in the absence of absorption, for example, was shown by Monte Carlo (MC) to be insensitive to the scattering changes as the fiberdiameter scaled dimensionless reduced-scattering reaches μ'darea >10. Similar limiting patterns are observed with steady-state CIC-A known to be scalable over steady-state SfR. To gain insights on the conditions of saturation in CIC-A as projectable to SfR, we revise a model of the steady-state photon remission of CIC-A geometry demonstrated in (Sun & Piao, 2022, App. Opt.), which predicts the onset of saturation as the dimensionless reduced scattering increases. The model-projected saturation-level and the corner-condition of the saturation, both being absorptiondependent, are examined against MC simulations. Experiments in non-contact CIC-A geometry were conducted. Diffuse reflectance was collected from two co-centric areas that differ by ~10 times in diameter when responding to the same centered illumination. The limiting-pattern indicative model was applicable to the diffuse reflectance from aqueous samples of the scattering increased to near-saturation followed by absorption increasing. The scattering-saturation may be useful for simplifications such as implementing differential pathlength factor towards real-time assessment of absorption.
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Nanoparticle-based drug delivery systems have shown potential for cancer treatment. Due to their size, nanoparticles penetrate into tumors, facilitate drug uptake, enable efficient drug delivery and ensure efficacy at the target site. They present good pharmacokinetics, precisely target tumor cells, reduce side effects and drug resistance. Recently, patient-derived organoids (PDOs) of colorectal cancer (CRC) patients proved to be a valuable tool to study the pathological, physiological, and genetic characteristics of CRC. In particular, they can be employed to test drugs in-vitro in a complex heterogeneous environment. We use UiO-66-NH2 nanoparticles as drug delivery carriers for 5-Fluorouracil (5-FU) to PDOs of CRC to study interactions between organoids and nanoparticles. Since PDOs preserve tumor heterogeneity to a significant extent, we investigate the structural, metabolic, and molecular changes in PDOs across different CRC and the impact of UiO-66-NH2 as carrier for 5-FU with our custom multimodal optical microscope offering complementary co-registered contrast from Optical Coherence Tomography (OCT), Multiphoton Microscopy (MPM), and Raman Microscopy (RM). We examine the interactions between nanoparticles and PDOs of CRC as well as their response to 5-FU. We assess the influence of the drug by extracting morphological features from OCT/MPM images, distinguish PDOs phenotypic differences through RM molecular analysis, identifying alterations in relevant biomarkers such as lipids, proteins, collagen, DNA, further analyze molecular metabolic differences and conduct sample classification. We demonstrate the significant potential of our multimodal imaging system in the analysis of PDOs and nano-medicine effects, enabling high-precision morpho-molecular metabolic analysis and classification.
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Thin-film interference is commonly used for characterizing some physical properties of thin-film such as the thickness of it. Thin-film interference under broad-band light manifests spectrally varying periodicity, with the fringe becoming sparser towards longer wavelength, that informs the optical thickness of the thin-film. A non-scattering substrate of thin-film shall not alter the spectral periodicity of the thin-film interference due to no change to the pathlengths of interfering photons. A scattering substrate, however, may affect the fringe due to the contribution by photons of longer pathlengths than normal. And the proportion of the photons of longer pathlengths than normal may be affected by the diffusivity of the substrate. We observed that the spectrally varying periodicity of thin-film interference was affected by the substrate’s diffusivity. Spectral thin-film interference was acquired from regular household food-wrap (Bakers & Chefs) placed in good-contact with planar materials, by diffuse reflectance spectroscopy in a center-illuminatedcentral- acquired (CICA) geometry over 550-850nm. Spectral thin-film interference was compared among that acquired from film-attached Spectralon reflectance standards (40%, 60%, 80%, 99%) and film-covered solid tissue phantoms (near-identical reduced scattering with the absorption scaled 1:2:4). The variation of the spectral periodicity of thin-film interference can be associated with the scattering and absorption properties of the diffusive substrate. The effect of the diffusivity of the substrate on the spectral periodicity of thin-film interference may become a confounding issue for thin-film characterization but could provide information for probing thin-film covered materials towards applications including assessing surface and below-surface formation of metmyoglobin.
