The conservation of the native state during sample preparation is mandatory for a correct interpretation of any micrograph. Particularly for EM, the preservation of the pristine architecture is challenging. If not imaged in situ (1), two different strategies can be followed to prepare hydrated samples for electron microscopy: the conventional and the cryogenic routine. Conventional preparation protocols typically rely on the chemical fixation and staining of the sample material, whereby both steps are known to induce artefacts (2). The second preparation routine, introduced by Moor (3) and Dubochet (4), circumvents chemical artefacts. The basic principle of every cryogenic (cryo) preparation protocol is the physical immobilization of the sample in a frozen-hydrated solid state, by vitrifying the sample within milliseconds. Due to the excellent structural preservation cryo imaging techniques have gained increasing popularity (5, 6).

However, the handling of a vitrified sample becomes rather delicate. Two prerequisites must be fulfilled during the entire handling in order to maintain the artefact free conservation of the specimen: 1) the sample must be kept well below the de-vitrification temperature of water (approximately -137°C) in order to avoid structural rearrangements due to ice crystal growth (5). As a consequence of the permanent cooling, the sample material acts as a cold trap and is therefore prone to contamination. 2) Thus, the sample must be transferred within an anhydrous environment all the time. In the case of the latest preparation protocols or imaging strategies this has been proven to be particularly challenging, since these methods include several transfer steps, either due to their extensive post-processing or complex workflow (7).

In the past, several cryo-transfer concepts were introduced mainly for cryo SEM, with bulk add-ons and cryo-stages inside the microscope to allow e.g. high-vacuum cryo-transfer (8), which is not feasible for “in-lens” systems like S/TEM’s. Standard cryo-transfer systems for cryo-TEM, however, work under ambient pressure from liquid nitrogen direct into the load lock of cryo-TEM.

Here, we present a high-vacuum cryo-transfer system that overcomes the limitations of existing systems. The system includes four parts: 1) sample cartridge (Fig 1a), 2) storage device (Fig 1b), 3) high-vacuum cryo shuttle (Fig 1c) and 4) a side-entry TEM cryo-stage. Moreover, our solution offers connectivity between different kind of instruments not limited to post-lens systems (8), enabling new types of multimodal imaging approaches by transferring cryo-samples between different imaging and manipulation devices (Fig 1d). The performance of the developed system is demonstrated on an “in-lens” cryo-STEM. In order to determine the quality of the transfer process, the temperature and pressure level were recorded during the entire transfer. Moreover, prior and subsequent to the cryo-transfer, the mass of the TMV as well as the thickness of a carbon film were measured and compared. Here, any possible contamination would falsify the scattering characteristics of the sample material, and consequently cause an apparent increase in mass or thickness, see also (9).

REFERENCES

(1) M. J. Dukes et al, Microsc. Microanal. 20 (2014) p.338.

(2) M. Pilhofer et al, Environ. Microbiol.16 (2014) p.417.

(3) H. Moor, and K. Mühlethaler, J. Cell Biol. 17 (1963) p.609.

(4) J. Dubochet, and A.W. McDowall, J. Microsc. 124 (1983) p.3.

(5) A. Al-Amoudi et al, EMBO J., 23 (2004) p. 3583.

(6) W. Kühlbrandt, Science 6178 (2014) p. 1443.

(7) A. Rigort and J. M. Plitzko, Arch. Biochem. Biophys. 581 (2015), p. 122.

(8) M. Ritter et al, Microsc. Microanal. 5 Suppl. 2 (1999) p. 424.

(9) S.Tacke et al, Biophys. J. 110 (2016), p. 758.

(10) Rudolf Reichelt initiated this project. Unfortunately, he passed away on 2^nd October 2010, too early to see the final results. This research was supported by the DFG Grant RE 782/11. V. Krzyzanek acknowledges the support by the grant 14-20012S (GACR).

Figures:

Figure 1 a) Sample cartridge. b) Storage device. c) High-vacuum cryo transfer. d) Proposed working routine.

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/a-versatile-high-vacuum-cryo-transfer-system-for-cryo-microscopy-and-analytics/