Multimodal spectral histopathology for quantitative diagnosis of residual tumour during basal cell carcinoma surgery

Basal cell carcinoma (BCC) is the most common type of cancer in the world. The preferred method of treating BCC is surgical excision followed by histopathological investigation of the removed tissue. Mohs micrographic surgery is a highly specialised surgical procedure which involves the removal of sequential layers of skin until a tumour is completely excised. Each removed skin layer is analysed through frozen section histopathology to assess whether residual BCC is present on the resection margin and if so, a subsequent tissue layer is removed. Mohs surgery is only used to treat BCCs in high risk locations (such as the head and neck area) or recurrences. Mohs surgery is not available universally, as it requires highly specialised personnel, is labour intensive and expensive. New imaging modalities are therefore being developed to investigate whether they can accompany or replace frozen section histopathology in assessing the surgical margin of removed skin specimens within the Mohs clinical workflow. This thesis describes the development and optimisation of a prototype instrument that utilises a multimodal spectral histopathology (MSH) approach to investigate the surgical margins of excised skin tissue samples. Multimodal spectral histopathology combines autofluorescence confocal microscopy and Raman spectroscopy to objectively determine whether the margins of an investigated sample are BCC-positive. The prototype instrument was developed to measure tissue samples automatically (without requiring user input), and to display the measurement results as easy-to-interpret tissue maps. The maps do not require specialist interpretation, meaning that MSH can be used by non- spectroscopy users, resulting in improved objectivity of diagnosis. The MSH measurement and data analysis software, which enables the automated operation of the instrument, was optimised by measuring 97 frozen skin samples obtained after Mohs surgery of 70 patients. The MSH software was shown to be transferable between similar instruments, demonstrating the potential to produce such devices at scale. The performance of the prototype instrument for frozen skin samples was tested by measuring an independent set of 10 samples from 9 patients. The prototype was shown to correctly diagnose all samples based on their corresponding adjacent histopathology sections. The prototype instrument was then moved into the Mohs clinic where it was used to measure fresh tissue samples intra-operatively. In order to adapt the prototype instrument to the clinical setting, 50 fresh tissue samples from 18 patients were investigated. The MSH software and tissue handling procedure were adapted to account for the abundant presence of blood and surgical positioning ink. The measurement time was also shortened to 30 minutes per Mohs layer, to increase compatibility with the Mohs workflow. The performance of the prototype instrument for fresh samples was assessed by measuring an independent set of 30 samples from 12 patients, after the software changes were finalised. Fresh sample diagnoses were shown to be in accordance with Mohs frozen sections for 96.4% of the test samples. These results were enabled by the use of a multinomial logistic regression classification model capable of distinguishing between BCC and healthy spectra with a 94.3% sensitivity and a 95.3% specificity. The prototype described in this thesis is, therefore, the first fully-automated instrument based on Raman spectroscopy for intra-operative microscopic imaging of surgical margins during cancer surgery, suitable to be used by a non-specialist user in a clinical environment

Time-gated Raman spectroscopy for biomedical application under ambient or strong background light conditions

Many biomedical applications require measurements of Raman spectra of tissue under ambient lighting conditions. However, the background light often swamps the weaker Raman signal. The use of time-gated (TG) Raman spectroscopy based on a single photon avalanche diode (SPAD) operating in time-correlated single photon counting and near-infrared laser excitation was investigated for acquisition of Raman spectra and spectral images of biological tissue. The results obtained using animal tissue samples (adipose tissue and muscle) show that the time gating modality enables measurement of Raman spectra under background light conditions of similar quality as conventional continuous wave Raman spectroscopy in the absence of background light. Optimal suppression of the background light was observed for time gate widths of 300–1000 ps. The results also showed that TG Raman spectroscopy was able to detect subtle spectral differences required for medical diagnostics, such as differences in Raman spectra of cancer and normal tissue. While the current instrument required scanning of the grating in order to obtain full Raman spectra, leading to impractical times for multi-wavenumber Raman mapping, imaging time could be drastically reduced by spectral multiplexing (compressed detection) using digital micromirror devices or by using SPAD arrays

Selective-sampling Raman imaging techniques for ex vivo assessment of surgical margins in cancer surgery

