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.

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.

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%.

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.

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.

Detection and differentiation of causative organisms of onychomycosis in an ex vivo nail model by means of Raman spectroscopy.

BACKGROUND: Onychomycosis is worldwide the most prevalent infection of the nail. It is mainly caused by the dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes and to a lesser extent Trichophyton tonsurans. The yeast Candida albicans and the mould Scopulariopsis brevicaulis can also cause onychomycosis. Management of these nail conditions may require appropriate treatment methods and therefore the identification of the causative species can be of importance. However, the determination of agents causing onychomycosis is still not optimal. OBJECTIVES: To detect and differentiate causative organisms of onychomycosis in an ex vivo nail model by means of Raman spectroscopy. The work focusses is on the discriminative power of Raman spectroscopy for detection of differences between T. rubrum, T. mentagrophytus and T. tonsurans on human nail and distinguishing these dermatophytic from the non-dermatophytic species S. brevicaulis and C. albicans. METHODS: Raman spectra (200/sample) were taken from 50-µm human nail slices infected with T. rubrum, T. mentagrophytus, T. tonsurans, S. brevicaulis or C. albicans using a 2500 High-Performance Raman Module and 785-nm diode laser. Processed spectra were analysed by sorting the correlation matrix and presented as dendrogram and heat map. Raman spectra from suspended dermatophytic microconidia were taken for mutual comparisons. RESULTS: Spectral differences between the dermatophytes T. rubrum, T. mentagrophytus and T. tonsurans (635-795, 840-894, 1018-1112, 1206-1372, 1566-1700/cm) and the non-dermatophytes S. brevicaulis and C. albicans (442-610, 692-758, 866-914, 1020-1100, 1138-1380,1492-1602/cm) growing on nail were confirmed by clustering correlation showing two main clusters. Dissimilarities between tested dermatophytes were also found with T. rubrum being most different. Raman spectra of the dermatophytic microconidia varied over the whole tested 400-1800/cm range. CONCLUSION: Important dermatophytic and non-dermatophytic agents of onychomycosis growing on ex vivo human nail can be distinguished specifically and non-invasively by Raman spectroscopy.

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.

Autofluorescence and Raman microspectroscopy of tissue sections of oral lesions.

Autofluorescence spectroscopy and Raman spectroscopy have been suggested for lesion diagnostics. We investigate the information contained in autofluorescence and Raman spectra recorded from oral tissue slices of various lesion types. Thirty-seven human oral mucosa lesions were biopsied and freeze-dried. Complete autofluorescence images and spectra were recorded from 20 microm sections. Raman spectra were acquired from the same positions for 12 of the sections. Cluster analysis was applied to find any relationship between spectral shape and lesion type or cell layer. Autofluorescence images showed high intensities for keratin layers and connective tissue, but hardly any for the epithelium. Autofluorescence spectra were centered around 520 nm and did not show specific spectral features. No clustering with regard to lesion type or cell layer was observed. Raman spectra allowed for reliable classification into cell layers, but differences between lesion types were not significant in this study. Autofluorescence spectra of freeze-dried oral mucosa sections did not contain useful information. A more comprehensive study is required for Raman spectra.

On-line detection of cholesterol and calcification by catheter based Raman spectroscopy in human atherosclerotic plaque ex vivo.

BACKGROUND: Raman spectroscopy has the unique potential to detect and quantify cholesterol and calcification in an atherosclerotic plaque in vivo. OBJECTIVE: To evaluate the sensitivity and specificity of this technique for detecting cholesterol or calcification in human coronary artery and aorta specimens ex vivo, using a compact clinical fibreoptic based Raman system developed for in vivo applications. DESIGN: From nine coronary arteries and four aorta specimens, 114 sites were evaluated for the presence of cholesterol and calcification by Raman spectroscopy and standard histology. Raman spectra were acquired and evaluated on-line in around five seconds. RESULTS: The correlation between Raman spectroscopy and histology was r = 0.68 for cholesterol and r = 0.71 calcification in the plaque (p < 0.0001). Sensitivity and specificity for detecting cholesterol and calcification were excellent: receiver operating characteristic (ROC) analysis for each of the components revealed areas under the curves of > 0.92 (p < 0.0001). At the optimal cut-off values determined by ROC analysis, positive predictive values of > 80% and negative predictive values of > 90% were obtained. CONCLUSIONS: On-line real time catheter based Raman spectroscopy detects accumulation of cholesterol and calcification in atherosclerotic plaque with high sensitivity and specificity.

Identification of bladder wall layers by Raman spectroscopy.

