Non-Gated Laser Induced Breakdown Spectroscopy Provides a Powerful Segmentation Tool on Concomitant Treatment of Characteristic and Continuum Emission

We demonstrate the application of non-gated laser induced breakdown spectroscopy (LIBS) for characterization and classification of organic materials with similar chemical composition. While use of such a system introduces substantive continuum background in the spectral dataset, we show that appropriate treatment of the continuum and characteristic emission results in accurate discrimination of pharmaceutical formulations of similar stoichiometry. Specifically, our results suggest that near-perfect classification can be obtained by employing suitable multivariate analysis on the acquired spectra, without prior removal of the continuum background. Indeed, we conjecture that pre-processing in the form of background removal may introduce spurious features in the signal. Our findings in this report significantly advance the prior results in time-integrated LIBS application and suggest the possibility of a portable, non-gated LIBS system as a process analytical tool, given its simple instrumentation needs, real-time capability and lack of sample preparation requirements.National Institute for Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1

Raman Spectroscopy Provides a Powerful Diagnostic Tool for Accurate Determination of Albumin Glycation

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.National Center for Research Resources (U.S.) (Grant No. P41-RR02594)Massachusetts Institute of Technology. Laser Biomedical Research Cente

Spectroscopic approach for dynamic bioanalyte tracking with minimal concentration information

Vibrational spectroscopy has emerged as a promising tool for non-invasive, multiplexed measurement of blood constituents - an outstanding problem in biophotonics. Here, we propose a novel analytical framework that enables spectroscopy-based longitudinal tracking of chemical concentration without necessitating extensive a priori concentration information. The principal idea is to employ a concentration space transformation acquired from the spectral information, where these estimates are used together with the concentration profiles generated from the system kinetic model. Using blood glucose monitoring by Raman spectroscopy as an illustrative example, we demonstrate the efficacy of the proposed approach as compared to conventional calibration methods. Specifically, our approach exhibits a 35% reduction in error over partial least squares regression when applied to a dataset acquired from human subjects undergoing glucose tolerance tests. This method offers a new route at screening gestational diabetes and opens doors for continuous process monitoring without sample perturbation at intermediate time points.National Institute for Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-27)Kwansei Gakuin University (Grant 126004

Investigating Effects of Proteasome Inhibitor on Multiple Myeloma Cells Using Confocal Raman Microscopy

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

Three-Dimensional Holographic Refractive-Index Measurement of Continuously Flowing Cells in a Microfluidic Channel

The refractive index of biological specimens is a source of intrinsic contrast that can be explored without any concerns of photobleaching or harmful effects caused by extra contrast agents. In addition, the refractive index contains rich information related to the metabolism of cells at the cellular and subcellular levels. Here, we report a no-moving-parts approach that provides three-dimensional refractive-index maps of biological samples continuously flowing in a microfluidic channel. Specifically, we use line illumination and off-axis digital holography to record the angular spectra of light scattered from flowing samples at high speed. Applying the scalar diffraction theory, we obtain accurate refractive-index maps of the samples from the measured spectra. Using this method, we demonstrate label-free three-dimensional imaging of live RKO human colon cancer cells and RPMI8226 multiple myeloma cells, and obtain the volume, dry mass, and density of these cells from the measured three-dimensional refractive-index maps. Our results show that the reported method, alone or in combination with the existing flow cytometry techniques, shows promise as a quantitative tool for stain-free characterization of a large number of cells.National Institutes of Health (U.S.) (9P41EB015871-26A1)National Institutes of Health (U.S.) (P41 EB002503)Hamamatsu Corporatio

A graphene-based physiometer array for the analysis of single biological cells

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.National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)U.S. Army Research LaboratoryUnited States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant P41EB015871-27)Skolkovo Institute of Science and Technolog

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Errata: Observation of dynamic subdomains in red blood cells

This article was originally published online on 18 July 2006 without Michael S. Feld in the author list. In addition, there were errors in Eq. 3. The corrected equation appears below. All online versions of the article were corrected on October 3, 2006

Errata: Observation of dynamic subdomains in red blood cells

This article was originally published online on 18 July 2006 without Michael S. Feld in the author list. In addition, there were errors in Eq. 3. The corrected equation appears below. All online versions of the article were corrected on October 3, 2006