Effect of tissue fixation on the optical properties of structural components assessed by non-linear microscopy imaging

Abstract

Fixation methods such as formalin are commonly used for the preservation of tissue with the aim of keeping their structure as close as possible to the native condition. However, fixatives chemically interact with tissue molecules, such as collagen in the extracellular matrix (ECM) or myosin, and may thus modify their structure. Taking advantage of the second- and third-harmonic generation (SHG and THG) emission capabilities of such components, we used nonlinear two-photon microscopy (NL2PM) to evaluate the effect that preservation methods, such as chemical fixatives, have on the nonlinear capabilities of protein components within mouse tissues. Our results show that depending on the preservation technique used, the nonlinear capabilities of collagen, lipid droplets and myosin microarchitecture are strongly affected. Parameters of collagen fibers, such as density and branch points, especially in collagen-sparse regions, e.g., in kidneys, were found to be altered upon formalin fixation. Moreover, cryo-freezing drastically reduced SHG signals from myosin. Our findings provide valuable information to select the best tissue fixation method for visualization and quantification of structural proteins, such as collagen and myosin by advanced NL2PM imaging techniques. This may advance the interpretation of the role these proteins play in disease.

Visible Wavelength Beam Steering in Silicon Nitride Nanophotonic Phased Arrays with a Supercontinuum Laser

“…However, we found that the beam profiles measured in the mouse cortex were significantly larger than the beam width predicted in the scattering simulation presented in Section S2. We speculate that one reason for this difference is the use of fixed tissue, which can have a higher optical scattering than fresh samples, as reported in [41,42]. Further evidence of this hypothesis is provided by the improved agreement between the experimental and simulated beam widths when using higher scattering coefficient settings in the simulation.…”

X-ray phase-contrast 3D virtual histology characterises complex tissue architecture in colorectal cancer

“…This method generates specific signals for structures such as collagen and myosin without the need for labelling. Depending on the properties of the excitation laser, the maximum possible imaging depth can range between 100 µm and 1,000 µm; however, this method performs poorly in paraffinembedded tissue (32). While the contrast produced in SRµCT is not specific to histologic features, the placement of the histologic slice into the 3D µCT context, as shown here, provides sufficient "starting point" information for extrapolation, which is a good alternative to the aforementioned methods.…”