Spatiotemporal Observation of Monosodium Urate Crystals Deposition in Synovial Organoids Using Label-Free Stimulated Raman Scattering

Abstract

Gout, a common form of arthritis, is characterized by the deposition of monosodium urate (MSU) crystals in joints. MSU deposition in synovial tissues would initiate arthritis flares and recurrence, causing irreversible joint damage. However, the dynamic deposition of MSU crystals in tissues lacks experimental observation. In this study, we used chemical-specific, label-free stimulated Raman scattering (SRS) microscopy to investigate the spatiotemporal deposition and morphological characteristics of MSU crystals in human synovial organoids. Our findings revealed a critical 12-h window for MSU deposition in the lining layer of gouty synovium. Moreover, distinctive inflammatory reactions of the lining and sublining synovial layers in gout using SRS microscopy were further verified by immunofluorescence. Importantly, we identified a crucial proinflammatory role of sublining fibroblast-like synoviocytes, indicating a need for targeted medication treatment on these cells. Our work contributes to the fundamental understanding of MSU-based diseases and offers valuable insights for the future development of targeted gout therapies.

Fig. 1.

Overview of the experimental procedure. (A) Experimental setup: Illustration of SRS signal detection. The femtosecond pump and stokes beams were elongated by 2 SF57 glass rods to achieve spectral resolution. The coherent pump and stokes beams were introduced to the sample, inducing stimulated emission. The modulation of the stokes beam was transferred to the pump beam and demodulated by a lock-in amplifier. The abbreviations used are as follows: R, SF57 glass rod; DL, delay line; EOM, electro-optical modulator; DM, dichroic mirror; GM, galvo mirror; BPF, band-pass filter; PD, photodiode; PMT, photomultiplier tube; LIA, lock-in amplifier. (B) Energy diagram and detected process: In the SRS process, the pump and stokes beams interact with the organoid sample simultaneously. When the beating frequency matched the Raman vibrational mode Ω, a coherently amplified energy transfer process occurred, resulting in the annihilation of the pump photon (SRL) and the generation of the stokes photon (stimulated Raman gain). In our experiment, the stimulated Raman gain signal was filtered out, and the SRL signal was detected. (C) Organoid cultivation: Human synovial tissue was harvested from patients with meniscus injuries through arthroscopic surgery. Synovial organoids were established after a 21-d culture period. Subsequently, MSU crystals were added to the organoids to create an acute-gout synovial organoid model. (D) Data collection and processing: The appropriate pump and stokes wavelength combination was selected for imaging lipids, proteins, and MSU crystals in the synovium organoid. In situ images of lipids and proteins were decomposed according to the SRS spectra displayed in the lower panel. By moving the objective along the z-axis, 3D information about the organoid was acquired, and lining and sublining layers could be differentiated based on cell density. ImageJ and MATLAB were then used for data processing. The abbreviations used are as follows: OA, oleic acid; BSA, bovine serum albumin.

Fig. 2.

Establishment of synovial organoids. (A to C) HE, reticular fiber, and lubricin IHC staining of human 3D synovial organoids. The left panel shows the lining layer, and the right panel shows the sublining layer. Scale bar: 20 μm. (D) Spontaneous and SRS analysis of standard OA, BSA, and MSU (upper panel) and lower panel presents the SRS spectra of MSU, synovium tissue, and synovium organoid. The SRS spectra were consistent with the spontaneous Raman (SR) spectra and had characteristic features that distinguished MSU crystals in the synovium organoid. The synovium organoid exhibited similar SRS spectra to fresh synovium tissue.

Fig. 3.

3D reconstruction using SRS microscopy. (A) Top, side, and sectional views at different depths of the organoid (left upper, right, and left lower panels, respectively) after MSU crystal supplementation for 6 h. Scale bar: 100 μm. (B to E) Top views of the organoid after MSU crystal addition for 0, 12, 24, and 48 h, respectively. MSU crystals (red, 630 cm⁻¹), lipid (green, 2,930 cm⁻¹), and protein (blue, 2,930 cm⁻¹). Scale bar: 100 μm.

Fig. 4.

Temporal deposition of MSU crystals in synovial organoids. (A) Heatmap illustrating MSU crystal intensity in synovial organoids after 6, 12, 24, and 48 h of MSU crystal exposure. Yellow dashed lines denote the organoid surface, while yellow arrows indicate areas with MSU crystals. Scale bar: 100 μm. (B) Distribution of MSU crystals in synovial organoids at 0, 6, 12, 24, and 48 h. (C) Intensity density of MSU crystal deposition in synovial organoids at 0, 6, 12, 24, and 48 h. * represents P < 0.05, and *** indicates P < 0.001.

Fig. 5.

Spatial deposition of MSU crystals in lining and sublining layers. (A) Stereoscopic view showing the lining (white) and sublining (blue) layers of a typical field of view (FoV) measuring 508.93 × 508.93 × 915.68 μm³. (B) Stereoscopic view displaying MSU crystals in synovial organoids within the same FoV as (A), with MSU crystals marked in white and blue to represent deposition in the lining and sublining layers, respectively. (C) Intensity density of MSU crystal deposition in the lining and sublining layers of synovial organoids at 0, 6, 12, 24, and 48 h.

Fig. 6.

Spatial deposition of MSU crystals in cells and ECM. (A) Typical stereoscopic views of MSU crystals in synovial organoids at 6, 12, 24, and 48 h. MSU crystals in lining and sublining cells are marked in yellow and cyan, respectively, while those in the ECM are marked in magenta. (B to D) MSU deposition intensity density (B), average length (C), and aspect ratio (D) of MSU crystals in cells and ECM of synovial organoids at 0, 6, 12, 24, and 48 h. (E to G) MSU deposition intensity density (E), average length (F), and aspect ratio (G) of MSU crystals in lining and sublining cells of synovial organoids at 0, 6, 12, 24, and 48 h. * denotes P < 0.05, ** denotes P < 0.01, and *** denotes P < 0.001.

Fig. 7.

Response of lining and sublining cells to MSU crystals in synovial organoids. (A and B) Top views (A) and sectional views (B) of cells in acute-gout synovial organoids at 0, 6, 12, 24, and 48 h. Lining cells are marked in cyan, while sublining cells are marked in magenta. Yellow dashed lines indicate the organoid surface. Scale bar: 100 μm. (C) Aspect ratio of lining and sublining cells in acute-gout synovial organoids at 0, 6, 12, 24, and 48 h. (D and E) Representative images of double immunofluorescent staining of IL-1β and TNF-α in the lining (D) and sublining (E) layers of synovial organoids with or without MSU crystal addition. DAPI, 4′,6-Diamidino-2′-phenylindole. (F and G) Relative fluorescence intensity of IL-1β (F) and TNF-α (G) in the lining and sublining layers of synovial organoids with or without MSU crystal addition. Scale bar: 50 μm. ** denotes P < 0.01, and *** denotes P < 0.001.