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

M. Pradeep , S. Das Gupta , T. Zhang , T. Liimatainen , V.M. Pohjanen , P. Lehenkari , S. Palosaari , M. Finnilä
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引用次数: 0

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.
滑膜炎的新镜头:全贴装人病理滑膜的多光子显微镜无标记成像
骨关节炎进展的典型标志之一是滑膜炎症,也称为滑膜炎。病理性滑膜炎评估通常通过传统的二维组织病理学进行,它提供有限的方向依赖信息,需要化学标记,并且本质上是破坏性的。使用多光子显微镜(MPM)对整个滑膜组织活检和非破坏性光学切片进行组织清除可以克服二维组织学方法的局限性。MPM提供高空间分辨率,并利用二次谐波信号(SHG)提供胶原纤维的特定信息。本研究旨在建立一种组织清除方法,利用无标记MPM分析病理人滑膜组织。本研究的目的是:1)通过比较亲水试剂(CUBIC方案)和疏水试剂肉桂酸乙酯(ECi)的清除性能,优化滑膜活检的无标记MPM方案。2)定量评价滑膜的自体荧光(AF)和SHG信号,了解滑膜组织形态、细胞结构和纤维化情况。方法为优化组织清除方案,将滑膜活检切片切成两段。福尔马林固定后,一个切片进行CUBIC清除方案,另一个脱水并浸泡在ECi中。在MPM研究中,12例滑膜活检(6例OA, 6例类风湿关节炎[RA])用福尔马林固定,脱水,并用ECi溶液清除。所有样本均来自芬兰奥卢大学医院的全膝关节置换手术。使用900 nm激光器进行MPM,捕获450 nm处的SHG信号和470-600 nm处的AF信号。成像采用16X/0.6 NA水浸物镜,像元尺寸0.7 µm。首先,在距离样品表面600、1000和1300 µm的深度处获取整个样品的马赛克。随后,获得深度为1mm;步长:200微米)的AF通道(包括衬里层)被收集并用于3D细胞分割。通过强度阈值分割、二值掩蔽和分水岭分割,对z叠加的最大强度投影进行处理。只有面积小于500 µm²的粒子才被认为是单个细胞。此外,从二维马赛克图像中手动识别亚衬层内的脂肪细胞和血管。进一步,计算了地震强度和面积分数的热图。最后保留组织清理,进行滑膜炎的标准组织病理学评估(Krenn评分系统)。结果seci清除在3天内实现了滑膜活检的完全透明(清除约1.2 mm),而CUBIC方案即使在3周后仍然是部分不透明的组织(清除约500µm),如图1所示。在光学切片的马赛克图像中,OA样本比RA组织具有更大的脂肪细胞,但在亚衬里层内血管化较少(图2)。分节细胞的数量大多跟随Krenn评分,特别是在RA样本中。SHG分析通过强度分析和面积分数计算,在组织清除的样本中发现了纤维化区域,并通过组织学图像证实了这一点(图2)。在这里,我们提出了一个工作流程,允许光学清除和无标签评估整个滑膜活检。MPM提供了组织自身荧光和胶原特异性SHG信号分析的详细和可量化的检查。这可以评估滑膜炎症和重塑(纤维化),使该方案成为标准滑膜组织病理学的补充工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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Osteoarthritis imaging
Osteoarthritis imaging Radiology and Imaging
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