The complementary value of intraoperative fluorescence imaging and Raman spectroscopy for cancer surgery: combining the incompatibles.

A clear margin is an important prognostic factor for most solid tumours treated by surgery. Intraoperative fluorescence imaging using exogenous tumour-specific fluorescent agents has shown particular benefit in improving complete resection of tumour tissue. However, signal processing for fluorescence imaging is complex, and fluorescence signal intensity does not always perfectly correlate with tumour location. Raman spectroscopy has the capacity to accurately differentiate between malignant and healthy tissue based on their molecular composition. In Raman spectroscopy, specificity is uniquely high, but signal intensity is weak and Raman measurements are mainly performed in a point-wise manner on microscopic tissue volumes, making whole-field assessment temporally unfeasible. In this review, we describe the state-of-the-art of both optical techniques, paying special attention to the combined intraoperative application of fluorescence imaging and Raman spectroscopy in current clinical research. We demonstrate how these techniques are complementary and address the technical challenges that have traditionally led them to be considered mutually exclusive for clinical implementation. Finally, we present a novel strategy that exploits the optimal characteristics of both modalities to facilitate resection with clear surgical margins.

Raman spectroscopic analysis of the molecular composition of oral cavity squamous cell carcinoma and healthy tongue tissue.

A Raman tissue spectrum is a quantitative representation of the overall molecular composition of that tissue. Raman spectra are often used as tissue fingerprints without further interpretation of the specific information that they contain about the tissue's molecular composition. In this study, we analyzed the differences in molecular composition between oral cavity squamous cell carcinoma (OCSCC) and healthy tissue structures in tongue, based on their Raman spectra. A total of 1087 histopathologically annotated spectra (142 OCSCC, 202 surface squamous epithelium, 61 muscle, 65 adipose tissue, 581 connective tissue, 26 gland, and 10 nerve) were obtained from Raman maps of 44 tongue samples from 21 patients. A characteristic, average spectrum of each tissue structure was fitted with a set of 55 pure-compound reference spectra, to define the best library of fit-spectra. Reference spectra represented proteins, lipids, nucleic acids, carbohydrates, amino acids and other miscellaneous molecules. A non-negative least-squares algorithm was used for fitting. Individual spectra per histopathological annotation were then fitted with this selected library in order to determine the molecular composition per tissue structure. The spectral contribution per chemical class was calculated. The results show that all characteristic tissue-type spectra could be fitted with a low residual of <4.82%. The content of carbohydrates, proteins and amino acids was the strongest discriminator between OCSCC and healthy tissue. The combination of carbohydrates, proteins and amino acids was used for a classification model of 'tumor' versus 'healthy tissue'. Validation of this model on an independent dataset showed a specificity of 93% at a sensitivity of 100%.

Determination of natural moisturizing factors in the skin: Raman microspectroscopy versus HPLC.

BACKGROUND: Natural moisturizing factor (NMF) is used as genotypic and phenotypic biomarker in diagnostics. This study is a side-to-side comparison of two different methods to determine NMF in atopic dermatitis patients: Raman microspectroscopy and stratum corneum tape stripping followed by HPLC. RESULTS: Measured NMF values were significantly correlated (R2 = .61; p < .0001), both methods demonstrated a concentration-depth dependence of NMF and reduced NMF levels in the carriers of filaggrin null mutations. Good agreement between measurements of left and right arms indicated robustness and good reproducibility of both methods. CONCLUSIONS: Both methods showed comparable performance, choice of method will rather be influenced by practical consideration.

Discrimination between oral cancer and healthy tissue based on water content determined by Raman spectroscopy.

Tumor-positive resection margins are a major problem in oral cancer surgery. High-wavenumber Raman spectroscopy is a reliable technique to determine the water content of tissues, which may contribute to differentiate between tumor and healthy tissue. The aim of this study was to examine the use of Raman spectroscopy to differentiate tumor from surrounding healthy tissue in oral squamous cell carcinoma. From 14 patients undergoing tongue resection for squamous cell carcinoma, the water content was determined at 170 locations on freshly excised tongue specimens using the Raman bands of the OH-stretching vibrations (3350-3550 cm(-1)) and of the CH-stretching vibrations (2910-2965 cm(-1)). The results were correlated with histopathological assessment of hematoxylin and eosin stained thin tissue sections obtained from the Raman measurement locations. The water content values from squamous cell carcinoma measurements were significantly higher than from surrounding healthy tissue (p-value < 0.0001). Tumor tissue could be detected with a sensitivity of 99% and a specificity of 92% using a cutoff water content value of 69%. Because the Raman measurements are fast and can be carried out on freshly excised tissue without any tissue preparation, this finding signifies an important step toward the development of an intraoperative tool for tumor resection guidance with the aim of enabling oncological radical surgery and improvement of patient outcome.

