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Figure 1 : HPM live µ, CryoCapCell
par Aurélien Dupont, MRic-TEM platform
The HPM live µ from CryoCapCell (fig.1) recently arrived on the MRic-TEM platform. This device is used to freeze samples such as cell culture or small tissues (dimensions of the freezing room : A circle of 2 mm diamateter and up to 200 µm thickness). The fixation technic, so called High Pressure Freezing (HPF), is used in TEM & Cryo-TEM for different purposes: getting ultrastructure closer to native state than with chemical fixation or for enabling Correlated Light & Electron Microscopy (CLEM) experiments.
The first results of freezing samples using our HPM live µ came, last Month, from O. Nicolle (M3PE team, IGDR) who froze WT C. elegans worms. The ultrastructure is well preserved with minimal artifacts (fig. 2).
Figure 2 : C. elegans WT, O. Nicolle
A dedicated LSM 900 inverted confocal microscope from Zeiss is connected to the HPM live µ (fig. 3 A). This configuration allows to freeze the sample within less than 2 sec after capturing, using fluorescence microscopy (fig. 3 B), a rare and fast event and to find it back at the electron microscope level.
This technic has just been tested by R. Gibeaux’s team (MiToS, IGDR) and the results are promising. The fluorescent image before freezing (fig. 4 A) and the fluorescent image in cryo conditions (fig. 4 B) were taken at a different focus but many structures can be found again after the high pressure freezing. Next step, a real CLEM experiment !
Figure 3: (A) LSM 900 inverted confocal from Zeiss connected to the HPM live µ, CryoCapCell. (B) Imaging position for the samples
This technic has just been tested by R. Gibeaux’s team (MiToS, IGDR) and the results are promising. The fluorescent image before freezing (fig. 4 A) and the fluorescent image in cryo conditions (fig. 4 B) were taken at a different focus but many structures can be found again after the high pressure freezing. Next step, a real CLEM experiment !
Figure 4 : (A) Fluorescent image of microtubules before HPF. (B) Fluorescent image of microtubules, in cryo condition, after HPF.
More projects to come soon ! If you want to know more about the devices, the technique or just talk about science, please feel free to contact Aurélien Dupont (aurelien.dupont@univ-rennes.fr) & Romain Gibeaux (romain.gibeaux@univ-rennes.fr).
