Advancing tissue clearing and expansion methods for high-resolution volumetric imaging of biological samples
Tid: Må 2023-01-23 kl 14.00
Plats: Sal Air/fire, Science for Life Laboratory, Tomtebodavägen 23A, Solna
Språk: Engelska
Ämnesområde: Biologisk fysik
Respondent: Steven Edwards , Biofysik, Brismar
Opponent: Professor Etsuo Susaki, Juntendo University
Handledare: Professor Hjalmar Brismar, KTH
QC 221214
Abstract
The development of advanced light microscopes, capable of imaging samples at ever-higher spatial resolution and increasing speeds is an ongoing endeavour. The sample itself is an integral part of the microscope and, unlike the intricately positioned and highly polished lenses, it is an optically unpredictable component. Composed of a mixture of biological polymers, lipids, inorganic ions, the sample is a hindrance to the otherwise predictable path of light and frequently degrades the microscope’s performance. The optical properties of the sample are therefore of equal importance to those of the microscope hardware. Preparing a sample for microscopy involves tuning these optical properties to maintain or in some cases, enhance the microscope’s performance.
Optical tissue clearing includes a wide range of protocols aiming at making large, opaque biological samples optically transparent. This in turn facilitates volumetric imaging of whole organ systems and negates the requirement for physical sectioning of the sample. Expansion microscopy is a technique in which biological samples can be physically magnified. This method not only clears the sample but improves the effective resolution that can be achieved in a microscope. Optical tissue clearing and expansion microscopy protocols must be further adapted and developed to address the variety of biological samples, ranging from single cells to complex tissues and model organisms.
In Paper I, we developed a clearing protocol, termed CUBIC-f, which was optimised for fragile samples. We used this method to quantify neuronal cell density and trace neuronal projections in the salamander brain. In Paper II, we explored the use of expansion microscopy on 3D cell cultures to perform high-resolution imaging with improved labelling and signal-to-background ratio, resulting in more accurate image segmentation. In paper III, expansion microscopy was used in combination with light-sheet and STED microscopy to reveal the role of cerebrospinal fluid-contacting neurons in the central canal of the lamprey spinal cord. Finally, in Paper IV we combined non-canonical amino acid fluorescent labelling with expansion microscopy, demonstrating two colour super-resolution imaging of the alpha and beta subunit of the sodium pump with minimal fluorophore linkage error.