Dual modal spectroscopic tissue scanner for colorectal cancer diagnosis.

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.

Spectrochemical Probing of MicroRNA Duplex Using Spontaneous Raman Spectroscopy for Biosensing Applications.

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.

Analysis of subcutaneous swine fat via deep Raman spectroscopy using a fiber-optic probe.

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.

Direct observation of glucose fingerprint using in vivo Raman spectroscopy.

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.

In vivo detection of drug-induced apoptosis in tumors using Raman spectroscopy.

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.

Label-free spectrochemical probe for determination of hemoglobin glycation in clinical blood samples.

Glycated hemoglobin, HbA1c, is an important biomarker that reveals the average value of blood glucose over the preceding 3 months. While significant recent attention has been focused on the use of optical and direct molecular spectroscopic methods for determination of HbA1c, a facile test that minimizes sample preparation needs and turnaround time still remains elusive. Here, we report a label-free approach for identifying low, mid and high-HbA1c groups in hemolysate and in whole blood samples featuring resonance Raman (RR) spectroscopy and support vector machine (SVM)-based classification of spectral patterns. The diagnostic power of RR measurements stems from its selective enhancement of hemoglobin-specific features, which simultaneously minimizes the blood matrix spectral interference and permits detection in the native solution. In this pilot study, our spectroscopic observations reveal that glycation of hemoglobin results in subtle but reproducible changes even when detected in the whole blood matrix. Leveraging SVM analysis of the principal component scores determined from the RR spectra, we show high degree of accuracy in classifying clinical specimen. We envisage that the promising findings will pave the way for more extensive clinical specimen investigations with the ultimate goal of translating molecular spectroscopy for routine point-of-care testing.

Differential diagnosis of otitis media with effusion using label-free Raman spectroscopy: A pilot study.

Otitis media with effusion (OME) is an important and common condition affecting hearing in pediatric patients characterized by the presence of fluid in the middle ear space. The fluid is normally described as serous or mucoid based on differences in the fluid viscosity. The differential diagnosis of two OMEs, namely serous and mucoid is of significant clinical value because while the former is self-limiting, surgical procedure is commonly required for the latter. However, accurate identification of fluid types remains a challenging target unattainable with current clinical modalities due to unavailability of nonperturbative molecular tools. Here, we report an emerging spectroscopy approach featuring Raman scattering and multivariate analysis of spectral patterns to discern serous and mucoid fluids, obtained from pediatric patients undergoing myringotomy and tube placement, by providing information of differentially expressed molecules with high specificity. We demonstrate the feasibility of Raman spectroscopy-based approach to categorize middle ear effusion based on the characteristic spectral markers, notably of mucin, with classification accuracy of 91% and 93% for serous and mucoid, respectively. Our findings pave the way for further development of such a tool for fully noninvasive application that will lead to objective and accurate diagnosis thereby reducing unnecessary visits and surgical procedures.

Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy.

Unlike most optical coherence microscopy (OCM) systems, dynamic speckle-field interferometric microscopy (DSIM) achieves depth sectioning through the spatial-coherence gating effect. Under high numerical aperture (NA) speckle-field illumination, our previous experiments have demonstrated less than 1 µm depth resolution in reflection-mode DSIM, while doubling the diffraction limited resolution as under structured illumination. However, there has not been a physical model to rigorously describe the speckle imaging process, in particular explaining the sectioning effect under high illumination and imaging NA settings in DSIM. In this paper, we develop such a model based on the diffraction tomography theory and the speckle statistics. Using this model, we calculate the system response function, which is used to further obtain the depth resolution limit in reflection-mode DSIM. Theoretically calculated depth resolution limit is in an excellent agreement with experiment results. We envision that our physical model will not only help in understanding the imaging process in DSIM, but also enable better designing such systems for depth-resolved measurements in biological cells and tissues.

Ticagrelor Removal From Human Blood.

The authors devised an efficient method for ticagrelor removal from blood using sorbent hemadsorption. Ticagrelor removal was measured in 2 sets of in vitro experiments. The first set was a first-pass experiment using bovine serum albumin (BSA) solution pre-incubated with ticagrelor, whereas the second set, performed in a recirculating manner, used human blood mixed with ticagrelor. Removal of ticagrelor from BSA solution reached values >99%. The peak removal rate was 99% and 94% from whole blood and 99.99% and 90% from plasma during 10 h and 3 to 4 h of recirculating experiments, respectively. In conclusion, hemadsorption robustly removes ticagrelor from BSA solution and human blood samples.

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.

Raman Spectroscopy Differentiates Each Tissue from the Skin to the Spinal Cord: A Novel Method for Epidural Needle Placement?

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.

Digital micromirror device-based laser-illumination Fourier ptychographic microscopy.

We report a novel approach to Fourier ptychographic microscopy (FPM) by using a digital micromirror device (DMD) and a coherent laser source (532 nm) for generating spatially modulated sample illumination. Previously demonstrated FPM systems are all based on partially-coherent illumination, which offers limited throughput due to insufficient brightness. Our FPM employs a high power coherent laser source to enable shot-noise limited high-speed imaging. For the first time, a digital micromirror device (DMD), imaged onto the back focal plane of the illumination objective, is used to generate spatially modulated sample illumination field for ptychography. By coding the on/off states of the micromirrors, the illumination plane wave angle can be varied at speeds more than 4 kHz. A set of intensity images, resulting from different oblique illuminations, are used to numerically reconstruct one high-resolution image without obvious laser speckle. Experiments were conducted using a USAF resolution target and a fiber sample, demonstrating high-resolution imaging capability of our system. We envision that our approach, if combined with a coded-aperture compressive-sensing algorithm, will further improve the imaging speed in DMD-based FPM systems.

