miércoles, 30 de agosto de 2017

Stowers researchers solve 3D structure of critical protein complex using microscopy techniques

Stowers researchers solve 3D structure of critical protein complex using microscopy techniques



Cryo-CLEM Techniques

Correlative light and electron microscopy (CLEM) is a technology that combines Fluorescence microscopy (FM) and Electron microscopy (EM). In FM, the marker of interest is tagged or engineered with fluorophores like green fluorescent protein (GFP).
When the sample is illuminated with the light of a specific wavelength, the fluorophores absorb the light and emit light of longer wavelengths to help the detector identify the fluorophore tagged-marker of interest in a dense pool of unlabeled background. Poor spatial resolution accompanied with the inability to provide the structural details of the fluorescently tagged marker of interest are some of the most significant limitations of FM.
As compared to optical microscopes that use visible light, transmission electron microscopy (TEM) utilizes an electron beam to map the local electron density differences in a sample to visualize objects. When the electron beam hits the ultra-thin specimen in vacuum, the accelerated electrons are either absorbed or scattered. A sophisticated system of electromagnetic lenses form an image based on the electron scattering pattern1. TEM sample preparation includes the removal of all the water from the sample by using a series of alcohol concentrations.
After the removal of water, the sample is passed through a transition solvent, such as propylene oxide, infiltrated and embedded in resin (such as Epoxy and LR White) that is then polymerized into a block. The resin block is then trimmed to expose the sample and cut into very thin sections, typically within the 50-70 nm size range, by a diamond or glass knife using an ultra-microtome.
In Cryo-electron microscopy (Cryo EM), preparation involves rapidly freezing the sample to below -140 °C in order to vitrify all the water in it. The samples are then cut into thin sections of 40 – 100 nm by diamond or glass knife in a cryo ultra-microtome, which maintains the temperature of the sample below -140 °C while cutting sections2. Similar to the TEM, specimens are collected on an EM grid with a diameter of 3.05 mm.
Single images with resolutions of less than 5 nm are possible with Cryo EM and computational averaging methods can further help to attain resolutions of 0.3 nm. In the case of EM, it is particularly difficult to differentiate between the specific kinds of molecules in a specimen.
CLEM can be used in different modalities depending upon the type of EM sample prepared and the type of FM being performed. The first approach is to perform live fluorescence imaging prior to preparing samples for EM. This allows a time delay of at least a few seconds in transitioning from FM to EM and in fixing or immobilizing the sample, which makes it unsuitable to identify very dynamic processes or a very precise localization of signal.
In the second approach, the sample is prepared for EM, then both FM and EM are performed on the same sample, allowing no time delay and thus, helps in achieving high correlation accuracy.
Cryo-CLEM technique uses the low temperature FM (Cryo FM) to identify the target of interest and provides the structure or morphological information at high resolution using cryo EM. The most significant challenge with Cryo EM is that the sample should remain at a temperature that is lower than -140 °C from initial sample collection to the final imaging process to prevent the formation of crystalline ice3.
Samples should not be exposed to humidity, as this can lead to water condensation. A cryo-stage ensures that the sample is kept at low temperatures while imaging. Several cryo stage and workflow configurations have recently emerged, making cryo-CLEM available in upright and inverted setups with different working distances (WD) and numerical apertures (NA). High-resolution optical systems with high sensitivity utilize fiducial gold beads, ensuring accurate coordinate transformation to provide high-accuracy correlation3.
In summary, Cryo-CLEM is a useful microscopy technique that helps in the identification and high resolution structural elucidation of rare and dynamic molecules of interest in biological samples and other microscopic organisms. In fact, recent advances in cryo-CLEM techniques are allowing Scientists to study how viruses infect and modify the cells they infect4.
Image Credit:
imredesiuk/ Shutterstock.com
  1. Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M, and Briggs JAG: Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. JCB 192 (1): 111–19 (2011)
  2. Schorb M, Briggs JAG: Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity. Ultramicroscopy 143: 24–32 (2014).
  3. "Cryo CLEM – the Combination of Cryo Fluorescence Microscopy with Cryo Electron Microscopy." Leica Sciencelab. Web. http://www.leica-microsystems.com/science-lab/clem/cryo-clem-the-combination-of-cryo-fluorescence-microscopy-with-cryo-electron-microscopy/.
  4. "Advancement in Microscopy Gives Scientists Clearer Picture Of Viruses." Counsel & Heal. 09 Jan. 2017. Web. http://www.counselheal.com/articles/29818/20170109/advancement-in-microscopy-gives-scientists-clearer-picture-of-viruses.htm.
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