Diagnosis of tumors during tissue-conserving surgery with integrated autofluorescence and Raman scattering microscopy

Abstract

Tissue-conserving surgery is used increasingly in cancer treatment. However, one of the main challenges in this type of surgery is the detection of tumor margins. Histopathology based on tissue sectioning and staining has been the gold standard for cancer diagnosis for more than a century. However, its use during tissue-conserving surgery is limited by time-consuming tissue preparation steps (1-2 h) and the diagnostic variability inherent in subjective image interpretation. Here, we demonstrate an integrated optical technique based on tissue autofluorescence imaging (high sensitivity and high speed but low specificity) and Raman scattering (high sensitivity and high specificity but low speed) that can overcome these limitations. Automated segmentation of autofluorescence images was used to select and prioritize the sampling points for Raman spectroscopy, which then was used to establish the diagnosis based on a spectral classification model (100% sensitivity, 92% specificity per spectrum). This automated sampling strategy allowed objective diagnosis of basal cell carcinoma in skin tissue samples excised during Mohs micrographic surgery faster than frozen section histopathology, and one or two orders of magnitude faster than previous techniques based on infrared or Raman microscopy. We also show that this technique can diagnose the presence or absence of tumors in unsectioned tissue layers, thus eliminating the need for tissue sectioning. This study demonstrates the potential of this technique to provide a rapid and objective intraoperative method to spare healthy tissue and reduce unnecessary surgery by determining whether tumor cells have been removed.

Raman spectroscopy: techniques and applications in the life sciences

“…Using data analysis techniques that will be discussed in Section 5, Raman spectroscopy has been used to classify bacteria [14,15], nano-bio-materials [138][139][140], cells [141,142], and animal and human tissues [143,144]. Because Raman spectroscopy is non-labeling and non-destructive, repeated measurements can be made of the same sample.…”

“…Although fewer Raman measuremens are acquired, which reduces the acquisition time by two orders of magnitude, the spatial resolution of the SSRS images are determined by the first imaging modality. Kong et al showed that it is possible to use integrated confocal auto-fluorescence imaging and Raman spectroscopy for fast detection of tumors in surgical resessions of skin [144] and breast tissue [446]. The auto-fluorescence image provided high resolution, while Raman spectroscopy provided chemical specificity for accuracy diagnosis.…”

“…34. Example of selective Raman scanning of (a) skin (scale bar is 0.5 mm) [144] and (b) breast tissue [446] using integrated autofluorescence and Raman microscopy. The series of images for each tissue type includes: auto-fluorescence image(s), segmented image with automated Raman sampling points, Raman diagnosis image(s), and an H&E image.…”

High sensitivity non‐invasive detection of calcifications deep inside biological tissue using Transmission Raman Spectroscopy

“…Raman spectroscopy has been proposed as a non-invasive and label free analytical method with a very consistent and prolific track record of in-vitro biomedical applications [22][23][24] for histopathological screening [25][26][27][28][29] as well as probing of lymph node pathology using fibre probes [30][31][32].…”

A Diagnostic Accuracy Comparison Between Tzanck Smear Test and Frozen Section Examination in Basal Cell Carcinoma

“…The MMS or FS have been recommended for difficult tumors such as recurrent tumors, perineural invasion or size > 2 cm, aggressive histological subtypes and located in anatomically sensitive areas (7). The MMS is a suitable treatment (8) and FS examination is the "gold standard" of surgical margins in tumoral skins (9), but FS is a timeconsuming test and expensive tool (10). On the other hand, Tzanck smear test (TST) is simple, easy to perform, inexpensive and a rapid test for cytological study to confirm or exclude malignancy.…”