Raman Spectroscopic Imaging of Human Bladder Resectates towards Intraoperative Cancer Assessment

Abstract

Raman spectroscopy offers label-free assessment of bladder tissue for in vivo and ex vivo intraoperative applications. In a retrospective study, control and cancer specimens were prepared from ten human bladder resectates. Raman microspectroscopic images were collected from whole tissue samples in a closed chamber at 785 nm laser excitation using a 20× objective lens and 250 µm step size. Without further preprocessing, Raman images were decomposed by the hyperspectral unmixing algorithm vertex component analysis into endmember spectra and their abundancies. Hierarchical cluster analysis distinguished endmember Raman spectra that were assigned to normal bladder, bladder cancer, necrosis, epithelium and lipid inclusions. Interestingly, Raman spectra of microplastic particles, pigments or carotenoids were detected in 13 out of 20 specimens inside tissue and near tissue margins and their identity was confirmed by spectral library surveys. Hypotheses about the origin of these foreign materials are discussed. In conclusion, our Raman workflow and data processing protocol with minimal user interference offers advantages for future clinical translation such as intraoperative tumor detection and label-free material identification in complex matrices.

Design of a Dispersive 1064 nm Fiber Probe Raman Imaging Spectrometer and Its Application to Human Bladder Resectates

“…The 785 nm excited Raman spectra from the same specimens are included in Figure 2E-G for comparison [17]. The overall signature and band positions agree well.…”

“…After finishing the 785 nm data collection, the samples were stored again in the −80 • C freezer until the 1064 nm Raman experiments. The size of each sample was approximately 10 × 10 mm 2 , and measurements were performed in a closed sample chamber without any sample preparation as described previously [17].…”

“…The lower resolution broadens the bands, which is particularly evident for the phenylalanine band at 1004 cm −1 . Careful inspection of 785 nm Raman spectra resolves more bands of aromatic amino acids at 621, 643, 756, 1031, 1209, 1548, 1584, 1606, and 1620 cm −1 (see also Table S1 in the Supplementary Materials and spectra in the main text of Krafft et al [17]). Another fine structure is better resolved in 785 nm Raman spectra of lipids at 1065 (C-C vibration), 1082 (phosphate vibration), and 1122 (C-C vibration) cm −1 , which gives a broad envelope at 1077 cm −1 in 1064 nm Raman spectra.…”

“…Raman spectroscopic-based techniques have been suggested as a complementary diagnostic tool to assess bladder tissues ex vivo in thin sections or biopsies and in vivo during cystoscopy. Raman papers in the context of bladder cancer between 2005 and 2022 have recently been summarized [17]. Of particular relevance for the current work was the characterization of bladder tissue using probe-based Raman spectroscopy [18].…”

“…The sample cohort consists of non-tumor and tumor specimens that were prepared from ten human bladder resectates. The samples were already studied by 785 nm Raman microscopic imaging [17] and the previous results are compared with the new results. As most bladder specimens showed an unusually low background in the 785 nm Raman study, the Raman analysis of four kidney stones were included in the discussion section.…”