Samples of biological specimens infected with class 3 pathogens can be prepared for observations by Transmission Electron Microscopy, including:Â
â Morphological analyzes, using high-resolution images (0.4 nm) of viruses, bacteria, organelles, cells. Pre-observation techniques are adapted to the nature of the specimen.
â Immuno-labeling (with labeling before or after inclusion in coating resins) enabling precise localization of one or more specific epitopes inside a virus, organelle or a cell
These techniques allow to follow a particular molecule from its synthesis to the sites of storage and secretion or on the contrary its entry and its intracellular path.
â Observations in conditions of negative staining, reserved for small biological objects (less than 100 nm) which are deposited directly on a grid covered with a very thin layer of plastic or carbon. Several dyes can be used and the choice depends on the nature of the sample (viruses, bacteria etc.)
The CEMIPAI platform proposes a unique opportunity to image biological samples throughÂ Photoactivation Localization Microscopy (PALM) installed in the BSL3 during 2017.Â
PALM achieves nanometre scale resolution by exploiting the properties of photoswitchable fluorophores. Â Exposing the fluorophores to a certain wavelength results in a change in emission spectra â photoactivatable and reversible photoswitachable fluorophores become fluorescent, and reversible photoswitchable fluorophores change from one fluorescent state to another.
The basic principle behind PALM is that a low-power activating laser beam will âstochasticallyâ convert the fluorophores, so that only a few will be in their âactiveâ or âonâ state. These molecules are then imaged by a high-power illuminating laser beam to immediately convert them back to their âinactiveâ or âoffâ state, e. g. by photobleaching. This process of activation and inactivation is then repeated over thousands of frames to ensure all molecules have been imaged.Â
Multiple images are taken with total internal reflection fluorescence (TIRF) microscopy, and then combined to yield a single, high-resolution image with a high density of features.
TIRF-PALM microscopeÂ ModelÂ Nikon Ti-E
Diodes Laser:Â 405nm, 488nm, 561nm, 641nm,Â Objectives:Â 20x, 40x, 100x
EMCCD Camera IXON 897Â (512Ă512 chip, pixel 16Â”m)
Motorized stage XYZ with autofocus system PFS
Atomic Force Microscopy (Bio AFM)Â
Â courtesy fromÂ M. Baclayon et al., 2010. DOI:Â 10.1039/B923992H
Planned for late 2017 or early 2018