Laser wavelength dependence of background fluorescence in Raman spectroscopic analysis of synovial fluid from symptomatic joints

Abstract

Gout is a disease process where the nucleation and growth of crystals in the synovial fluid of joints elicit painful arthritis-like symptoms. Raman spectroscopy is evolving as a potential diagnostic tool in identifying such crystals; however, attainment of sufficient Raman signal while overcoming the background fluorescence remains as a major challenge. The current study focused on assessing whether excitation in 532–700 nm range will provide greater signal intensity than the standard 785 nm while not being impeded by background fluorescence. We characterized the fluorescence spectra, absorption spectra and Raman spectra of synovial fluid from patients who presented “gout-like symptoms” (symptomatic) and controls (asymptomatic). A digestion and filtration method was developed to isolate crystals from synovial fluid while reducing the organic burden. Spectral profile and photobleaching dynamics during Raman spectroscopy were observed under an excitation wavelength range spanning 532 to 785 nm. Absorbance and fluorescence profiles indicated the digestion and filtration worked effectively to extract crystals from symptomatic synovial fluid without introducing additional fluorescence. Raman spectral analyses at 532 nm, 660 nm, 690 nm and 785 nm indicated that both asymptomatic and symptomatic samples had significant levels of fluorescence at excitation wavelengths below 700 nm, which either hindered the collection of Raman signal or necessitated prolonged durations of photobleaching. Raman-based diagnostics were more feasible at the longest excitation wavelength of 785 nm without employing photobleaching. This study further demonstrated that a near-infrared OEM based lower-cost Raman system at 785 nm excitation has sufficient sensitivity to identify crystals isolated from the synovial fluid. In conclusion, while lower excitation wavelengths provide greater signal, the fluorescence necessitates near-infrared wavelengths for Raman analysis of crystal species observed in synovial aspirates.

A customized Raman system for point-of-care detection of arthropathic crystals in the synovial fluid

“…The process eliminates subjectivity in the diagnosis of gout, an advantage over the PLM-based diagnosis which is operator dependent 28 . Previously we reported that the fluorescence spectra of raw and digested synovial samples to be comparable, indicating that digestion did not increase the background fluorescence 29 . Although filtration had discarded the organic material from the synovial fluid and digestive enzymes, there was residual organic matter in the crystals deposit, leading to background fluorescence in the Raman spectra.…”

Synthesis and Characterization of Graphene Oxide (GO) and Reduced Graphene Oxide (rGO) for Gas Sensing Application

“…However at 532 nm excitation, significant levels of fluorescence may be present hindering the Raman signal. This problem can be solved by using higher wavelength (785 nm excitation) laser which avoids fluorescence and still produces sufficient Raman signal …”

Detection of glioma by surface‐enhanced Raman scattering spectra with optimized mathematical methods

“…Thus, Raman spectra obtained with 633 and 532 nm were interfered by different fluorescence background, leading to small weak peaks. Although the Raman shift showed negligible variations with these lasers (785, 633, and 532 nm), the fluorescence noise changed with the alteration of laser and most fluorescence interference could be avoided by the laser at 785 nm . The results pointed out that 785‐nm laser was more appropriate for spectrum collection of the samples in this study.…”