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Single-cell RNA sequencing and other profiling assays have helped interrogate cells at unprecedented resolution and scale, but are inherently destructive. Raman microscopy reports on the vibrational energy levels of proteins and metabolites in a label-free and nondestructive manner at subcellular spatial resolution, but it lacks genetic and molecular interpretability. Here we present Raman2RNA (R2R), a method to infer single-cell expression profiles in live cells through label-free hyperspectral Raman microscopy images and domain translation. We predict single-cell RNA sequencing profiles nondestructively from Raman images using either anchor-based integration with single molecule fluorescence in situ hybridization, or anchor-free generation with adversarial autoencoders. R2R outperformed inference from brightfield images (cosine similarities: R2R >0.85 and brightfield <0.15). In reprogramming of mouse fibroblasts into induced pluripotent stem cells, R2R inferred the expression profiles of various cell states. With live-cell tracking of mouse embryonic stem cell differentiation, R2R traced the early emergence of lineage divergence and differentiation trajectories, overcoming discontinuities in expression space. R2R lays a foundation for future exploration of live genomic dynamics., Competing Interests: Competing interests A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was a scientific advisory board member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics and Asimov until 31 July 2020. A.R. is an employee of Genentech from 1 August 2020 with equity in Roche. T.B., S.G. and T.J. are employees of Genentech from 1 Feburary 2021, 29 March 2021 and 5 June 2023, respectively. J.S. is a scientific advisor for Arcadia Science. A patent application has been filed by the Broad Institute related to this work. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Optical fiber probe-based Raman spectroscopy systems are widely used for in situ measurements ranging from material characterization to biomedical applications. However, small Raman cross sections necessitate the use of high-power lasers or long exposure times that limit Raman's larger application to multiple research fields. This limitation can be overcome by collecting more Raman photons through additional collection fibers with taller detectors. This system configuration requires replacement of the detector and modification of the spectrograph to incorporate larger optical components, making it a costly and cumbersome option. In probe-based Raman systems, a typical detector image shows stacked collection fibers on the vertical axis and Raman spectra on the horizontal axis. While the vertical pixels are fully packed with multiple collection fibers, horizontal pixels have broad silent regions due to the narrow bandwidth of Raman peaks, potentially wasting valuable detector pixels. Here, we propose a new approach utilizing horizontally shifted collection fibers rather than vertically stacked ones. We designed and fabricated a novel collection fiber bundle that has horizontally shifted optical fibers in two vertical lines at the spectrograph entrance. This custom-made fiber bundle was incorporated into the imaging spectrograph to provide multiple horizontally shifted spectra on the detector. Through deconvolution, the original spectra can be recovered with an improved detection limit from greater photon collection. We demonstrate an enhanced limit of detection on various bioanalytes, such as glucose, urea, and lactate. Further, we applied the probe to measure tissue Raman spectra and successfully decomposed them into basis spectra, demonstrating the potential application of high-throughput in vivo tissue diagnosis. Our approach provides a simple, cost-effective, and universal method to increase the throughput without modifying existing Raman spectrometers.

Accurate, rapid and non-invasive cancer cell phenotyping is a pressing concern across the life sciences, as standard immuno-chemical imaging and omics require extended sample manipulation. Here we combine Raman micro-spectroscopy and phase tomography to achieve label-free morpho-molecular profiling of human colon cancer cells, following the adenoma, carcinoma, and metastasis disease progression, in living and unperturbed conditions. We describe how to decode and interpret quantitative chemical and co-registered morphological cell traits from Raman fingerprint spectra and refractive index tomograms. Our multimodal imaging strategy rapidly distinguishes cancer phenotypes, limiting observations to a low number of pristine cells in culture. This synergistic dataset allows us to study independent or correlated information in spectral and tomographic maps, and how it benefits cell type inference. This method is a valuable asset in biomedical research, particularly when biological material is in short supply, and it holds the potential for non-invasive monitoring of cancer progression in living organisms., (© 2024. The Author(s).)

Li-ion batteries stand out among energy storage systems due to their higher energy and power density, cycle life, and high-rate performance. Development of advanced, high-capacity anodes is essential for enhancing their performance, safety, and durability, and recently, two-dimensional materials have garnered extensive attention in this regard due to distinct properties, particularly their ability to modulate van der Waals gap through intercalation. Covalently intercalated Graphene oxide interlayer galleries with mono-Boc-ethylenediamine (GO-EnBoc) was synthesized via the ring opening of epoxide, forming an amino alcohol moiety. This creates three coordination sites for Li ion exchange on the graphene oxide nanosheets' surface. Consequently, the interlayer d -spacing expands from 8.47 Å to 13.17 Å, as anticipated. When explored as an anode, Li-GO-EnBoc shows a significant enhancement in the stable and reversible capacity of 270 mA h g -1 at a current density of 25 mA g -1 compared to GO (80 mA h g -1 ), without compromising the mechanical or chemical stability. Through 13 C, 7 Li and 6 Li MAS NMR, XPS, IR, Raman microscopy, and density functional theory (DFT) calculations, we confirm the positioning of Li + ions at multiple sites of the interlayer gallery, which enhances the electrochemical performance. Our findings suggest that these novel systematically modulated van der Waals gap GO-engineered materials hold promise as efficient anodes for Li-ion batteries., Competing Interests: The authors are not involved in conflict of interest., (This journal is © The Royal Society of Chemistry.)

