Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy.
The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.
Lu
Zonghuan
Z
Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Shaikh
Tanvir R
TR
Barnard
David
D
Meng
Xing
X
Mohamed
Hisham
H
Yassin
Aymen
A
Mannella
Carmen A
CA
Agrawal
Rajendra K
RK
Lu
Toh-Ming
TM
Wagenknecht
Terence
T
D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D007700:Kinetics; D012270:Ribosomes / Q000648:ultrastructure
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