One of the main challenges in cancer surgery is to ensure the complete excision of the tumour while sparing as much healthy tissue as possible. Histopathology, the gold-standard technique used to assess the surgical margins on the excised tissue, is often impractical for intra-operative use because of the time-consuming tissue cryo-sectioning and staining, and availability of histopathologists to assess stained tissue sections. Raman micro-spectroscopy is a powerful technique that can detect microscopic residual tumours on ex vivo tissue samples with accuracy, based entirely on intrinsic chemical differences. However, raster-scanning Raman micro-spectroscopy is a slow imaging technique that typically requires long data acquisition times wich are impractical for intra-operative use. Selective-sampling Raman imaging overcomes these limitations by using information regarding the spatial properties of the tissue to reduce the number of Raman spectra. This paper reviews the latest advances in selective-sampling Raman techniques and applications, mainly based on multimodal optical imaging. We also highlight the latest results of clinical integration of a prototype device for non-melanoma skin cancer. These promising results indicate the potential impact of Raman spectroscopy for providing fast and objective assessment of surgical margins, helping surgeons ensure the complete removal of tumour cells while sparing as much healthy tissue as possible

Diagnostic accuracy of autofluorescence-Raman spectroscopy for surgical margin assessment during Mohs micrographic surgery of basal cell carcinoma

Autofluorescence (AF)-Raman spectroscopy has been shown to identify residual basal cell carcinoma (BCC) on frozen skin specimens and fresh skin specimens immediately after excision by Mohs surgery. This first diagnostic test of accuracy of AF-Raman on 130 full-face Mohs tissue layers (130 patients) shows that with improvement in tissue processing, the AF-Raman instrument is viable technique for intra-operative assessment of surgical margins

Ex vivo assessment of basal cell carcinoma surgical margins in Mohs surgery by autofluorescence‐Raman spectroscopy: A pilot study

Background: Autofluorescence (AF)‐Raman spectroscopy is a technology that can detect tumour tissue in surgically excised skin specimens. The technique does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps within 30 min. Objectives: To explore the clinical application of AF‐Raman microscopy to detect residual basal cell carcinoma (BCC) positive margins in ex vivo skin specimens excised during real‐time Mohs surgery. To investigate the ability to analyse skin specimens from different parts of the head‐and‐neck areas and detect nodular, infiltrative and superficial BCC. Methods: Fifty Mohs tissue layers (50 patients) were investigated: 27 split samples (two halves) and 23 full‐face samples. The AF‐Raman results were compared to frozen section histology, carried out intraoperatively by the Mohs surgeon and postoperatively by dermatopathologists. The latter was used as the standard of reference. Results: The AF‐Raman analysis was completed within the target time of 30 min and was able to detect all subtypes of BCC. For the split specimens, the AF‐Raman analysis covered 97% of the specimen surface area and detected eight out of nine BCC positive layers (similar to Mohs surgeons). For the full‐face specimens, poorer contact between tissue and cassette coverslip led to lower coverage of the specimen surface area (92%), decreasing the detection rate (four out of six positives for BCC). Conclusions: These preliminary results, in particular for the split specimens, demonstrate the feasibility of AF‐Raman microscopy for rapid assessment of Mohs layers for BCC presence. However, for full‐face specimens, further work is required to improve the contact between the tissue and the coverslip to increase sensitivity

Combined total internal reflection AF spectral-imaging and Raman spectroscopy for fast assessment of surgical margins during breast cancer surgery

The standard treatment for breast cancer is surgical removal mainly through breast conserving surgey (BCS). We developed a new technique based on auto-fluorescence (AF) spectral imaging and Raman spectroscopy for fast intraoperative assessment of excision margins in BCS. A new wide-field AF imaging unit based on total internal reflection (TIR) was combined with a Raman spectroscopy microscope equipped with a 785 nm laser. The wavelength of the AF excitation was optimized to 365 nm in order to maximize the discrimination of adipose tissue. This approach allows for the non-adipose regions of tissue, which are at higher-risk of containing tumor, to be targeted more efficiently by the Raman spectroscopy measurements. The integrated TIR-AF-Raman was tested on small tissue samples as well as fresh wide local excisions, delivering the analysis of the entire cruciate surface of BCS specimens (5.1 × 7.6 cm 2) in less than 45 minutes and also providing information regarding the location of the tumour in the specimen. Full automation of the instrument and selection of a faster translation stage would allow for the measurement of BCS specimens within intraoperative time scale (20 minutes). This study demonstrates that the TIR-AF Raman microscope represents a feasible step towards the development of a technique for intraoperative assessment of large WLE within intraoperative timescales

Towards quantitative molecular mapping of cells by Raman microscopy: using AFM for decoupling molecular concentration and cell topography

Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in-vitro. However, obtaining quantitative molecular information from the Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least square fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 256 mg/m, while proteins were distributed more uniformly and reaching concentrations as high as ~5012 mg/ml. The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman map of fixed cells (n=10), we found a linear relationship between the scores corresponding to the first component (PC1) and cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n=10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were obtained concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in-vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing Raman spectra of cells with large morphological differences

