Linkam CMS196 - CRYO-Correlative Microscopy Stage
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Linkam CMS196 Cryo-CLEM stage : overview
The Linkam CMS196 is a cryo-CLEM instrument to enable the full workflow of CLEM. Apart from maintaining the sample vitrified by means of liquid nitrogen cooling it provides proven capabilities to safely handle and transfer your cryo samples and image them with optical microscopy while keeping them free of contamination at all times. The integrated motorized, encoded stage of the latest model, together with the new LINK software enables high resolution maps of the grid to be produced quickly and easily. This provides a straight forward method of coordinate mapping required to locate the same sample in the fluorescence microscope as well as in the EM.
Although the Linkam CMS196 was designed specifically to solve the problem of how to get vitrified EM grids from the fluorescent microscope into the cryo TEM without devitrification and contamination through condensation, the stage also had to be optimized optically to enable the use of high NA lenses.
Up to 3 grids are loaded into a specially designed cassette which is transported from your plunge freezer in a small sealed container. The container is loaded into a pre-cooled dry sample loading chamber fitted on your upright fluorescent microscope. The cassette is then easily loaded onto the viewing bridge using special manipulation tools.
Electron microscopy (EM) provides structural information at very high resolution. However, it can give only restricted insight into biological and chemical processes due to limitations in staining and sample preparation processes. Fluorescence microscopy on the other hand is a very sensitive method to detect biological, chemical and genetic processes and events inside living cells.
Cryo-CLEM brings it all together: it is a new and emerging technique to combine the individual advantages from both Fluorescence and EM by imaging the same sample location with both techniques and superimposing the complementing information.
“We are using the cryo-correlative stage to screen cells trapped in a thin layer of ice prior to imaging the samples at synchrotrons in Oxford, Barcelona and Berlin. The team at Linkam Scientific have helped to drive forward our research, quickly adapting and refining the technology to deliver an elegant, flexible, user-friendly stage.” Dr. Lucy Collinson –Head of Electron Microscopy, London Research Institute
Imaging of vitrified samples under cry-conditions
In order to make a biological sample compatible with the vacuum conditions found in EM and preserve the structural detail, samples are embedded in vitrified “glass like” ice and need to be kept below -140°C. Any contact with moisture contained in the air has to be avoided since ice crystals would form immediately and contaminate the sample. Under cryo-conditions the fluorescence signals structural detail is preserved and photobleaching is significantly reduced.
Developed in collaboration with Prof. Bram Koster’s team at Leiden University Medical Centre (LUMC) in the Netherlands, the CMS196 is the result of 4 years of R&D.
“The correlative stage was developed out of the necessity for a tool that uses fluorescence microscopy to help localize structures of interest in samples for high resolution cryo electron microscopy imaging. Over the years the system has evolved into an effective and efficient instrument that enables the imaging of cryo electron microscope images after light microscopy characterization routinely.” Prof. A.J. Koster – LUMC
Features / Highlights of the Linkam CMS196
- Self-contained cryo sample observation system with automatic top-up of the sample chamber with liquid nitrogen, keeping the sample vitrified at constant -196°C and avoiding contamination
- Short startup time of only 10 minutes, no mechanical connection between sample mount and dewar, very good stability, low drift in nm range, good long-term stability
- Integrated motorised XY stage with optical encoders for high precision movement and position readout of better than 1um
- Fully Automated XY Mapping
- Mounting option for most upright microscopes from all major manufacturers
- Self-aligning magnetic sample cassette system for up to three samples
- Support for EM-grids, Auto-Grids (FEI), Membrane carriers (Leica HPM100), Bessy grids, planchettes, CryoCapsule from CryoCapCell and custom sample holders, no need to handle bare grids
- Sample cassette holder for safe and contamination-free sample loading, storage and transfer
- Connects to plunge-freezing and High-Pressure Freezing sample preparation protocols
- Integrated sensors for sample temperature monitoring
- Integrated condenser optics for transmitted light brightfield and phase contrast
- Fluorescence observation with NA of up to 0.9
- LN2 refill cycle of 35 minutes with integrated dewar, full day of independent operation with linked 3l autofill dewar
- LCD display with button control, joystick operation (option) and control via USB interface
- Automated heating cycle to drive out moisture
- More than 25 systems proven in the field worldwide, market leader in Cryo-CLEM