Lipid to protein ratio plays an important role in the skin barrier function in patients with atopic eczema.

BACKGROUND: The barrier function of the skin is primarily provided by the stratum corneum (SC), the outermost layer of the skin. Skin barrier impairment is thought to be a primary factor in the pathogenesis of atopic eczema (AE). Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, the role of filaggrin mutations in the decreased skin barrier in AE is not fully understood. It was recently shown that changes in SC lipid composition and organization play a role in the reduced skin barrier in AE. OBJECTIVES: To determine whether the lipid/protein ratio and the total dry SC mass per surface area are related to the skin barrier function of controls and patients with AE. METHODS: A case-control study was performed to compare nonlesional and lesional skin of AE with skin of controls. The dry SC mass was determined by tape-stripping and Squamescan(™) . The ratio between lipid and protein bands in the Raman spectrum was used to determine the lipid/protein ratio. Skin barrier function was assessed by transepidermal water loss. RESULTS: The results show that the dry SC mass per skin area is altered only in lesional SC of patients with AE compared with control subjects. The observed reduction in the lipid/protein ratio in SC of patients with AE was more pronounced, both in lesional and nonlesional SC and correlated strongly with the skin barrier function and disease severity. CONCLUSIONS: The lipid/protein ratio plays a role in the reduced skin barrier function in AE.

Raman spectroscopy with an integrated arrayed-waveguide grating.

An integrated arrayed-waveguide grating fabricated in silicon-oxynitride technology is applied to Raman spectroscopy. After its validation by reproducing the well-known spectrum of cyclohexane, polarized Raman spectra are measured of extracted human teeth containing localized initial carious lesions. Excellent agreement is obtained between the spectra of healthy and carious tooth enamel measured with our integrated device and spectra recorded using a conventional Raman spectrometer. Our results represent a step toward the realization of compact, hand-held, integrated spectrometers, e.g. for the detection of dental caries at an early stage.

Experimental study on needle insertion force to minimize tissue deformation in tongue tissue.

This study reports on the effects of insertion velocity, needle tip geometry and needle diameter on tissue deformation and maximum insertion force. Moreover, the effect of multiple insertions with the same needle on the maximum insertion force is reported. The tissue deformation and maximum insertion force strongly depend on the insertion velocity and the tip geometry. No correlation was found between the outer diameter and the maximum insertion force for small needles (30G - 32G). The endurance experiments showed no remarkable difference in the maximum insertion force during 100 insertions.

Proof of principle for successful characterization of methicillin-resistant coagulase-negative staphylococci isolated from skin by use of Raman spectroscopy and pulsed-field gel electrophoresis.

Coagulase-negative staphylococci (CNS) are among the most frequently isolated bacterial species in clinical microbiology, and most CNS-related infections are hospital acquired. Distinguishing between these frequently multiple-antibiotic-resistant isolates is important for both treatment and transmission control. In this study we used isolates of methicillin-resistant coagulase-negative staphylococci (MR-CNS) that were selected from a large surveillance study of the direct spread of MR-CNS. This strain collection was used to evaluate (i) Raman spectroscopy as a typing tool for MR-CNS isolates and (ii) diversity between colonies with identical and different morphologies. Reproducibility was high, with 215 of 216 (99.5%) of the replicate samples for 72 isolates ending up in the same cluster. The concordance with pulsed-field gel electrophoresis (PFGE)-based clusters was 94.4%. We also confirm that the skin of patients can be colonized with multiple MR-CNS types at the same time. Morphological differences between colonies from a single patient sample correlated with differences in Raman and PFGE types. Some morphologically indistinguishable colonies revealed different Raman and PFGE types. This indicates that multiple MR-CNS colonies should be examined to obtain a complete insight into the prevalence of different types and to be able to perform an accurate transmission analysis. Here we show that Raman spectroscopy is a reproducible typing system for MR-CNS isolates. It is a tool for screening variability within a collection of isolates. Because of the high throughput, it enables the analysis of multiple colonies per patient, which will enhance the quality of clinical and epidemiological studies.

Rapid identification of mycobacteria by Raman spectroscopy.

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.

Evaluation of bone resection margins of segmental mandibulectomy for oral squamous cell carcinoma.