Clinical integration of fast Raman spectroscopy for Mohs micrographic surgery of basal cell carcinoma

We present the first clinical integration of a prototype device based on integrated auto-fluorescence imaging and Raman spectroscopy (Fast Raman device) for intra-operative assessment of surgical margins during Mohs micrographic surgery of basal cell carcinoma (BCC). Fresh skin specimens from 112 patients were used to optimise the tissue pre-processing and the Fast Raman algorithms to enable an analysis of complete Mohs layers within 30 minutes. The optimisation allowed >95% of the resection surface area to be investigated (including the deep and epidermal margins). The Fast Raman device was then used to analyse skin layers excised from the most relevant anatomical sites (nose, temple, eyelid, cheek, forehead, eyebrow and lip) and to detect the three main types of BCC (nodular, superficial and infiltrative). These results suggest that the Fast Raman technique is a promising tool to provide an objective diagnosis “tumour clear yes/no” during Mohs surgery of BCC. This clinical integration study is a key step towards a larger scale diagnosis test accuracy study to reliably determine the sensitivity and specificity in a clinical setting

Multimodal spectral histopathology for quantitative diagnosis of residual tumour during basal cell carcinoma surgery

Basal cell carcinoma (BCC) is the most common type of cancer in the world. The preferred method of treating BCC is surgical excision followed by histopathological investigation of the removed tissue. Mohs micrographic surgery is a highly specialised surgical procedure which involves the removal of sequential layers of skin until a tumour is completely excised. Each removed skin layer is analysed through frozen section histopathology to assess whether residual BCC is present on the resection margin and if so, a subsequent tissue layer is removed. Mohs surgery is only used to treat BCCs in high risk locations (such as the head and neck area) or recurrences. Mohs surgery is not available universally, as it requires highly specialised personnel, is labour intensive and expensive. New imaging modalities are therefore being developed to investigate whether they can accompany or replace frozen section histopathology in assessing the surgical margin of removed skin specimens within the Mohs clinical workflow. This thesis describes the development and optimisation of a prototype instrument that utilises a multimodal spectral histopathology (MSH) approach to investigate the surgical margins of excised skin tissue samples. Multimodal spectral histopathology combines autofluorescence confocal microscopy and Raman spectroscopy to objectively determine whether the margins of an investigated sample are BCC-positive. The prototype instrument was developed to measure tissue samples automatically (without requiring user input), and to display the measurement results as easy-to-interpret tissue maps. The maps do not require specialist interpretation, meaning that MSH can be used by non- spectroscopy users, resulting in improved objectivity of diagnosis. The MSH measurement and data analysis software, which enables the automated operation of the instrument, was optimised by measuring 97 frozen skin samples obtained after Mohs surgery of 70 patients. The MSH software was shown to be transferable between similar instruments, demonstrating the potential to produce such devices at scale. The performance of the prototype instrument for frozen skin samples was tested by measuring an independent set of 10 samples from 9 patients. The prototype was shown to correctly diagnose all samples based on their corresponding adjacent histopathology sections. The prototype instrument was then moved into the Mohs clinic where it was used to measure fresh tissue samples intra-operatively. In order to adapt the prototype instrument to the clinical setting, 50 fresh tissue samples from 18 patients were investigated. The MSH software and tissue handling procedure were adapted to account for the abundant presence of blood and surgical positioning ink. The measurement time was also shortened to 30 minutes per Mohs layer, to increase compatibility with the Mohs workflow. The performance of the prototype instrument for fresh samples was assessed by measuring an independent set of 30 samples from 12 patients, after the software changes were finalised. Fresh sample diagnoses were shown to be in accordance with Mohs frozen sections for 96.4% of the test samples. These results were enabled by the use of a multinomial logistic regression classification model capable of distinguishing between BCC and healthy spectra with a 94.3% sensitivity and a 95.3% specificity. The prototype described in this thesis is, therefore, the first fully-automated instrument based on Raman spectroscopy for intra-operative microscopic imaging of surgical margins during cancer surgery, suitable to be used by a non-specialist user in a clinical environment

Autofluorescence-Raman spectroscopy for ex-vivo mapping colorectal liver metastases and liver tissue

Background: 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. Aims: 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 8-folds 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 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. Conclusion: AF imaging and Raman spectroscopy can discriminate CRLM from normal liver tissue in an ex-vivo setting. These results suggest the potential for developing an integrated multimodal AF-Raman imaging techniques for intra-operative assessment of surgical margins