Less is more: Avoiding the LIBS dimensionality curse through judicious feature selection for explosive detection.

Despite its intrinsic advantages, translation of laser induced breakdown spectroscopy for material identification has been often impeded by the lack of robustness of developed classification models, often due to the presence of spurious correlations. While a number of classifiers exhibiting high discriminatory power have been reported, efforts in establishing the subset of relevant spectral features that enable a fundamental interpretation of the segmentation capability and avoid the 'curse of dimensionality' have been lacking. Using LIBS data acquired from a set of secondary explosives, we investigate judicious feature selection approaches and architect two different chemometrics classifiers -based on feature selection through prerequisite knowledge of the sample composition and genetic algorithm, respectively. While the full spectral input results in classification rate of ca.92%, selection of only carbon to hydrogen spectral window results in near identical performance. Importantly, the genetic algorithm-derived classifier shows a statistically significant improvement to ca. 94% accuracy for prospective classification, even though the number of features used is an order of magnitude smaller. Our findings demonstrate the impact of rigorous feature selection in LIBS and also hint at the feasibility of using a discrete filter based detector thereby enabling a cheaper and compact system more amenable to field operations.

High resolution live cell Raman imaging using subcellular organelle-targeting SERS-sensitive gold nanoparticles with highly narrow intra-nanogap.

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.

Emerging trends in optical sensing of glycemic markers for diabetes monitoring.

In the past decade, considerable attention has been focused on the measurement of glycemic markers, such as glycated hemoglobin and glycated albumin, that provide retrospective indices of average glucose levels in the bloodstream. While these biomarkers have been regularly used to monitor long-term glucose control in established diabetics, they have also gained traction in diabetic screening. Detection of such glycemic markers is challenging, especially in a point-of-care setting, due to the stringent requirements for sensitivity and robustness. A number of non-separation based measurement strategies were recently proposed, including photonic tools that are well suited to reagent-free marker quantitation. Here, we critically review these methods while focusing on vibrational spectroscopic methods, which offer highly specific molecular fingerprinting capability. We examine the underlying principles and the utility of these approaches as reagentless assays capable of multiplexed detection of glycemic markers and also the challenges in their eventual use in the clinic.

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

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, RI 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 RI maps of the samples from the measured spectra. Using this method, we demonstrate label-free 3-D 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 3-D refractive index maps. Our results show that the reported method, alone or in combination with the existing flow cytometry techniques, promises as a quantitative tool for stain-free characterization of large number of cells.

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.

Dynamic speckle illumination wide-field reflection phase microscopy.

We demonstrate a quantitative reflection-phase microscope based on time-varying speckle-field illumination. Due to the short spatial coherence length of the speckle field, the proposed imaging system features superior lateral resolution, 520 nm, as well as high-depth selectivity, 1.03 µm. Off-axis interferometric detection enables wide-field and single-shot imaging appropriate for high-speed measurements. In addition, the measured phase sensitivity of this method, which is the smallest measurable axial motion, is more than 40 times higher than that available using a transmission system. We demonstrate the utility of our method by successfully distinguishing the motion of the top surface from that of the bottom in red blood cells. The proposed method will be useful for studying membrane dynamics in complex eukaryotic cells.

Multiwavelength fluorescence otoscope for video-rate chemical imaging of middle ear pathology.

A common motif in otolaryngology is the lack of certainty regarding diagnosis for middle ear conditions, resulting in many patients being overtreated under the worst-case assumption. Although pneumatic otoscopy and adjunctive tests offer additional information, white light otoscopy has been the main tool for diagnosis of external auditory canal and middle ear pathologies for over a century. In middle ear pathologies, the inability to avail high-resolution structural and/or molecular imaging is particularly glaring, leading to a complicated and erratic decision analysis. Here, we propose a novel multiwavelength fluorescence-based video-rate imaging strategy that combines readily available optical elements and software components to create a novel otoscopic device. This modified otoscope enables low-cost, detailed and objective diagnosis of common middle ear pathological conditions. Using the detection of congenital cholesteatoma as a specific example, we demonstrate the feasibility of fluorescence imaging to differentiate this proliferative lesion from uninvolved middle ear tissue based on the characteristic autofluorescence signals. Availability of real-time, wide-field chemical information should enable more complete removal of cholesteatoma, allowing for better hearing preservation and substantially reducing the well-documented risks, costs and psychological effects of repeated surgical procedures.

Three-dimensional imaging of cavity vacuum with single atoms localized by a nanohole array.

Zero-point electromagnetic fields were first introduced to explain the origin of atomic spontaneous emission. Vacuum fluctuations associated with the zero-point energy in cavities are now utilized in quantum devices such as single-photon sources, quantum memories, switches and network nodes. Here we present three-dimensional (3D) imaging of vacuum fluctuations in a high-Q cavity based on the measurement of position-dependent emission of single atoms. Atomic position localization is achieved by using a nanoscale atomic beam aperture scannable in front of the cavity mode. The 3D structure of the cavity vacuum is reconstructed from the cavity output. The root mean squared amplitude of the vacuum field at the antinode is also measured to be 0.92±0.07 V cm(-1). The present work utilizing a single atom as a probe for sub-wavelength imaging demonstrates the utility of nanometre-scale technology in cavity quantum electrodynamics.