3D Histological Evaluation of Growth Plate Cartilage using Confocal Laser Scanning Microscopy

Abstract

Traditional histological processing techniques can subject tissue to distortion and artifacts and provide a limited view of cell dynamics. An evolving histological technique is the use of optical microscopy to acquire high resolution image stacks to visualize tissue in‐depth. Using the confocal laser scanning microscope (CLSM), it is possible to collect an image stack to process into a 3‐D perspective. In bone, autofluorescence is largely due to the collagen component. We aimed to use the autofluorescent properties of the collagen in the growth plate of murine femurs to characterize and evaluate the resting, proliferating and hypertrophic zones of articular cartilage. Murine distal femurs were sectioned along their longitudinal axis. The bones were allowed to incubate in refractive index mating solution (RIMS) media for two days prior to imaging. Z stacks of approximately 80–100 μm in depth were captured using ACS APO 40x/1.15 oil on Leica SPE confocal microscope. Acquisition of the z‐stacks were obtained in the blue‐green and green‐yellow emission spectrum. Incubation of tissue in RIMS solution reduced the opacity of the tissue and facilitated in‐depth optical imaging. Imaging was focused on the growth plate (Fig 1). Sequential image stacks were acquired with confocal microscope and Leica LAS software algorithms were used to compose 3‐D images. Image stacks were further manipulated in ImageJ and ICY to isolate and count the chondrocytes, respectively. 3D histology can be accomplished with minimal processing cost and can provide an alternate perspective of cell dynamics. Intrinsic biomolecules in tissue possess endogenous fluorophores that can at times be advantageous when evaluating intact tissue architecture. Confocal microscopy in conjunction with a refractive index matching solution rendered the murine femurs less opaque and allowed for greater photon penetration and thus resolution of spatial‐temporal structures in the bone matrix. Examination of the matrix and morphology of the cells within, provided an indication of the development of the cells and progression of ossification.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.