Resection margins are frequently studied in patients with oral squamous cell carcinoma and are accepted as a constant prognostic factor. While most evidence is based on soft tissue margins, reported data for bone resection margins are scarce. The aim of this retrospective study was to evaluate and determine the utility of surgical margins in bone resections for oral cavity squamous cell carcinoma (OCSCC). The status of bone resection margins and their impact on survival was investigated in patients who had undergone segmental mandibulectomy for OCSCC. Medical records were retrieved for the years 2000-2012; 127 patients were identified and included in the study. Tumour-positive bone resection margins were found in 21% of the patients. The 5-year overall survival was significantly lower in this group (P<0.005). Therefore, there is a need for intraoperative feedback on the status of bone resection margins to enable immediate additional resection where necessary. Although the lack of intraoperative methods for the evaluation of bone tissue has been addressed by many authors, there is still no reliable method for widespread use. Future research should focus on an objective, accurate, and rapid method of intraoperative assessment for the entire bone resection margin to optimize patient outcomes.

Local variations in protein structure in the human eye lens: a Raman microspectroscopic study.

Confocal Raman microspectroscopy was used to monitor local and age-related changes in protein conformation in human eye lenses. In clear human lenses of varying age (range 17-80 years) spectra were recorded along the visual axis, using laser light of 660 nm wavelength. The Raman vibrations in the 650-1750 cm-1 spectral region were analyzed. Difference spectra between central core and different positions along the visual axis were calculated after calibration for protein content using the I(1450) cm-1 CH2/CH3 vibration peak. Tryptophan content was quantified using the peak at 760 cm-1 calibrated for protein. Changes in the 'exposed' vs. 'buried' position of tryptophan were analyzed using the peak heights at I(880) and I(760) cm-1. The difference spectra revealed an excess of tryptophan, tyrosine, phenylalanine, beta-sheet conformation and molecules or molecular groups responsible for a 1425 cm-1 peak in the core region in all lenses investigated. The excess peaks disappeared at about 0.6-0.9 mm below the surface. The tryptophan content increased from superficial to deep layers, levelling off between 0.4-0.8 mm below the surface. Upon aging, the tryptophan content increases in the core not in the cortex. No changes in the 'exposed' vs. 'buried' position of tryptophan were observed. Changes in tryptophan and tyrosine probably reflect the maturational shift from cortex to core in the relative content of alpha, beta and gamma crystallines. The age-related increase in tryptophan in the core may reflect the preferential breakdown by endo- and exopeptidases of alpha-crystallins damaged upon aging. The increase in beta-sheet conformation may indicate a post-translational shift in secondary conformation upon aging. These changes in protein conformation are largely completed in a small superficial zone, i.e., in the early life span of the crystallins.

Rapid identification of Candida spp. in peritonitis patients by Raman spectroscopy.

This prospective study evaluated Raman spectroscopy for the identification of clinically relevant Candida spp. in peritonitis patients. A Raman database was developed by measuring spectra from 93 reference strains belonging to ten different Candida spp. Clinical samples were obtained from the surgical department and intensive care unit of a tertiary university hospital. In total, 88 peritoneal specimens from 45 patients with primary, secondary or tertiary peritonitis were included. Specimens were cultured initially on a selective Sabouraud medium that contained gentamicin to suppress bacterial growth. For conventional identification, a chromogenic medium was used for presumptive identification, followed by use of the Vitek 2 system for definitive identification (requiring a total time of 48-96 h). Raman measurements were taken on overnight cultures from Sabouraud-gentamicin medium. Thirty-one samples were positive for Candida by culture. Using multivariate statistical analyses, a prediction accuracy of 90% was obtained for Raman spectroscopy, which appears to offer an accurate and rapid (12-24 h) alternative for the identification of Candida spp. in peritonitis patients. The reduced turn-around time is of great clinical importance for the treatment of critically ill patients with invasive candidiasis in intensive care units.

Tissue characterization using high wave number Raman spectroscopy.

Raman spectroscopy is a powerful diagnostic tool, enabling tissue identification and classification. Mostly, the so-called fingerprint (approximately 400-1800 cm(-1)) spectral region is used. In vivo application often requires small flexible fiber-optic probes, and is hindered by the intense Raman signal that is generated in the fused silica core of the fiber. This necessitates filtering of laser light, which is guided to the tissue, and of the scattered light collected from the tissue, leading to complex and expensive designs. Fused silica has no Raman signal in the high wave number region (2400-3800 cm(-1)). This enables the use of a single unfiltered fiber to guide laser light to the tissue and to collect scattered light in this spectral region. We show, by means of a comparison of in vitro Raman microspectroscopic maps of thin tissue sections (brain tumors, bladder), measured both in the high wave number region and in the fingerprint region, that essentially the same diagnostic information is obtained in the two wave number regions. This suggests that for many clinical applications the technological hurdle of designing and constructing suitable fiber-optic probes may be eliminated by using the high wave number region and a simple piece of standard optical fiber.