Systems that can image in three dimensions at cellular resolution and across different locations within an organism may enable insights into complex biological processes, such as immune responses, for which a single location measurement may be insufficient. In this Letter, we describe an in vivo two-site imaging probe (TIP) that can simultaneously image two anatomic sites with a maximum separation of a few centimeters. The TIP consists of two identical bendable graded index (GRIN) lenses and is demonstrated by a two-photon two-color fluorescence imaging system. Each GRIN lens has a field of view of 162 ×   162 ×   170 µm 3 , a nominal numerical aperture of 0.5, a magnification of 0.7, and working distances of 0.2 mm in air for both ends. A blind linear unmixing algorithm is applied to suppress bleedthrough between channels. We use this system to successfully demonstrate two-site two-photon two-color imaging of two biomedically relevant samples, i.e., (1) a mixture of two autofluorescent anti-cancer drugs and (2) a live hybrid tumor consisting of two spectrally distinct fluorescent cell lines.

Significance: Raman spectroscopy has been used as a powerful tool for chemical analysis, enabling the noninvasive acquisition of molecular fingerprints from various samples. Raman spectroscopy has proven to be valuable in numerous fields, including pharmaceutical, materials science, and biomedicine. Active research and development efforts are currently underway to bring this analytical instrument into the field, enabling in situ Raman measurements for a wider range of applications. Dispersive Raman spectroscopy using a fixed, narrowband source is a common method for acquiring Raman spectra. However, dispersive Raman spectroscopy requires a bulky spectrometer, which limits its field applicability. Therefore, there has been a tremendous need to develop a portable and sensitive Raman system., Aim: We developed a compact swept-source Raman (SS-Raman) spectroscopy system and proposed a signal processing method to mitigate hardware limitations. We demonstrated the capabilities of the SS-Raman spectroscopy by acquiring Raman spectra from both chemical and biological samples. These spectra were then compared with Raman spectra obtained using a conventional dispersive Raman spectroscopy system., Approach: The SS-Raman spectroscopy system used a wavelength-swept source laser (822 to 842 nm), a bandpass filter with a bandwidth of 1.5 nm, and a low-noise silicon photoreceiver. Raman spectra were acquired from various chemical samples, including phenylalanine, hydroxyapatite, glucose, and acetaminophen. A comparative analysis with the conventional dispersive Raman spectroscopy was conducted by calculating the correlation coefficients between the spectra from the SS-Raman spectroscopy and those from the conventional system. Furthermore, Raman mapping was obtained from cross-sections of swine tissue, demonstrating the applicability of the SS-Raman spectroscopy in biological samples., Results: We developed a compact SS-Raman system and validated its performance by acquiring Raman spectra from both chemical and biological materials. Our straightforward signal processing method enhanced the quality of the Raman spectra without incurring high costs. Raman spectra in the range of 900 to 1200    cm - 1 were observed for phenylalanine, hydroxyapatite, glucose, and acetaminophen. The results were validated with correlation coefficients of 0.88, 0.84, 0.87, and 0.73, respectively, compared with those obtained from dispersive Raman spectroscopy. Furthermore, we performed scans across the cross-section of swine tissue to generate a biological tissue mapping plot, providing information about the composition of swine tissue., Conclusions: We demonstrate the capabilities of the proposed compact SS-Raman spectroscopy system by obtaining Raman spectra of chemical and biological materials, utilizing straightforward signal processing. We anticipate that the SS-Raman spectroscopy will be utilized in various fields, including biomedical and chemical applications., (© 2024 The Authors.)

We studied the in vitro rate of fluorescent advanced glycation end products (fAGEs) formation with multiphoton microscopy in different porcine tissues (aorta, cornea, kidney, dermis, and tendon). These tissues were treated with d-glucose, d-galactose, and d-fructose, three primary monosaccharides found in human diets. We found that the use of d-fructose resulted in the highest glycation rate, followed by d-galactose and then d-glucose. Moreover, compared to non-collagen tissue constituents such as elastic fibers and cells, the rate of tissue glycation was consistently higher in collagen, suggesting that collagen is a more sensitive target for fAGE formation. However, we also found that collagen in different tissues exhibits different rates of fAGE formation, with slower rates observed in tightly packed tissues such as cornea and tendon. Our study suggests that for fAGE to be developed into a long-term glycemic biomarker, loosely organized collagen tissues located in the proximity of vasculature may be the best targets., (© 2023 Wiley-VCH GmbH.)