PURPOSE: We explored the applicability of Raman spectroscopy to in situ investigation of bladder wall tissue. MATERIALS AND METHODS: Bladder wall tissue was obtained from a guinea pig model and frozen sections were used for Raman spectroscopic investigations. From each section 500 to 700 spectra were obtained in a 2-dimensional grid spanning the urothelium, lamina propria and muscle layer. The data set of spectra was subdivided into groups of similar spectra by a cluster analysis algorithm. With each group assigned a different color Raman maps of frozen sections were constructed based on group membership of measured spectra. These maps were then compared with histological and histochemical data obtained from hematoxylin and eosin and immunohistochemical staining for collagen I and III and for smooth muscle actin to correlate Raman spectral features with bladder wall structure and molecular composition. RESULTS: Urothelium, lamina propria and muscle layers could be clearly distinguished based on Raman spectra. Lamina propria spectra were dominated by signal contributions of collagen and the smooth muscle layer showed strong signal contributions of actin. The urothelium had a relatively strong lipid signal contribution. CONCLUSIONS: These results and the fact that Raman spectroscopy is rapidly evolving into a technology that can be applied in vivo by thin, flexible fiberoptic catheters indicate that prospects are good for in vivo analysis of the molecular composition of the normal and pathological bladder without biopsies.

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.

In vivo detection of dysplastic tissue by Raman spectroscopy.

The detection of dysplasia and early cancer is important because of the improved survival rates associated with early treatment of cancer. Raman spectroscopy is sensitive to the changes in molecular composition and molecular conformation that occur in tissue during carcinogenesis, and recent developments in fiber-optic probe technology enable its application as an in vivo technique. In this study, the potential of Raman spectroscopy for in vivo classification of normal and dysplastic tissue was investigated. A rat model was used for this purpose, in which dysplasia in the epithelium of the palate was induced by topical application of the carcinogen 4-nitroquinoline 1-oxide. High quality in vivo spectra of normal and dysplastic rat palate tissue, obtained using signal integration times of 100 s were used to create tissue classification models based on multivariate statistical analysis methods. These were tested with an independent set of in vivo spectra, obtained using signal collection times of 10 s. The best performing model, in which signal variance due to signal contributions of the palatal bone was eliminated, was able to distinguish between normal tissue, low-grade dysplasia, and high-grade dysplasia/carcinoma in situ with a selectivity of 0.93 and a sensitivity of 0.78 for detecting low-grade dysplasia and a specificity of 1 and a sensitivity of 1 for detecting high-grade dysplasia/ carcinoma in situ.

Intracellular carotenoid levels measured by Raman microspectroscopy: comparison of lymphocytes from lung cancer patients and healthy individuals.

Most studies concerning a possible protective role of carotenoids against cancer focus on serum carotenoid levels. We have used Raman microspectroscopy to study the intracellular amounts of carotenoids in lymphocytes of lung cancer patients and of healthy individuals. Our results indicate a significant decrease of carotenoids in lung carcinoma patients compared with healthy individuals, particularly in adenocarcinoma patients. Carotenoid supplementation raised the serum concentration in 2 lung cancer patients up to normal levels, whereas intracellular content remained significantly lower. This indicates that carotenoid uptake by lymphocytes is not only dependent on serum carotenoid concentration. Our findings indicate that Raman microspectroscopy, a recently developed technique to measure intracellular levels of drugs, is also well suited to obtain quantitative data on carotenoid amounts inside cells.

ANALYSIS: software for graphical analysis of multidimensional flow cytometric list mode data.

A computer program for graphical analysis of multidimensional flow cytometric list mode data is described. The program offers one-, two-, and three-dimensional inspection of an amount of data that is only limited by disk space. Subpopulations within the original data set can be identified by setting one or more two-dimensional AND gates around them. The order of measurement can be used as a parameter for evaluation of time-dependent processes. Other new parameters can be made by zooming in on a parameter, logarithmic transformation, or division of two parameters. The program is written in Turbo Pascal and it can run on any MS-DOC PC with an EGA/VGA resolution screen.

Selective electrofusion of conjugated cells in flow.

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.

Signal processing in slit-scan flow cytometry of cell conjugates.

The design and implementation of a real-time signal processing system for slit-scan flow cytometry is described. The system is used to measure the separate scatter and fluorescence peak heights of 2 adherent cells. Preliminary measurements of changes in the membrane potential induced by interactions between natural killer (NK) cells and their target cells are presented.

Cluster analysis of flow cytometric list mode data on a personal computer.

A cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS-DOS personal computer. It uses k-means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce the large number of subclusters. Thus we combine the advantage of the k-means (speed) with that of the nearest neighbor technique (accuracy). In order to achieve a rapid analysis, no complex data transformations such as principal components analysis were used. Results of the cluster analysis on both real and artificial flow cytometric data are presented and discussed. The results show that it is possible to get very good cluster analysis partitions, which compare favorably with manually gated analysis in both time and in reliability, using a personal computer.

A new principle of cell sorting by using selective electroporation in a modified flow cytometer.

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 microseconds and resulting field strengths of 2.0 and 3.2 x 10(6) V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.