Raman spectroscopic evaluation of the effects of diet and lipid-lowering therapy on atherosclerotic plaque development in mice.

Quantitative characterization of atherosclerotic plaque composition with standard histopathological methods remains limited to sectioned plaques. Raman spectroscopy enables nondestructive quantification of atherosclerotic plaque composition. We used Raman spectroscopy to study the effects of diet and lipid-lowering therapy on plaque development in apolipoprotein (APO) E*3-Leiden transgenic mice. Raman spectra were obtained over the full width and entire length of the ascending aorta and aortic arch. Spectra were modeled to calculate the relative dry weights of cholesterol and calcium salts, and quantitative maps of their distribution were created. In male mice (n=20) that received a high-fat/high-cholesterol (HFC) diet for 0, 2, 4, or 6 months, Raman spectroscopy showed good correlation between cholesterol accumulation and total serum cholesterol exposure (r approximately 0.87, P<0.001). In female mice (n=10) that were assigned to an HFC diet, with or without 0.01% atorvastatin, a strong reduction in cholesterol accumulation (57%) and calcium salts (97%) (P<0.01) was demonstrated in the atorvastatin-treated group. In conclusion, Raman spectroscopy can be used to quantitatively study the size and distribution of depositions of cholesterol and calcification in APOE*3-Leiden transgenic mice. This study encourages Raman spectroscopy for the quantitative investigation of atherosclerosis and lipid-lowering therapy in larger animals or humans in vivo.

Design and validation of an analyser to measure sulphur hexafluoride gas during respiration.

The study presents the results of the development of an analyser to measure sulphur hexafluoride (SF6) gas in breathing circuits, for application is studies of lung function. The analyser consists of an in-line breathing circuit measurement transducer and a compact unit for signal treatment. The detector unit of the analyser consists of a near-infrared light source, a bandpass filter and a pyro-electrical detector. When incremental steps of SF6 gas between 0 and 2% were presented to the analyser, the maximum deviation from the theoretical calibration curve was calculated to be 0.01% SF6. The step response of the analyser (10-90%) was 250 ms. The sensitivity of the analyser to ambient temperature was 0.01% SF6 degrees C(-1) in the range between 0 and 2% SF6. It is concluded that the analyser presented is accurate, and has a sufficient response speed to be used in clinical measurement settings. Furthermore, the analyser is resistant to changes in temperature, gas flow, orientation and movement, which are likely to occur in clinical measurement settings.

In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles.

Confocal Raman spectroscopy is introduced as a noninvasive in vivo optical method to measure molecular concentration profiles in the skin. It is shown how it can be applied to determine the water concentration in the stratum corneum as a function of distance to the skin surface, with a depth resolution of 5 microm. The resulting in vivo concentration profiles are in qualitative and quantitative agreement with published data, obtained by in vitro X-ray microanalysis of skin samples. Semi-quantitative concentration profiles were determined for the major constituents of natural moisturizing factor (serine, glycine, pyrrolidone-5-carboxylic acid, arginine, ornithine, citrulline, alanine, histidine, urocanic acid) and for the sweat constituents lactate and urea. A detailed description is given of the signal analysis methodology that enables the extraction of this information from the skin Raman spectra. No other noninvasive in vivo method exists that enables an analysis of skin molecular composition as a function of distance to the skin surface with similar detail and spatial resolution. Therefore, it may be expected that in vivo confocal Raman spectroscopy will find many applications in basic and applied dermatologic research.

Raman spectroscopy for quantifying cholesterol in intact coronary artery wall.

