A simple flash and freeze system for cryogenic time-resolved electron microscopy
As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-03-01
|
| Series: | Frontiers in Molecular Biosciences |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2023.1129225/full |
| _version_ | 1811158911689949184 |
|---|---|
| author | Biddut Bhattacharjee Md Mahfuzur Rahman Ryan E. Hibbs Michael H. B. Stowell |
| author_facet | Biddut Bhattacharjee Md Mahfuzur Rahman Ryan E. Hibbs Michael H. B. Stowell |
| author_sort | Biddut Bhattacharjee |
| collection | DOAJ |
| description | As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies. |
| first_indexed | 2024-04-10T05:32:24Z |
| format | Article |
| id | doaj.art-77420cf0a25c45588bee127d55b8db47 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-04-10T05:32:24Z |
| publishDate | 2023-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-77420cf0a25c45588bee127d55b8db472023-03-07T05:57:08ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2023-03-011010.3389/fmolb.2023.11292251129225A simple flash and freeze system for cryogenic time-resolved electron microscopyBiddut Bhattacharjee0Md Mahfuzur Rahman1Ryan E. Hibbs2Michael H. B. Stowell3University of Colorado Boulder, Boulder, United StatesUniversity of Texas Southwestern Medical Center, Dallas, United StatesUniversity of Texas Southwestern Medical Center, Dallas, United StatesUniversity of Colorado Boulder, Boulder, United StatesAs the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.https://www.frontiersin.org/articles/10.3389/fmolb.2023.1129225/fullCryoEMtime resolvedLEDoptogenetic activationcaged compoundsplunge freezer |
| spellingShingle | Biddut Bhattacharjee Md Mahfuzur Rahman Ryan E. Hibbs Michael H. B. Stowell A simple flash and freeze system for cryogenic time-resolved electron microscopy Frontiers in Molecular Biosciences CryoEM time resolved LED optogenetic activation caged compounds plunge freezer |
| title | A simple flash and freeze system for cryogenic time-resolved electron microscopy |
| title_full | A simple flash and freeze system for cryogenic time-resolved electron microscopy |
| title_fullStr | A simple flash and freeze system for cryogenic time-resolved electron microscopy |
| title_full_unstemmed | A simple flash and freeze system for cryogenic time-resolved electron microscopy |
| title_short | A simple flash and freeze system for cryogenic time-resolved electron microscopy |
| title_sort | simple flash and freeze system for cryogenic time resolved electron microscopy |
| topic | CryoEM time resolved LED optogenetic activation caged compounds plunge freezer |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2023.1129225/full |
| work_keys_str_mv | AT biddutbhattacharjee asimpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy AT mdmahfuzurrahman asimpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy AT ryanehibbs asimpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy AT michaelhbstowell asimpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy AT biddutbhattacharjee simpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy AT mdmahfuzurrahman simpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy AT ryanehibbs simpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy AT michaelhbstowell simpleflashandfreezesystemforcryogenictimeresolvedelectronmicroscopy |