In recent times, the utilization of three-dimensional (3D) printing technology, particularly a variant using digital light processing (DLP), has gained increasing fascination in the realm of microfluidic research because it has proven advantageous and expedient for constructing microscale 3D structures. The surface wetting characteristics (e.g., contact angle and contact angle hysteresis) of 3D-printed microstructures are crucial factors influencing the operational effectiveness of 3D-printed microfluidic devices. Therefore, this study systematically examines the surface wetting characteristics of DLP-based 3D printing objects, focusing on various printing conditions such as lamination (or layer) thickness and direction. We preferentially examine the impact of lamination thickness on the surface roughness of 3D-printed structures through a quantitative assessment using a confocal laser scanning microscope. The influence of lamination thicknesses and lamination direction on the contact angle and contact angle hysteresis of both aqueous and oil droplets on the surfaces of 3D-printed outputs is then quantified. Finally, the performance of a DLP 3D-printed microfluidic device under various printing conditions is assessed. Current research indicates a connection between printing parameters, surface roughness, wetting properties, and capillary movement in 3D-printed microchannels. This correlation will greatly aid in the progress of microfluidic devices produced using DLP-based 3D printing technology.

Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.

Background and Objectives: Determination of stomach tumor location and invasion depth requires delineation of gastric histological structure, which has hitherto been widely accomplished by histochemical staining. In recent years, alternative histochemical evaluation methods have been pursued to accelerate intraoperative diagnosis, often by bypassing the time-consuming step of dyeing. Owing to strong endogenous signals from coenzymes, metabolites, and proteins, autofluorescence spectroscopy is a favorable candidate technique to achieve this aim., Materials and Methods: We investigated stomach tissue slices and block specimens using a fast fluorescence imaging scanner. To obtain histological information from broad and structureless fluorescence spectra, we analyzed tens of thousands of spectra with multiple machine-learning algorithms and built a tissue classification model trained with dissected gastric tissues., Results: A machine-learning-based spectro-histological model was built based on the autofluorescence spectra measured from stomach tissue samples with delineated and validated histological structures. The scores from a principal components analysis were employed as input features, and prediction accuracy was confirmed to be 92.0%, 90.1%, and 91.4% for mucosa, submucosa, and muscularis propria, respectively. We investigated the tissue samples in both sliced and block forms using a fast fluorescence imaging scanner., Conclusion: We successfully demonstrated differentiation of multiple tissue layers of well-defined specimens with the guidance of a histologist. Our spectro-histology classification model is applicable to histological prediction for both tissue blocks and slices, even though only sliced samples were trained., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Graded index (GRIN) lens endoscopy has broadly benefited biomedical microscopic imaging by enabling accessibility to sites not reachable by traditional benchtop microscopes. It is a long-held notion that GRIN lenses can only be used as rigid probes, which may limit their potential for certain applications. Here, we describe bendable and long-range GRIN microimaging probes for a variety of potential micro-endoscopic biomedical applications. Using a two-photon fluorescence imaging system, we have experimentally demonstrated the feasibility of three-dimensional imaging through a 500-µm-diameter and ∼11 cm long GRIN lens subject to a cantilever beam-like deflection with a minimum bend radius of ∼25 cm. Bend-induced perturbation to the field of view and resolution has also been investigated quantitatively. Our development alters the conventional notion of GRIN lenses and enables a range of innovative applications. For example, the demonstrated flexibility is highly desirable for implementation into current and emerging minimally invasive clinical procedures, including a pioneering microdevice for high-throughput cancer drug selection.

Metastatic prostate cancer colonizes the bone to pave the way for bone metastasis, leading to skeletal complications associated with poor prognosis and morbidity. This study demonstrates the feasibility of Raman imaging to differentiate between cancer cells at different stages of tumorigenesis using a nanoclay-based three-dimensional (3D) bone mimetic in vitro model that mimics prostate cancer bone metastasis. A comprehensive study comparing the classification of as received prostate cancer cells in a two-dimensional (2D) model and cancer cells in a 3D bone mimetic environment was performed over various time intervals using principal component analysis (PCA). Our results showed distinctive spectral differences in Raman imaging between prostate cancer cells and the cells cultured in 3D bone mimetic scaffolds, particularly at 1002, 1261, 1444, and 1654 cm -1 , which primarily contain proteins and lipids signals. Raman maps capture sub-cellular responses with the progression of tumor cells into metastasis. Raman feature extraction via cluster analysis allows for the identification of specific cellular constituents in the images. For the first time, this work demonstrates a promising potential of Raman imaging, PCA, and cluster analysis to discriminate between cancer cells at different stages of metastatic tumorigenesis., (© 2022. The Author(s).)

Digital microfluidics (DMF) has garnered considerable interest as a straightforward, rapid, and programmable technique for controlling microdroplets in various biological, chemical, and medicinal research disciplines. This study details the construction of compact and low-cost 3D DMF platforms with programmable contact charge electrophoresis (CCEP) actuations by employing electrode arrays composed of a small commercial pin socket and a 3D-printed housing. We demonstrate basic 3D droplet manipulation on the platform, including horizontal and vertical transport via lifting and climbing techniques, and droplet merging. Furthermore, phenolphthalein reaction and precipitation process are evaluated using the proposed 3D DMF manipulations as a proof of concept for chemical reaction-based analysis and synthesis. The threshold voltage (or electrical field) and maximum vertical transport velocity are quantified as a function of applied voltage and electrode distance to determine the CCEP actuation conditions for 3D droplet manipulations. The ease of manufacturing and flexibility of the proposed 3D DMF platform may provide an effective technique for programmable 3D manipulation of droplets in biochemical and medical applications, such as biochemical analysis and medical diagnostics.

