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.

Correction to: Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

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.

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.

Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

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.

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.

Organ-specific isogenic metastatic breast cancer cell lines exhibit distinct Raman spectral signatures and metabolomes.

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.

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.

Identification of morphological and biochemical changes in keratin-8/18 knock-down cells using Raman spectroscopy.

Accurate understanding of cellular processes and responses to stimuli is of paramount importance in biomedical research and diagnosis. Raman spectroscopy (RS), a label-free and nondestructive spectroscopic method has the potential to serve as a novel 'theranostics' tool. Both fiber-optic and micro-Raman studies have demonstrated efficacy in diagnostics and therapeutic response monitoring. In the present study, we have evaluated the potential of micro-Raman spectroscopic maps in identifying changes induced by loss of K8/18 proteins in a tongue cancer cell line. Furthermore, we also evaluated the efficacy of less expensive and commercially available fiber probes to identify K8/18 wild and knock-down cell pellets, in view of the utility of cell pellet-based studies. The findings suggest that major differences in the cellular morphology and biochemical composition can be objectively identified and can be utilized for classification using both micro-Raman and fiber-probe-based RS. These findings highlight the potential of fiber-optic probe-based RS in noninvasive cellular phenotyping for diagnosis and therapeutic response monitoring, especially in low-resource settings.

Noninvasive Monitoring of Blood Glucose with Raman Spectroscopy.

The successful development of a noninvasive blood glucose sensor that can operate reliably over sustained periods of time has been a much sought after but elusive goal in diabetes management. Since diabetes has no well-established cure, control of elevated glucose levels is critical for avoiding severe secondary health complications in multiple organs including the retina, kidney and vasculature. While fingerstick testing continues to be the mainstay of blood glucose detection, advances in electrochemical sensing-based minimally invasive approaches have opened the door for alternate methods that would considerably improve the quality of life for people with diabetes. In the quest for better sensing approaches, optical technologies have surfaced as attractive candidates as researchers have sought to exploit the endogenous contrast of glucose, notably its absorption, scattering, and polarization properties. Vibrational spectroscopy, especially spontaneous Raman scattering, has exhibited substantial promise due to its exquisite molecular specificity and minimal interference of water in the spectral profiles acquired from the blood-tissue matrix. Yet, it has hitherto been challenging to leverage the Raman scattering signatures of glucose for prediction in all but the most basic studies and under the least demanding conditions. In this Account, we discuss the newly developed array of methodologies that address the key challenges in measuring blood glucose accurately using Raman spectroscopy and unlock new prospects for translation to sustained noninvasive measurements in people with diabetes. Owing to the weak intensity of spontaneous Raman scattering, recent research has focused on enhancement of signals from the blood constituents by designing novel excitation-collection geometries and tissue modulation methods while our attempts have led to the incorporation of nonimaging optical elements. Additionally, invoking mass transfer modeling into chemometric algorithms has not only addressed the physiological lag between the actual blood glucose and the measured interstitial fluid glucose values but also offered a powerful tool for predictive measurements of hypoglycemia. This framework has recently been extended to provide longitudinal tracking of glucose concentration without necessitating extensive a priori concentration information. These findings are advanced by the results of recent glucose tolerance studies in human subjects, which also hint at the need for designing nonlinear calibration models that can account for subject-to-subject variations in skin heterogeneity and hematocrit levels. Together, the emerging evidence underscores the promise of a blood withdrawal-free optical platform-featuring a combination of high-throughput Raman spectroscopic instrumentation and data analysis of subtle variations in spectral expression-for diabetes screening in the clinic and, ultimately, for personalized monitoring.

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.

Objective identification of dental abnormalities with multispectral fluorescence imaging.

Sensitive methods that can enable early detection of dental diseases (caries and calculus) are desirable in clinical practice. Optical spectroscopic approaches have emerged as promising alternatives owing to their wealth of molecular information and lack of sample preparation requirements. In the present study, using multispectral fluorescence imaging, we have demonstrated that dental caries and calculus can be objectively identified on extracted tooth. Spectral differences among control, carious and calculus conditions were attributed to the porphyrin pigment content, which is a byproduct of bacterial metabolism. Spectral maps generated using different porphyrin bands offer important clues to the spread of bacterial infection. Statistically significant differences utilizing fluorescence intensity ratios were observed among three groups. In contrast to laser induced fluorescence, these methods can provide information about exact spread of the infection and may aid in long term dental monitoring. Successful adoption of this approach for routine clinical usage can assist dentists in implementing timely remedial measures.

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.

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay.

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.

Discerning the differential molecular pathology of proliferative middle ear lesions using Raman spectroscopy.

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.