Sample Preparation
Samples often require preparation prior to viewing under light and electron microscopes. The type of sample preparation depends on the sample and what you want to investigate.
Biological samples often require more complex sample preparation. We recommend you discuss sample collection and preparation with an Adelaide Microscopy staff member before proceeding. Good sample preparation is the key to success.
Please contact Adelaide Microscopy for more details regarding sample preparation equipment booking or usage.
SEM
In most cases biological samples must be dehydrated for SEM. This generally involves processing through solvents and/or use of a Critical Point Dryer, and mounting on a stub.
Most samples are then coated with a thin layer of carbon or platinum to prevent charging under the electron beam in the SEM. Ensure your samples are individually labelled before you bring them to Adelaide Microscopy for coating. Allow at least 3 business days prior to your microscope booking. Coating is included in the per-sample processing fee. If you plan to do your microscopy offsite, there are additional fees for coating and staff assistance.
Some samples can be viewed uncoated or hydrated (wet) on the Quanta 450 in ESEM mode. Cryo techniques can also be used to preserve the structure of the sample in its natural state on the Philips XL30 SEM.
TEM
There are many sample preparation techniques for TEM; solutions can be dried on a TEM grid which has a thin film support, FIB or milling of a thin sample, etc. Biological samples for TEM generally require fixation, dehydration, embedding, and sectioning on an ultramicrotome prior to viewing in the microscope. Adelaide Microscopy staff can assist you to decide on the best approach for your aims.
BIOLOGICAL
Optical Clearing of Tissues
Clearing samples renders tissues transparent, reduces the light scattering associated with refractive index mismatches and results in an improvement of imaging depth. Optical clearing techniques has improved and multiplied in recent years with the adoption of Lightsheet microscope technology. This page aims to give an overview of some of these techniques and what clearing techniques Adelaide Microscopy recommends within the facility.
There are a number of different techniques to clear tissues and the type of optical clearing method depends on a number of factors, including size of sample and fluorescent labelling.
General reviews of different clearing techniques:
Clarifying Tissue Clearing
Douglas S. Richardson1,2,* and Jeff W. Lichtman1,2,3,* 1Harvard Center for Biological Imaging 2Department of Molecular and Cellular Biology 3Center for Brain Science Harvard University, Cambridge, MA 02138, USA *Correspondence: drichardson@fas.harvard.edu (D.S.R.), jeff@mcb.harvard.edu (J.W.L.)
High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging
Weizhe Li, Ronald N. Germain & Michael Y. Gerner Nature Protocols volume 14, pages 1708–1733 (2019)
Zeiss Tissue Clearing Seminar, Lightsheet 7, University of Adelaide
Specific methods available in Adelaide Microscopy:
X-Clarity (Note that ETC differs depending on tissue)
Ce3D
Solvent based clearing
Our Lightsheet can also accommodate RI from 1.33-1.56 so other clearing methods are compatible. Clearing methods, including alternatives where applicable, will be discussed with staff in the initial meetings.