Cardiovascular diseases pose a serious health risk and have a high mortality rate of 31% worldwide. Digoxin is the most commonly prescribed pharmaceutical preparation to cardiovascular patients particularly in developing countries. The effectiveness of the drug critically depends on its presence in the therapeutic range (0.8-2.0 ng mL -1 ) in the patient's serum. We fabricated immunoassay chips based on QD photoluminescence (QDs-ELISA) and AuNP Surface Enhanced Raman Scattering (SERS-ELISA) phenomena to detect digoxin in the therapeutic range. Digoxin levels were monitored using digoxin antibodies conjugated to QDs and AuNPs employing the sandwich immunoassay format in both the chips. The limit of detection (LOD) achieved through QDs-ELISA and SERS-ELISA was 0.5 ng mL -1 and 0.4 ng mL -1 , respectively. It is demonstrated that the sensitivity of QDs-ELISA was dependent on the charge transfer mechanism from the QDs to the antibody through ionic media, which was further explored using electrochemical impedance spectroscopy. We demonstrate that QDs-ELISA was relatively easy to fabricate compared to SERS-ELISA. The current study envisages replacement of conventional methodologies with small immunoassay chips using QDs and/or SERS-based tags with fast turnaround detection time as compared to conventional ELISA., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (This journal is © The Royal Society of Chemistry.)

Advances in the intratumor measurement of drug responses have included a pioneering biomedical microdevice for high throughput drug screening in vivo, which was further advanced by integrating a graded-index lens based two-dimensional fluorescence micro-endoscope to monitor tissue responses in situ across time. While the previous system provided a bulk measurement of both drug delivery and tissue response from a given region of the tumor, it was incapable of visualizing drug distribution and tissue responses in a three-dimensional (3D) way, thus missing the critical relationship between drug concentration and effect. Here we demonstrate a next-generation system that couples multiplexed intratumor drug release with continuous 3D spatial imaging of the tumor microenvironment via the integration of a miniaturized two-photon micro-endoscope. This enables optical sectioning within the live tissue microenvironment to effectively profile the entire tumor region adjacent to the microdevice across time. Using this novel microimaging-microdevice (MI-MD) system, we successfully demonstrated the four-dimensional imaging (3 spatial dimensions plus time) of local drug delivery in tissue phantom and tumors. Future studies include the use of the MI-MD system for monitoring of localized intra-tissue drug release and concurrent measurement of tissue responses in live organisms, with applications to study drug resistance due to nonuniform drug distribution in tumors, or immune cell responses to anti-cancer agents.

Background: Margin status is an important prognostic factor for treating colorectal cancer. This study aimed to investigate the usefulness of a multimodal spectroscopic tissue scanner for real-time cancer diagnosis without tissue staining., Patients and Methods: Diffuse reflectance spectra (DRS) and fluorescence spectra (FS) of < 1-mm-sized paired cancer and normal mucosa tissue were acquired using custom-built spectroscopic tissue scanners. For FS, we analyzed wavelengths and intensities at peaks and highest intensities near (± 1.25 nm) the known fluorescence spectral peaks of collagen (380 nm), reduced nicotinamide adenine dinucleotide (NADH, 460 nm), and flavin adenine dinucleotide (FAD, 550 nm). For DRS, we performed a similar analysis near the peaks of strong absorbers, oxyhemoglobin (oxyHb; 414 nm, 540 nm, and 576 nm) and deoxyhemoglobin (deoxyHb; 432 nm and 556 nm). Logistic regression analysis for these parameters was performed in the testing set., Results: We acquired 17,735 spectra of cancer tissues and 9438 of normal tissues from 30 patients. Intensity peaks of representative normal spectra for FS and DRS were higher than those of representative cancer spectra. Logistic regression analysis showed wavelength and intensity at peaks, and the intensities of the peak wavelength of NADH, FAD, deoxyHb, and oxyHb had significant coefficients. The area under the receiver operating characteristic curve was 0.927. The scanner had 100%, 64.3%, and 85.3% sensitivity, specificity, and accuracy, respectively., Conclusions: The spectroscopic tissue scanner has high sensitivity and accuracy and provides real-time intraoperative resection margin assessments and should be further investigated as an alternative to frozen section.

Label-free live cell imaging was performed using a custom-built high-speed confocal Raman microscopy system. For various cell types, cell-intrinsic Raman bands were monitored. The high-resolution temporal Raman images clearly delineated the intracellular distribution of biologically important molecules such as protein, lipid, and DNA. Furthermore, optical phase delay measured using quantitative phase microscopy shows similarity with the image reconstructed from the protein Raman peak. This reported work demonstrates that Raman imaging is a powerful label-free technique for studying various biomedical problems in vitro with minimal sample preparation and external perturbation to the cellular system.

