Ice Contamination
Table 1: A summary of the ice contamination issues studied in our survey, the conventional cryo-ET practices, and the solutions targeting the cryo-ET ice contamination issues that were recently made commercially available.
Ice contamination category
Sample mounting Conventional approach
C-clipping, shuttle loading, and cassette loading performed in a bench-top preparation station open to the atmosphere, risking crystalline ice contamination.
Sample transfer Cryo-FLM
Use of a sample transfer device that transfers at low vacuum, risking amorphous ice contamination.
Stand-alone FLM equipped with a cryo stage requiring an extra sample transfer step to the cryo-FIB/SEM; cryo-FLM imaging takes place at atmospheric pressure, risking extra ice contamination.
Lamella preparation Cryogenic samples are unprotected from the residual moisture in the SEM chamber and suffer from amorphous ice contamination at a rate of 30–50 nm h-1
. To mitigate the sample-mounting ice contamination issues,
Tacke et al. designed a glovebox (GB) to minimize sample mount- ing-related ice contamination [10]. Teir GB is purged with dry nitrogen gas and thereby prevents moisture condensation on the cryogenic samples. Te GB is equipped with a LN2
-filled prepara-
tion table with C-clipping, shuttle, and cassette loading modules. Te user can slide the table on a rail to position the appropriate module in front of the user for ease of use. Te rubber gloves are holed and allow for the users’ fingers to protrude through to retain the full agility needed for manipulating delicate samples. To mitigate the transfer-related ice contamination issues, Tacke et al. fitted their GB with a load lock, to which a high-vacuum cryo transfer (HVCT) can be docked. Te sample can be trans- ferred from the preparation table into the HVCT without expo- sure to humid air, and the HVCT can be pumped to high vacuum (1 × 10-6
sample chamber wall is furthermore cooled by LN2
mbar) and thereby extremely low moisture. Te HVCT to keep the
sample vitrified during transfer. Using both their GB and HVCT, Tacke et al. demonstrated a decrease from 20% crystal ice contami- nation to 9% [10]—more than 2-fold better. In 2021, Delmic Cryo B.V. (Delſt, Te Netherlands) commercialized the GB and HVCT as the CERES Clean Station and CERES Vitri-Lock, respectively. Cryo-FLM-associated ice contamination. Cryo-FLM
imaging can be used to identify the sample ROIs that are fluo- rescently labeled and improve lamella targeting. However, 32% of our survey participants pointed to the cryo-FLM steps being problematic. Until recently, fluorescence-guided lamella targeting was done by cryo-FLM imaging of the sample in a stand-alone cryo-FLM equipped with a cryogenic stage, and subsequent transfer of the sample to a cryo-FIB/SEM for lamella milling. Tis practice risked increasing ice contamination on the samples due to the extra transfer steps added to the work- flow. Moreover, the samples were imaged at ambient pressure, further presenting opportunities for ice contamination. Gorelick et al. demonstrated an in situ cryo-correlative light and electron microscopy (cryo-CLEM) workflow by integrating
34
Recently made commercially available solutions targeting cryo-EM ice contamination issues
CERES Clean Station allows for sample mounting within a <1 ppm water enclosure, minimizing chances for crystalline ice contamination.
CERES Vitri-Lock transfers cryo samples at high vacuum, minimizing amorphous ice contamination.
METEOR, which can be integrated in TFS, Zeiss, and Tescan cryo-FIB/SEMs, and TFS iFLM, which can be integrated in TFS Aquilos 2, reduce the number of transfer steps in the cryo-ET workflow and allow for cryo-FLM imaging at high vacuum, thereby reduce ice contamination.
CERES Ice Shield is designed to protect the cryogenic samples from amorphous ice growth, especially during FIB milling, and therefore keeps the amorphous ice growth to a minimum.
a FLM into a cryo-FIB/SEM [11]. In situ cryo-FLM reduces ice contamination associated with the transfer steps and imaging at ambient pressure and enables a more streamlined CLEM pro- cess as the same sample stage is used. In October 2020, Delmic Cryo B.V. (Delſt, Netherlands) launched METEOR [12], the first commercially available cryo-FLM, which can be integrated with Termo Fisher Scientific, Zeiss, and Tescan cryo-FIB/ SEMs to help cryo-ET users achieve better sample yields. Since 2021, another commercial cryo-FLM called the iFLM has also become available for the TFS Aquilos™ 2 cryo-FIB. Lamella preparation-associated ice contamination.
While the cryo-FIB/SEM chamber typically operates at a high vacuum (around 1 × 10-7
mbar) and the level of moisture in the
SEM chamber is very low, the residual moisture in the cham- ber can still form amorphous ice layers. Te ice contamination rates reported in the literature [10] and by our survey partici- pants are around 30 to 50 nm H-1
. Lamellae are typically rough-
milled to around 500–800 nm, aſter which they are polished to 150–300 nm. During a lamella milling session, the lamellae may be in the chamber for several hours during the polishing ses- sion. Te amorphous ice layers decrease TEM image contrast, resulting in significant data degradation, and limit the number of lamellae that can be prepared in one session. Tacke et al. developed a method to minimize amorphous ice
growth on the sample. Te method involves using a LN2-cooled
cryo-shutter inserted between the SEM column and the sample [10]. Te cryo-shutter type II described in their publication fea- tures a hole of 1 mm diameter, through which the FIB can mill the lamellae. At the same time, the cryo-shutter protects the sample from the direct line of sight of the SEM column, which is a source of ice contamination. Te LN2
cooling provides a cryo pumping
effect and lowers the partial pressure of water in the sample vicinity to 4 × 10-9
mbar [10]. Te type II cryo-shutter design successfully
eliminated the amorphous ice growth in the SEM such that the ice growth became undetectable [10]. Delmic Cryo B.V. has commer- cialized this cryo-shutter type II into the CERES Ice Shield.
www.microscopy-today.com • 2022 May
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72