The chemical composition of vascular lesions, an important determinant of plaque progression and rupture, can not presently be determined in vivo. Prior studies have shown that Raman spectroscopy can accurately quantify the amounts of major lipid classes and calcium salts in homogenized coronary artery tissue. This study determines how the relative cholesterol content, which is calculated from Raman spectra collected at the luminal surface of an artery, is related to its depth in an intact arterial wall. Raman spectra of human atherosclerotic plaques were measured after thin tissue layers were successively placed on them. From these spectra, relative cholesterol contents were calculated and used to determine how cholesterol signal strength is attenuated by overlaying tissue. Then, intact artery samples (n = 13) were examined spectroscopically, sectioned and stained specifically for cholesterol. Images of these sections were digitized, and image intensities were related to cholesterol content. These cholesterol amounts were weighed appropriately for depth into the tissue and area-integrated for comparison with spectroscopy results. A decaying exponential curve was fit to the layer study data (r2 = 0.97) and showed that approximately 300 microm of tissue attenuates cholesterol signals by 50%. In intact plaques, the spectroscopically-determined cholesterol amounts correlated strongly and linearly with those determined by digital microscopy (r2 = 0.94). With Raman spectroscopy techniques, the cholesterol content of a lesion can be determined by properly accounting for its depth into an arterial wall. Our results suggest that chemical concentrations in an artery wall could be mapped throughout its thickness, possibly by combining Raman spectroscopy methods with other techniques.

Identification of medically relevant microorganisms by vibrational spectroscopy.

In the recent years, vibrational spectroscopies (infrared and Raman spectroscopy) have been developed for all sorts of analyses in microbiology. Important features of these methods are the relative ease with which measurements can be performed. Furthermore, in order to obtain infrared or Raman spectra, there is only a limited amount of sample handling involved without the need for expensive chemicals, labels or dyes. Here, we review the potential application of vibrational spectroscopies for the use in medical microbiology. After describing some of the basics of the techniques, considerations on reproducibility and standardisation are presented. Finally, the use of infrared and Raman spectroscopy for the (rapid) identification of medically relevant microorganisms is discussed. It can be concluded that vibrational spectroscopies show high potential as novel methods in medical microbiology.

A fast, digitally controlled flow proportional gas injection system for studies in lung function.

The aim of this paper is to describe a device for flow proportional injection of tracer gas in the lungs of mechanically ventilated patients. This device may then be used for the study of the multiple breath indicator gas washout technique to determine the end-expiratory lung volume. Such a tracer gas injection device may also be used in the study of other techniques that rely on uptake and elimination of tracer gas by the lungs. In this paper, an injector is described which enables injection of indicator gas at a predetermined concentration in a breathing circuit independent of the type of breathing. The presented setup uses a control computer to produce steering signals to a multivalve array in proportion to the input breathing signals. The multivalve array consists of ten circular valves, each with a different diameter, which can be opened or closed individually according to the input signal of the array. By opening of a certain combination of valves an amount of sulphur hexafluoride gas proportional to the inspiratory breathing signal is released. The rate of transmission between the components of the injection system was 80 Hz. The injector has a full flow range between 0-10 L/min. The delay time between the breathing signal and the flow response was 70 ms. The aimed washin gas concentration of 1% SF6 was achieved after 0.5 s. The study describes the results of tests to determine valve-flow ratios, step response and dynamic response of the injector. The flow output response of the injector system was shown to increase in input frequencies above 3 Hz. The valve flow ratios showed the largest relative deviation in the two smallest valves of the 10 valve array, respectively 0.005 L/min (25%) and 0.002 L/min (20%). We conclude that the injector can achieve a stable concentration of indicator gas in a breathing system with an accuracy of 0.005 L/min to execute the multiple breath indicator washout test in human subjects. The results of the study indicate that the injector may be of use in other application fields in respiratory physiology in which breathing circuit injection of indicator gas is required.

Vibrational spectroscopic characterization of wild growing mushrooms and toadstools.

Recently, there has been increase of general interest in fungi because of the possible medical applications of their polysaccharide constituents called glucans, some of which are reported to have immunomodulatory properties. Since an extraction method can change the chemical composition of a substance, especially a delicate one such as fungal thallus, it is necessary and useful to know more about the studied matter in advance in order to choose the chemical procedure properly. We demonstrated the usefulness of vibrational spectroscopy in identifying different glucan types in various parts of intact fruiting bodies of Asco- and Basidiomycetes. Fourier transform-infrared (FT-IR) spectroscopy was used for obtaining vibrational spectra of spores and fruiting bodies of more than 70 species belonging to 37 different genera of wild growing mushrooms. The list of the bands in 750-950 cm(-1) interval, assigned to alpha- and beta-glucans, is provided for all species studied. Vibrational spectra in the interval 1000-1200 cm(-1) could serve as an indicator of mushroom genus, although particular species cannot be identified spectroscopically. Great similarities in spectra of spores of the same genus, but different species, e.g. Tricholoma album and Trichloma sulphureum, were observed. On the other hand, spectra of cap, stalk and spores of the same mushroom show great differences, indicating variety in the chemical composition of different parts of the same fruiting body.