Prolonged exposure of tissues to elevated blood sugar levels lead to the formation of advanced glycation end products (AGEs), thus contributing to diabetic complications. Since the vascular system is in immediate contact with blood, diabetic effects on aorta is a major health concern. However, the relative effect of the diffusion of sugar molecular through the vascular wall and the rate of AGE formation is not known. In this study, we aim to address this issue by incubating excised porcine aorta in D-glucose, D-galactose, and D-fructose solutions for different periods. The tissue specimens were then excised for multiphoton imaging of autofluorescence intensity profiles across the aorta wall. We found that for Days 4 to 48 incubation, autofluorescence is constant along the radial direction of the aorta sections, suggesting that monosaccharide diffusion is rapid in comparison to the rate of formation of fluorescent AGEs (fAGEs). Moreover, we found that in porcine aorta, the rate of fAGE formation of D-fructose and D-glucose are factors 2.08 and 1.14 that of D-galactose. Our results suggest that for prolonged exposure of the cardiovascular system to elevated monosaccharides 4 days or longer, damage to the aorta is uniform throughout the tissues., (© 2021 Wiley-VCH GmbH.)

We analytically investigate the feasibility of long graded-index (GRIN)-lens-based microendoscopes through wavefront shaping. Following the very well-defined ray trajectories in a GRIN lens, mode-dependent phase delay is first determined. Then, the phase compensation needed for obtaining diffraction limited resolution is derived. Finally, the diffraction pattern of the lens output is computed using the Rayleigh-Sommerfeld diffraction theory. We show that diffraction-limited resolution is obtained for a 0.5 mm diameter lens with a length over 1 m. It is also demonstrated that different imaging working distances (WDs) can be realized by modifying the phase compensation. When a short design WD is used, a large imaging numerical aperture (NA) higher than 0.4 is achievable even when a low NA lens (NA = 0.1) is used. The long- and thin-GRIN-lens-based microendoscope investigated here, which is attractive for biomedical applications, is being prioritized for use in a clinical stage microdevice that measures three-dimensional drug responses inside the body. The advance described in this work may enable superior imaging capabilities in clinical applications in which long and flexible imaging probes are favored.

MicroRNAs are emerging as both diagnostic and therapeutic targets in different human pathologies. An accurate understanding of the structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications that can transform into highly sensitive diagnostic devices and therapeutic molecules. In this proof-of-principle study, we have utilized label-free spontaneous Raman spectroscopy to understand the structural differences in sense and antisense microRNA-21 by hybridizing them with complementary RNA and DNA oligos. Overall, the results suggest that the changes in the Raman band at 785 cm -1 originating from the phosphodiester bond of the nucleic acid backbone, linking 5' phosphate of the nucleic acid with 3' OH of the other nucleotide, can serve as a marker to identify these structural variations. Our results support the application of Raman spectroscopy in discerning intramolecular (ssRNA and ssDNA) and intermolecular (RNA-RNA, RNA-DNA, and DNA-DNA hybrids) interactions of nucleic acids. This is potentially useful for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with robust functionality.

Since the fat content of pork is a deciding factor in meat quality grading, the use of a noninvasive subcutaneous probe for real-time in situ monitoring of the fat components is of importance to vendors and other interested parties. In this work, we developed a spectroscopic method using a fiber-optic probe for subcutaneous fat analysis that utilizes spatially offset Raman spectroscopy (SORS). Here, normalized Raman spectra were acquired as a function of spatial offset, and the relative composition of fat-to-skin was determined. We found that the Raman intensity ratio varied disproportionately depending on the fat content and that the variations of the slope were correlated to the thickness of the fat layer. Furthermore, ordinary least square (OLS) regression using two components indicated that the depth-resolved SORS spectra reflected the relative thickness of the fat layer. We concluded that the local distribution of subcutaneous fat could be measured noninvasively using a pair of fiber-optic probes.

Noninvasive blood glucose monitoring has been a long-standing dream in diabetes management. The use of Raman spectroscopy, with its molecular specificity, has been investigated in this regard over the past decade. Previous studies reported on glucose sensing based on indirect evidence such as statistical correlation to the reference glucose concentration. However, these claims fail to demonstrate glucose Raman peaks, which has raised questions regarding the effectiveness of Raman spectroscopy for glucose sensing. Here, we demonstrate the first direct observation of glucose Raman peaks from in vivo skin. The signal intensities varied proportional to the reference glucose concentrations in three live swine glucose clamping experiments. Tracking spectral intensity based on linearity enabled accurate prospective prediction in within-subject and intersubject models. Our direct demonstration of glucose signal may quiet the long debate about whether glucose Raman spectra can be measured in vivo in transcutaneous glucose sensing., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Cancers of the gastrointestinal (GI) tract are among the most frequent and most lethal cancers worldwide. An important reason for this high mortality is that early disease is typically asymptomatic, and patients often present with advanced, incurable disease. Even in high-risk patients who routinely undergo endoscopic screening, lesions can be missed due to their small size or subtle appearance. Thus, current imaging approaches lack the sensitivity and specificity to accurately detect incipient GI tract cancers. Here we report our finding that a single dose of a high-sensitivity surface-enhanced resonance Raman scattering nanoparticle (SERRS-NP) enables reliable detection of precancerous GI lesions in animal models that closely mimic disease development in humans. Some of these animal models have not been used previously to evaluate imaging probes for early cancer detection. The studies were performed using a commercial Raman imaging system, a newly developed mouse Raman endoscope, and finally a clinically applicable Raman endoscope for larger animal studies. We show that this SERRS-NP-based approach enables robust detection of small, premalignant lesions in animal models that faithfully recapitulate human esophageal, gastric, and colorectal tumorigenesis. This method holds promise for much earlier detection of GI cancers than currently possible and could lead therefore to marked reduction of morbidity and mortality of these tumor types.

