Highly specific and label-free histological identification of microcrystals in fresh human gout tissues with stimulated Raman scattering

Abstract

Gout is a common metabolic disease with growing burden, caused by monosodium urate (MSU) microcrystal deposition. In situ and chemical-specific histological identification of MSU is crucial in the diagnosis and management of gout, yet it remains inaccessible for current histological methods. Methods: Stimulated Raman scattering (SRS) microscopy was utilized to image MSU based on its fingerprint Raman spectra. We first tested SRS for the diagnosis capability of gout and the differentiation power from pseudogout with rat models of acute gout arthritis, calcium pyrophosphate deposition disease (CPDD) and comorbidity. Then, human synovial fluid and surgical specimens (n=120) were were imaged with SRS to obtain the histopathology of MSU and collagen fibers. Finally, quantitative SRS analysis was performed in gout tissue of different physiological phases (n=120) to correlate with traditional histopathology including H&E and immunohistochemistry staining. Results: We demonstrated that SRS is capable of early diagnosis of gout, rapid detection of MSU in synovial fluid and fresh unprocessed surgical tissues, and accurate differentiation of gout from pseudogout in various pathophysiological conditions. Furthermore, quantitative SRS analysis revealed the optical characteristics of MSU deposition at different pathophysiological stages, which were found to matched well with corresponding immunofluorescence histochemistry features. Conclusion: Our work demonstrated the potential of SRS microscopy for rapid intraoperative diagnosis of gout and may facilitate future fundamental researches of MSU-based diseases.

Type II collagen facilitates gouty arthritis by regulating MSU crystallisation and inflammatory cell recruitment

“…Details of the SRS microscope setup are provided in our previous work 45 46. Two laser beams of different frequencies, ω p and ω s , coincide on the sample; the difference in matches with a particular molecular vibrational frequency Ω, inducing amplification of Raman signals by a factor of ~10 5 .…”

“…The optical power of the pump and Stokes beams at the sample were kept around 80 mW and 160 mW, respectively. SRS was able to specifically and quantitatively evaluate MSU crystals and surrounding tissue background in human synovial fluids or tophi specimens 45…”

In Vitro Anticancer Drug Sensitivity Sensing through Single-Cell Raman Spectroscopy

“…The metabolic index is an indicator of cellular metabolic activity and is defined as the ratio of the integrated spectral intensity of the C-D (2040-2300 cm −1 ) band over the sum of the C-D and C-H bands (2800-3100 cm −1 ). The Raman spectra of biological samples reflect their fingerprints [22,23]; previous studies in which bacteria were cultured in medium containing deuterium suggested that the Raman band at 2170 cm −1 was due to the carbon-deuterium (C-D) bond in the newly synthesized lipids and protein [16]. The generation of biological building blocks containing a C-D bond was due to the incorporation of deuterium from D 2 O into biomolecules via NADPH regeneration by metabolically active cells; NADPH regeneration is an intracellular anabolic biochemical process (illustrated in Figure S3).…”

Label-Free Histology and Evaluation of Human Pancreatic Cancer with Coherent Nonlinear Optical Microscopy

“…Stimulated Raman scattering (SRS) offers a gain of Raman scattering efficiency by 3–5 orders of magnitude, enabling high-speed chemical imaging. − SRS microscopy hence overcomes the speed limit of Raman imaging while retaining the spectral identities for molecule-specific detection. Moreover, SRS microscopy could provide tissue histology similar to traditional H&E staining based on the chemical contrast of intrinsic biomolecules such as lipids, proteins, and DNA. − Compared with frozen H&E staining, SRS microscopy has the advantages of imaging fresh, unprocessed tissues to achieve near real-time tissue histology in the operating room. − In addition to lipid and protein contents, collagen fibers are known to correlate with cancer fibrosis and tumor cell intravasation , and may play a protective role in controlling pancreatic cancer progression . Hence, it is necessary to include collagen fiber-specific detection with second harmonic generation (SHG) during SRS imaging. − Up to now, SRS/SHG microscopy has shown potential in the diagnosis and evaluation of many types of human tumor tissues, including laryngeal carcinoma, brain tumor, and breast cancer. ,,− However, the possibility of use of SRS microscopy for human pancreatic tumor imaging and intraoperative guidance has never been investigated.…”