Autofluorescence-Raman Spectroscopy for Ex Vivo Mapping Colorectal Liver Metastases and Liver Tissue.

INTRODUCTION: Identifying colorectal liver metastases (CRLM) during liver resection could assist in achieving clear surgical margins, which is an important prognostic variable for both disease-free and overall survival. The aim of this study was to investigate the effect of auto-fluorescence (AF) and Raman spectroscopy for ex vivo label-free discrimination of CRLMs from normal liver tissue. Secondary aims include exploring options for multimodal AF-Raman integration with respect to diagnosis accuracy and imaging speed on human liver tissue and CRLM. METHODS: Liver samples were obtained from patients undergoing liver surgery for CRLM who provided informed consent (15 patients were recruited). AF and Raman spectroscopy was performed on CRLM and normal liver tissue samples and then compared to histology. RESULTS: AF emission spectra demonstrated that the 671 nm and 775/785 nm excitation wavelengths provided the highest contrast, as normal liver tissue elicited on average around eight-fold higher AF intensity compared to CRLM. The use of the 785 nm wavelength had the advantage of enabling Raman spectroscopy measurements from CRLM regions, allowing discrimination of CRLM from regions of normal liver tissue eliciting unusual low AF intensity, preventing misclassification. Proof-of-concept experiments using small pieces of CRLM samples covered by large normal liver tissue demonstrated the feasibility of a dual-modality AF-Raman for detection of positive margins within few minutes. CONCLUSIONS: AF imaging and Raman spectroscopy can discriminate CRLM from normal liver tissue in an ex vivo setting. These results suggest the potential for developing integrated multimodal AF-Raman imaging techniques for intraoperative assessment of surgical margins.

Fast Raman spectral mapping of highly fluorescing samples by time-gated spectral multiplexed detection.

We present a time-gated Raman micro-spectroscopy technique suitable for fast Raman mapping of samples eliciting large laser-induced fluorescence backgrounds. To achieve the required time resolution for effective fluorescence rejection, a picosecond pulsed laser and a single-photon avalanche diode were used. A module consisting of a spectrometer, digital micromirror device, and two prisms was used for high-resolution spectral filtering and multiplexing, which is required for a high chemical specificity and short integration times. With this instrument, we demonstrated time-gated Raman imaging of highly fluorescent samples, achieving acquisition times as short as 3 min for 40×40 pixel resolution images.

Sub-Surface Molecular Analysis and Imaging in Turbid Media Using Time-Gated Raman Spectral Multiplexing.

Obtaining molecular information deeper within optically turbid samples is valuable in many applications. However, in many cases this is challenging, in particular when the sample elicits strong laser-induced fluorescence emission. Here, we investigated the use of time-gated and micro-spatially offset Raman spectroscopy (micro-SORS) based on spectral multiplexing detection to obtain sub-surface molecular analysis and imaging for both fluorescing and non-fluorescing samples. The multiplexed spectral detection achieved with a digital micromirror device (DMD) allowed fast acquisition of the time-gated signals to enable three-dimensional Raman mapping (raster scanning in the lateral x,y plane and using time-of-flight calibration for the axial z-direction). Sub-millimeter resolution molecular depth mapping was achieved with dwell times on the order of seconds per pixel. To suppress fluorescence backgrounds and enhance Raman bands, time-gated Raman spectroscopy was combined with micro-SORS to recover Raman signals of red pigments placed behind a layer of optically turbid material. Using a defocusing micro-SORS approach, both fluorescence and Raman signals from the surface layers were further suppressed, which enhanced the Raman signals from the deeper sublayers containing the pigment. These results demonstrate that time-gated Raman spectroscopy based on spectral multiplexed detection, and in combination with micro-SORS, is a powerful technique for sub-surface molecular analysis and imaging, which may find practical applications in medical imaging, cultural heritage, forensics, and industry.