The authors would like to bring to the reader's attention that the Clarke error grid plot presented in Fig. 3 was generated using codes adapted from following reference.

Many disease states are associated with cellular biomechanical changes as markers. Label-free phase microscopes are used to quantify thermally driven interface fluctuations, which allow the deduction of important cellular rheological properties. Here, the spatio-temporal coherence of light was used to implement a high-speed reflection phase microscope with superior depth selectivity and higher phase sensitivity. Nanometric scale motion of cytoplasmic structures can be visualized with fine details and three-dimensional resolution. Specifically, the spontaneous fluctuation occurring on the nuclear membrane of a living cell was observed at video rate. By converting the reflection phase into displacement, the sensitivity in quantifying nuclear membrane fluctuation was found to be about one nanometer. A reflection phase microscope can potentially elucidate biomechanical mechanisms of pathological and physiological processes.

We describe a label-free approach based on Raman spectroscopy, to study drug-induced apoptosis in vivo. Spectral-shifts at wavenumbers associated with DNA, proteins, lipids, and collagen have been identified on breast and melanoma tumor tissues. These findings may enable a new analytical method for rapid readout of drug-therapy with miniaturized probes.

Optical monitoring of blood glucose levels for non-invasive diagnosis is a growing area of research. Recent efforts in this direction have been inclined towards reducing the requirement of calibration framework. Here, we are presenting a systematic investigation on the influence of variation in the ratio of calibration and validation points on the prospective predictive accuracy of spectral models. A fiber-optic probe coupled Raman system has been employed for transcutaneous measurements. Limit of agreement analysis between serum and partial least square regression predicted spectroscopic glucose values has been performed for accurate comparison. Findings are suggestive of strong predictive accuracy of spectroscopic models without requiring substantive calibration measurements. Graphical abstract.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in developed countries [1]. A subset of individuals with NAFLD progress to non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD which predisposes individuals to cirrhosis, liver failure and hepatocellular carcinoma. The current gold standard for NASH diagnosis and staging is based on histological evaluation, which is largely semi-quantitative and subjective. To address the need for an automated and objective approach to NASH detection, we combined Raman micro-spectroscopy and machine learning techniques to develop a classification model based on a well-established NASH mouse model, using spectrum pre-processing, biochemical component analysis (BCA) and logistic regression. By employing a selected pool of biochemical components, we identified biochemical changes specific to NASH and show that the classification model is capable of accurately detecting NASH (AUC=0.85-0.87) in mice. The unique biochemical fingerprint generated in this study may serve as a useful criterion to be leveraged for further validation in clinical samples., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Minimizing morbidities and mortalities associated with skin cancers requires sustained research with the goal of obtaining fresh insights into disease onset and progression under specific stimuli, particularly the influence of ultraviolet rays. In the present study, label-free profiling of skin fibroblasts exposed to time-bound ultra-violet radiation has been performed using quantitative phase imaging and Raman spectroscopy. Statistically significant differences in quantifiable biophysical parameters, such as matter density and cell dry mass, were observed with phase imaging. Accurate estimation of changes in the biochemical constituents, notably nucleic acids and proteins, was demonstrated through a combination of Raman spectroscopy and multivariate analysis of spectral patterns. Overall, the findings of this study demonstrate the promise of these non-perturbative optical modalities in accurately identifying cellular phenotypes and responses to external stimuli by combining molecular and biophysical information.

Molecular characterization of organ-specific metastatic lesions, which distinguish them from the primary tumor, will provide a better understanding of tissue specific adaptations that regulate metastatic progression. Using an orthotopic xenograft model, we have isolated isogenic metastatic human breast cancer cell lines directly from organ explants that are phenotypically distinct from the primary tumor cell line. Label-free Raman spectroscopy was used and informative spectral bands were ascertained as differentiators of organ-specific metastases as opposed to the presence of a single universal marker. Decision algorithms derived from the Raman spectra unambiguously identified these isogenic cell lines as unique biological entities - a finding reinforced through metabolomic analyses that indicated tissue of origin metabolite distinctions between the cell lines. Notably, complementarity of the metabolomics and Raman datasets was found. Our findings provide evidence that metastatic spread generates tissue-specific adaptations at the molecular level within cancer cells, which can be differentiated with Raman spectroscopy.

