A NEW LENS ON SYNOVITIS: LABEL-FREE IMAGING OF WHOLE-MOUNT HUMAN PATHOLOGICAL SYNOVIAL MEMBRANE WITH MULTIPHOTON MICROSCOPY

Abstract

INTRODUCTION

One of the typical hallmarks of osteoarthritis progression is the inflammation of the synovial membrane, also known as synovitis. Pathological synovitis assessment is usually performed with traditional 2D histopathology, which provides limited orientation-dependent information, requires chemical labeling, and is destructive in nature. Tissue clearing of the whole synovial biopsy and non-destructive optical sectioning using multiphoton microscopy (MPM) can overcome the limitations of 2D histological approaches. MPM offers high spatial resolution and utilizes the second harmonic signal (SHG) to provide specific information about collagen fibers. This study aims to establish a tissue-clearing approach to analyze pathological human synovial tissue using label-free MPM.

OBJECTIVE

The objectives of the study are: 1) to optimize a clearing-enabled label-free MPM protocol for synovial biopsies by comparing the clearing performance of a hydrophilic reagent (CUBIC protocol) and hydrophobic reagents Ethyl Cinnamate (ECi). 2) To quantitatively evaluate autofluorescence (AF) and SHG signals from synovium to understand synovial tissue morphology, cellularity, and fibrosis.

METHODS

For tissue-clearing protocol optimization, one synovial biopsy was cut into two sections. After formalin fixation, one section underwent CUBIC clearing protocol, and the other was dehydrated and immersed in ECi. For the MPM study, 12 synovial biopsies (6 OA, 6 rheumatoid arthritis [RA]) were formalin-fixed, dehydrated, and cleared with ECi solution. All samples were collected from total knee replacement surgeries at Oulu University Hospital, Finland. MPM was conducted using a 900 nm laser, capturing the SHG signal at 450 nm and the AF signal between 470–600 nm. A 16X/0.6 NA water-immersion objective was used for imaging, with a pixel size of 0.7 µm. At first, mosaics of the whole sample were acquired at depths of 600, 1000, and 1300 µm from the sample surface. Subsequently, Z-stack images (depth: 1mm; step size: 200 microns) of the AF channel that includes the lining layer were collected and used for 3D cell segmentation. Maximum intensity projections of the Z-stack were processed through intensity thresholding, binary masking, and watershed segmentation. Only particles with an area less than 500 µm² were considered individual cells. Moreover, adipocytes and vascularity within the sub-lining layer from the 2D mosaic images were manually identified. Further, the heat maps for SHG intensity and area fraction were calculated. Finally, the tissue clearing was reserved, and the standard histopathological assessment of synovitis (Krenn scoring system) was performed.

RESULTS

ECi clearing achieved complete transparency of a synovial biopsy in 3 days (cleared around 1.2 mm), while the CUBIC protocol was still partially opaque tissue even after 3 weeks (cleared around 500 µm), as shown in Figure 1. In the optically sectioned mosaic images, OA samples had larger adipocytes but less vascularization within the sub-lining layers than RA tissues (Figure 2). The number of segmented cells mostly followed Krenn scores, particularly in RA samples. SHG analysis revealed fibrotic regions in the tissue-cleared samples through intensity analysis and area fraction calculation, which were confirmed by histological images (Figure 2).

CONCLUSION

Here, we present a workflow that allows optical clearing and label-free assessment of whole synovial biopsy. MPM provides a detailed and quantifiable examination of tissue autofluorescence and collagen-specific SHG signal analysis. This can assess synovial inflammation and remodeling (fibrosis), making this protocol a complementary tool for standard synovial histopathology.