Subcellular Raman Microspectroscopy Imaging of Nucleic Acids and Tryptophan for Distinction of Normal Human Skin Cells and Tumorigenic Keratinocytes

Abstract

At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.

Challenges in application of Raman spectroscopy to biology and materials

“…47 Further improvements in the instrumentation have enabled studying of various problems concerning the structure and interaction in nucleic acids, nucleic acid hydration, interaction with metal ions, DNA melting, DNA damage etc. [48][49][50][51] Raman spectra of nucleic acids contain a large number of bands originating from various moieties like sugar, phosphate, bases etc. which provide very useful information about backbone conformation, base pairing etc.…”

Broadband Coherent Raman Scattering Microscopy

“…SR is able to identify selectively many types of biomolecules found in human tissues and cells . The detailed chemical information provided by SR enables a clear distinction between states of a dynamical process: there are already many encouraging reports of the diagnostic success of this technique in cancer research . SR can detect biochemical differences in irradiated cells and discriminate between normal and cancerous state for skin, bladder and gastric tissues, rat fibroblast cells, human bone, and human epithelial cells from a variety of organs.…”

Dynamic Sparse Sampling for Confocal Raman Microscopy

“…1 Because of its high chemical specificity and requirement for minimal sample preparation, Raman imaging has found broad adoption in both chemical and biological sample analysis, with applications ranging from the screening of pharmaceutical formulations, 2–5 to characterization of semiconductors, 6,7 to cancer diagnosis, 8–10 to forensic analysis. 11,12 Specifically, in the pharmaceutical industry, spontaneous Raman spectroscopy and microscopy have been established as a standard method for polymorphic characterization of active pharmaceutical ingredients (APIs).…”