Due to its label-free and non-destructive nature, applications of Raman spectroscopic imaging in monitoring therapeutic responses at the cellular level are growing. We have recently developed a high-speed confocal Raman microscopy system to image living biological specimens with high spatial resolution and sensitivity. In the present study, we have applied this system to monitor the effects of Bortezomib, a proteasome inhibitor drug, on multiple myeloma cells. Cluster imaging followed by spectral profiling suggest major differences in the nuclear and cytoplasmic contents of cells due to drug treatment that can be monitored with Raman spectroscopy. Spectra were also acquired from group of cells and feasibility of discrimination among treated and untreated cells using principal component analysis (PCA) was accessed. Findings support the feasibility of Raman technologies as an alternate, novel method for monitoring live cell dynamics with minimal external perturbation., Competing Interests: The authors declare no conflict of interest.

Background: Neuraxial anesthesia and epidural steroid injection techniques require precise anatomical targeting to ensure successful and safe analgesia. Previous studies suggest that only some of the tissues encountered during these procedures can be identified by spectroscopic methods, and no previous study has investigated the use of Raman, diffuse reflectance, and fluorescence spectroscopies. The authors hypothesized that real-time needle-tip spectroscopy may aid epidural needle placement and tested the ability of spectroscopy to distinguish each of the tissues in the path of neuraxial needles., Methods: For comparison of detection methods, the spectra of individual, dissected ex vivo paravertebral and neuraxial porcine tissues were collected using Raman spectroscopy (RS), diffuse reflectance spectroscopy, and fluorescence spectroscopy. Real-time spectral guidance was tested using a 2-mm inner-diameter fiber-optic probe-in-needle device. Raman spectra were collected during the needle's passage through intact paravertebral and neuraxial porcine tissue and analyzed afterward. The RS tissue signatures were verified as mapping to individual tissue layers using histochemical staining and widefield microscopy., Results: RS revealed a unique spectrum for all ex vivo paravertebral and neuraxial tissue layers; diffuse reflectance spectroscopy and fluorescence spectroscopy were not distinct for all tissues. Moreover, when accounting for the expected order of tissues, real-time Raman spectra recorded during needle insertion also permitted identification of each paravertebral and neuraxial porcine tissue., Conclusions: This study demonstrates that RS can distinguish the tissues encountered during epidural needle insertion. This technology may prove useful during needle placement by providing evidence of its anatomical localization.

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.

Despite its widespread prevalence, middle ear pathology, especially the development of proliferative lesions, remains largely unexplored and poorly understood. Diagnostic evaluation is still predicated upon a high index of clinical suspicion on otoscopic examination of gross morphologic features. We report the first technique that has the potential to non-invasively identify two key lesions, namely cholesteatoma and myringosclerosis, by providing real-time information of differentially expressed molecules. In addition to revealing signatures consistent with the known pathobiology of these lesions, our observations provide the first evidence of the presence of carbonate- and silicate-substitutions in the calcium phosphate plaques found in myringosclerosis. Collectively, these results demonstrate the potential of Raman spectroscopy to not only provide new understanding of the etiology of these conditions by defining objective molecular markers but also aid in margin assessment to improve surgical outcome.

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.

We report a method to achieve high speed and high resolution live cell Raman images using small spherical gold nanoparticles with highly narrow intra-nanogap structures responding to NIR excitation (785 nm) and high-speed confocal Raman microscopy. The three different Raman-active molecules placed in the narrow intra-nanogap showed a strong and uniform Raman intensity in solution even under transient exposure time (10 ms) and low input power of incident laser (200 μW), which lead to obtain high-resolution single cell image within 30 s without inducing significant cell damage. The high resolution Raman image showed the distributions of gold nanoparticles for their targeted sites such as cytoplasm, mitochondria, or nucleus. The high speed Raman-based live cell imaging allowed us to monitor rapidly changing cell morphologies during cell death induced by the addition of highly toxic KCN solution to cells. These results strongly suggest that the use of SERS-active nanoparticle can greatly improve the current temporal resolution and image quality of Raman-based cell images enough to obtain the detailed cell dynamics and/or the responses of cells to potential drug molecules.

By using the depth selective imaging method, we studied the UV induced change in a photomobile liquid crystalline polymer film. With 1 μm depth resolution, each slice inside the film was selectively observed. A network-like structure mixed with the ordered and disordered regions of molecules in the middle of the film, and a rubbed polymer layer at the bottom of the film were observed. In each slice of the film, the phase change induced by UV light was observed strongly dependent on the director direction, which indicates the ordering change of the liquid crystalline molecules in the director direction. It took several tens of seconds for the ordering change caused by the collaborative interaction between the molecules. Furthermore, it was suggested that the UV induced change travelled from the bottom layer to the middle layer on the micron order.

A simple and practical method to measure three-dimensional (3-D) refractive index (RI) distributions of biological cells is presented. A common-path self-reference interferometry consisting of a compact set of polarizers is attached to a conventional inverted microscope equipped with a beam scanning unit, which can precisely measure multiple 2-D holograms of a sample with high phase stability for various illumination angles, from which accurate 3-D optical diffraction tomograms of the sample can be reconstructed. 3-D RI tomograms of nonbiological samples such as polystyrene microspheres, as well as biological samples including human red blood cells and breast cancer cells, are presented.