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Ocular melanoma, albeit being rare, is an aggressive type of cancer, developing within the interior of a person’s eye. Location of this malignant growth increases severity of this type of cancer. It commonly develops in the middle layer of the eye, referred to as uvea. Melanoma develops from melanocytes, with a 90% of cases observed in posterior uvea (choroid). Complications from this type of cancer include metastasis in the liver. To avoid such outcome and improve treatments we are proposing application of fluorescence lifetime imaging microscopy (FLIM) to distinguish metastatic and non-metastatic melanomas. In the reported here preliminary study, we evaluate the system requirements for FLIM imaging of melanin-based tissues and structures.
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Cervical cancer is one of the leading causes of death in women across the globe. Among the several diagnosis techniques that have been explored, optics-based methods have demonstrated highly promising outcomes in detection of cervical cancer at early stages [1]. Mueller matrix imaging and spectroscopy is one of the polarizations based optical technique which has been used in classification of cervical cancer. Polarization parameters obtained from the Mueller matrix have indicated the changes in real and imaginary part of refractive indices between two orthogonal polarizations. In this study, we develop the Fourier domain Mueller matrix imaging (FMMI) system to look at correlations between the real and imaginary parts of the refractive indices. However, the dependence of both parts of refractive indices to one another needs to be established. Simulation studies were performed on 1-D sinusoidal functions and fractional Brownian motion signals as inputs of refractive indices. The correlations between these functions (refractive indices) obtained through Fourier domain Mueller matrix and pairwise linear correlation coefficients were found to be identical. Further, a FMMI system has been developed and calibrated using standard samples (like homogenous diattenuator and linear retarder) with an error <10 %. Currently, experiments are being performed on unstained cervical tissue sections to understand the correlations between refractive indices.
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Urine is one of the diagnostically important bio fluids, as it has many metabolites and some of them are native fluorophores. Riboflavin and its cofactors FMN and FAD which act as electron carriers participates in a diversity of redox reactions central to human metabolism. It has been reported that riboflavin plays a prominent role in progression of various cancers. It is well documented that, the fluorophore flavins that is not bound to proteins in the plasma is filtered by glomerulus and excreted in urine. Fluorescence spectroscopy has been considered as a promising tool to characterize the riboflavin present in the urine. The overall spectral data at 450 nm excitation were subjected to principal components based linear discriminant analysis. As a result, 100 % of the normal subjects and 90% of the cervical cancer subjects were correctly classified which shows that there exists significant difference between them.
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Pteridines and its derivatives are considered as important cofactors participating in cellular metabolism. Studies reported that, the distribution of pteridines and its derivatives may change when monocytes and macrophages are activated under interferon- γ stimulus by cancer. Also, there is a significant variation in the concentration and conformation of pteridines under different pathological conditions. It has been reported that during the transformation of normal cells into neoplasm, the metabolic end products of the cancer cells are released into the blood, thereby changing the components and contents of the biological molecules and their local environment. In this regard, the present study is aimed to characterize pteridines and its derivatives in the blood plasma of normal subjects and patients with confirmed oral cancer using fluorescence and Raman spectroscopy. Observed Fluorescence & Raman spectral characteristics of samples and subsequent discriminant analysis predicts that 75 % of the original and cross validated groups are correctly classified.
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Cervical cancer is of significant health concern globally, particularly in developing countries where access to advanced healthcare facilities and medical resources is limited, leading to increased mortality rates. The gold standard for diagnosing CIN (cervical intraepithelial neoplasia) and invasive cervical cancer involves performing a colposcopy-guided biopsy followed by a pathological diagnosis. However, its effectiveness is challenged by limited sensitivity in distinguishing between various stages of cervical cancer, especially in regions where there is a shortage of skilled colposcopists and insufficient access to medical resources. This study presents a method for categorizing infectious, pre-cancerous, and cancerous conditions through the application of multifractal analysis, specifically two dimensional multifractal detrended fluctuation analysis (2D MFDFA), using images obtained through colposcopy. The utilization of multifractal parameters, namely the generalized Hurst exponent and the width of the singularity spectrum, in the analysis distinctly demonstrated variations among the infectious, precancerous, and cancerous conditions. Therefore, it offers valuable insights to healthcare professionals, assisting in the accurate classification and effective management of cervical cancer using Hurst exponent and multifractal spectrum width.
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