SCIENCE CHINA Life Sciences, Volume 61 , Issue 11 : 1312-1319(2018) https://doi.org/10.1007/s11427-018-9380-3

Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy

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  • ReceivedAug 14, 2018
  • AcceptedSep 17, 2018
  • PublishedNov 2, 2018


Remarkable progress in correlative light and electron cryo-microscopy (cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope (cryo-FM). Here, we describe an ultra-stable super-resolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than 0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system (average single molecule localization accuracy of ~13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy (SMLM) and cryogenic super-resolution correlative light and electron microscopy (csCLEM).

Funded by

the National Key R&D Program of China(2016YFA0500203,2016YFA0502400,2017YFA0504700,2017YFA0505300)

the National Natural Science Foundation of China(31661143041,31127901)

Joint Program between Chinese Academy of Sciences and Peking University.


This work was supported by the National Key R&D Program of China (2016YFA0500203, 2016YFA0502400, 2017YFA0504700, 2017YFA0505300), the National Natural Science Foundation of China (31661143041, 31127901) and Joint Program between Chinese Academy of Sciences and Peking University.

Interest statement

The author (s) declare that they have no conflict of interest.



Figure S1 Calibration curve used for obtaining the axial position.

Figure S2 Preparation of the cryo-section.

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    The thermal stability of the objective lens temperature controller and sample stage. A and C, Schematic depiction of the objective lens and sample holder temperature control module. B and D, The temperature curve of the two units for 12 h.

  • Figure 2

    The mechanical stability of the ultra-stable super-resolution cryo-FM at 93 K. A, The first frame of the 36,000 frame dark red beads (diameter 200 nm) images with sampling frequency of 2 Hz (exposure time 500 ms) at 93 K. Scale bar, 5 μm. B and C, The average horizontal and vertical drift of the center position of the beads in the red circle in A for 5 h. D, The distribution of the uncorrected average center position of the beads in the blue circle in A in three dimensions for 5 h. E, The distribution of the corrected center position of the bead in the blue circle in A in three dimension for 5 h.

  • Figure 3

    The workstation and workflow for loading and transferring the vitrified samples. A, Overview of the workstation. B, Loading chamber. C, Operating platform. D, Transfer chamber. E, Workflow of loading and transferring the vitrified samples. First, transferring the sample box into loading chamber filled with LN2. Second, taking out the EM grid with vitrified samples from the sample box. Third, fixing the EM grid on the sample holder through the copper ring. Finally, putting the sample holder into transfer chamber.

  • Figure 4

    The super-resolution imaging capability of the ultra-stable super-resolution cryo-FM. A and B, Wide-field image (A) and PALM image (B) of the 150 nm thick cryo-section of a TOM20-Dronpa-labeled HEK293 cell. Scale bar: 1 μm. C, Distribution of the localization error of all single-molecule data in (B). D, Normalized intensity profiles along the white line in (A) (green solid line) and (B) (yellow solid line) and the corresponding Gaussian fitting curve (red and blue dotted lines, respectively).

  • Figure 5

    Schematic configuration of the ultra-stable super-resolution cryo-FM. A, Home-built upright fluorescence microscopy. B, Schematic drawing of the cryostat. C, Details of the “cage” structure. D, Details of the image splitter. M, mirror; DM, dichroic mirror; Tlens, tube lens; AOTF, acousto-optic tunable filter; AFC, fiber coupler; SFC, fiber adapter.

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