MAVIS: an integrated system for live microscopy and vitrification.
Cryo-electron microscopy of vitrified biological samples can provide three-dimensional reconstructions of macromolecules and organelles within bacteria and cells at nanometer scale resolution, even in native conditions. Localization of specific structures and imaging of cellular dynamics in cellular cryo-electron microscopy is limited by (i) the use of cryo-fixation to preserve cellular structures, (ii) the restricted availability of electron dense markers to label molecules inside cells and (iii) the inherent low contrast of cryo electron microscopy. These limitations can be mitigated to a large extend by correlative light and electron microscopy, where the sample is imaged by both light and electron microscopy. Here we present a Microscopy and Vitrification Integrated System (MAVIS) that combines a light microscope with a plunger to vitrify thin specimens. MAVIS provides the capability for fluorescence light microscopic imaging of living cells and bacteria that are adhered to an electron microscopy grid and subsequent vitrification within a time frame of seconds. The instrument allows targeting of dynamic biological events in time and space by fluorescence microscopy for subsequent cryo light and electron microscopy. Here we describe the design and performance of the MAVIS, illustrated with biological examples.
Department of Molecular Cell Biology, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: r.i.koning@lumc.nl.
Roman I
RI
Koning
Department of Molecular Cell Biology, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Frank G
FG
Faas
Department of Instrumental Development, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Michael
M
Boonekamp
Department of Instrumental Development, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Bram
B
de Visser
Department of Instrumental Development, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Jan
J
Janse
Department of Molecular Cell Biology, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Joop C
JC
Wiegant
Department of Infectious Diseases, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Anna
A
de Breij
Department of Molecular Cell Biology, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Joost
J
Willemse
Department of Infectious Diseases, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Peter H
PH
Nibbering
Department of Molecular Cell Biology, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Hans J
HJ
Tanke
Department of Molecular Cell Biology, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden University Medical Center, Leiden, The Netherlands.
Abraham J
AJ
Koster
D002460:Cell Line; D020285:Cryoelectron Microscopy / Q000379:methods*; D015925:Cryopreservation / Q000379:methods; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008859:Microscopy, Polarization / Q000379:methods*; D058989:Vitrification
D046911:Macromolecular Substances
Correlative microscopy; Cryo-microscopy; Electron microscopy; Light microscopy