Surface-enhanced Raman spectroscopy (SERS) has evolved from an esoteric physical phenomenon to a robust and effective analytical method recently. The need of addressing both the field enhancement and the extinction of nanoparticle suspensions, however, has been underappreciated despite its substantive impact on the sensing performance. A systematic experimental investigation of SERS enhancement and attenuation is performed in suspensions of gold nanostars, which exhibit a markedly different behavior in relation to conventional nanoparticles. The relationship is elucidated between the SERS enhancement and the localized surface plasmon resonance band, and the effect of the concentration of the gold nanostars on the signal propagation is investigated. It is shown that an optimal concentration of gold nanostars exists to maximize the enhancement factor (EF), and the maximum EF occurs when the LSPR band is blue-shifted from the excitation wavelength rather than at the on-resonance position., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer - a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene - in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation.

A simple approach to enhancing the activity and stability of organophosphorus hydrolase (OPH) is developed based on interactions between the hydrophobic poly(propylene oxide) (PPO) block of amphiphilic Pluronics and the enzyme. This strategy provides an efficient route to new formulations for decontaminating organophosphate neurotoxins.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

Carbon nanotube uptake was measured via high-speed confocal Raman imaging in live cells. Spatial and temporal tracking of two cell-intrinsic and nine nanotube-derived Raman bands was conducted simultaneously in RAW 264.7 macrophages. Movies resolved single (n, m) species, defects, and aggregation states of nanotubes transiently as well as the cell position, denoted by lipid and protein signals. This work portends the real-time molecular imaging of live cells and tissues using Raman spectroscopy, affording multiplexing and complete photostability.

We present an imaging modality capable of providing high-speed optical dispersion measurements of live cells. The technique permits wide-field measurement of the optical phase shifts introduced by a sample for multiple discrete wavelengths in a single image capture. Utilizing spatial modulation and the wavelength dependence of the interference-fringe spacing, average refractive index as a function of wavelength is obtained, yielding optical dispersion measurements of the sample under observation. Because of its simple and low-cost design, the technique can be readily integrated into a standard microscope to collect additional diagnostic information about biological cells.

In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the nonenzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and nonglycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision--with a limit of detection that is a factor of ~15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multicomponent mixtures and potentially in hemolysate and whole blood lysate samples.

There continues to be a significant clinical need for rapid and reliable intraoperative margin assessment during cancer surgery. Here we describe a portable, quantitative, optical fiber probe-based, spectroscopic tissue scanner designed for intraoperative diagnostic imaging of surgical margins, which we tested in a proof of concept study in human tissue for breast cancer diagnosis. The tissue scanner combines both diffuse reflectance spectroscopy (DRS) and intrinsic fluorescence spectroscopy (IFS), and has hyperspectral imaging capability, acquiring full DRS and IFS spectra for each scanned image pixel. Modeling of the DRS and IFS spectra yields quantitative parameters that reflect the metabolic, biochemical and morphological state of tissue, which are translated into disease diagnosis. The tissue scanner has high spatial resolution (0.25 mm) over a wide field of view (10 cm × 10 cm), and both high spectral resolution (2 nm) and high spectral contrast, readily distinguishing tissues with widely varying optical properties (bone, skeletal muscle, fat and connective tissue). Tissue-simulating phantom experiments confirm that the tissue scanner can quantitatively measure spectral parameters, such as hemoglobin concentration, in a physiologically relevant range with a high degree of accuracy (<5% error). Finally, studies using human breast tissues showed that the tissue scanner can detect small foci of breast cancer in a background of normal breast tissue. This tissue scanner is simpler in design, images a larger field of view at higher resolution and provides a more physically meaningful tissue diagnosis than other spectroscopic imaging systems currently reported in literatures. We believe this spectroscopic tissue scanner can provide real-time, comprehensive diagnostic imaging of surgical margins in excised tissues, overcoming the sampling limitation in current histopathology margin assessment. As such it is a significant step in the development of a platform technology for intraoperative management of cancer, a clinical problem that has been inadequately addressed to date.

We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications including nephropathy and retinopathy. In this article, we propose and evaluate the efficacy of Raman spectroscopy for determination of this important analyte. By utilizing the pre-concentration obtained through drop-coating deposition, we show that glycation of albumin leads to subtle, but consistent, changes in vibrational features, which with the help of multivariate classification techniques can be used to discriminate glycated albumin from the unglycated variant with 100% accuracy. Moreover, we demonstrate that the calibration model developed on the glycated albumin spectral dataset shows high predictive power, even at substantially lower concentrations than those typically encountered in clinical practice. In fact, the limit of detection for glycated albumin measurements is calculated to be approximately four times lower than its minimum physiological concentration. Importantly, in relation to the existing detection methods for glycated albumin, the proposed method is also completely reagent-free, requires barely any sample preparation and has the potential for simultaneous determination of glycated hemoglobin levels as well. Given these key advantages, we believe that the proposed approach can provide a uniquely powerful tool for quantification of glycation status of proteins in biopharmaceutical development as well as for glycemic marker determination in routine clinical diagnostics in the future.