Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing

Abstract

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.

Optical clearing of human dura mater by glucose solutions

“…Different definitions of the optical clearing efficiency are used depending on the measure parameters, e.g., the ratio of transport scattering coefficient values before the beginning of immersion and in some time after it [36], the ratio of the collimated transmittance values after the achievement of the maximal clearing and in the intact sample [37], the ratio of total transmission intensities, including the collimated component and the diffuse one, in some time after the beginning of clearing and at the initial moment [38], the maximal resolution of the object, visible through the tissue sample in vitro, and the relative variation of the parameter, characterising the signal-to-noise ratio by means of the contrast analysis of laser speckles in vivo [39].…”

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

“…This reduction is dependent on the flow rate used in the microfluidic channel, that is, higher flow rates lead to higher shrinkage, which may be related to the fluidic compressive forces or to the osmotic pressure. Nevertheless, the authors also observed that SeeDB solution increased the green fluorescence intensity of the spheroids (Grist et al, 2016), which was attributed to the formation of Maillard reaction products (Berke, Miola, David, Smith, & Price, 2016 occurred as a consequence of the clearing process (Boutin & Hoffman-Kim, 2015). An analysis of the average FUCCI fluorescence intensity as a function of penetration depth into the sample demonstrated that the SeeDB method allowed to image the spheroids at depths greater than 250 μm, while the imaging of noncleared spheroid was limited to about 150 μm (Grist et al, 2016).…”

“…An analysis of the average FUCCI fluorescence intensity as a function of penetration depth into the sample demonstrated that the SeeDB method allowed to image the spheroids at depths greater than 250 μm, while the imaging of noncleared spheroid was limited to about 150 μm (Grist et al, 2016). Nevertheless, the authors also observed that SeeDB solution increased the green fluorescence intensity of the spheroids (Grist et al, 2016), which was attributed to the formation of Maillard reaction products (Berke, Miola, David, Smith, & Price, 2016 occurred as a consequence of the clearing process (Boutin & Hoffman-Kim, 2015). Authors also observed that 4′,6-diamidino-2phenylindole (DAPI)-labeled nuclei were difficult to distinguish.…”

3‐dimensional visualization of implant‐tissue interface with the polyethylene glycol associated solvent system tissue clearing method

“…to nearly invisible after treatment. The favourable transparency achieved with PEGASOS can be attributed to the combination of decalcification, decolourization, delipidation and the high RI (1.543) of BB-PEG clearing medium, which is close to the average RI (1.53) of decalcified bone 30,42,43. The fluorescence preservation is critical for acquiring multi-channel 3-D images after tissue clearing and enables applications of multiple transgenic mouse models to investigate implant-tissue interface.Combining with fluorescent reporter mouse lines, multi-colour 3-D investigation of implant-bone interface is becoming possible.…”

“…The fluorescence preservation is critical for acquiring multi-channel 3-D images after tissue clearing and enables applications of multiple transgenic mouse models to investigate implant-tissue interface.Combining with fluorescent reporter mouse lines, multi-colour 3-D investigation of implant-bone interface is becoming possible. Although 3-D dynamic labelling was tested using BABB clearing method,43 PEGASOS remains the only technique capable of combining dynamic labelling with endogenous fluorescent labels due to its preservation of GFP or tdTomato signal.It remains a concern whether decalcification treatment compromises the implant-bone interface. The depth of imaging is mainly determined by tissue transparency and working distance of microscope objectives.…”