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. 2021 Oct 26;8(5):054302.
doi: 10.1063/4.0000129. eCollection 2021 Sep.

Microsecond melting and revitrification of cryo samples

Jonathan M Voss  1 , Oliver F Harder  1 , Pavel K Olshin  1 , Marcel Drabbels  1 , Ulrich J Lorenz  1
Affiliations

Microsecond melting and revitrification of cryo samples

Jonathan M Voss et al. Struct Dyn. .

Abstract

The dynamics of proteins that are associated with their function typically occur on the microsecond timescale, orders of magnitude faster than the time resolution of cryo-electron microscopy. We have recently introduced a novel approach to time-resolved cryo-electron microscopy that affords microsecond time resolution. It involves melting a cryo sample with a heating laser, so as to allow dynamics of the proteins to briefly occur in the liquid phase. When the laser is turned off, the sample rapidly revitrifies, trapping the particles in their transient configurations. Precise control of the temperature evolution of the sample is crucial for such an approach to succeed. Here, we provide a detailed characterization of the heat transfer occurring under laser irradiation as well as the associated phase behavior of the cryo sample. While areas close to the laser focus undergo melting and revitrification, surrounding regions crystallize. In situ observations of these phase changes therefore provide a convenient approach for assessing the temperature reached in each melting and revitrification experiment and for adjusting the heating laser power on the fly.

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Figures

FIG. 1.
FIG. 1.
Rapid melting and revitrification of cryo samples, experimental concept, and demonstration. (a) Illustration of the geometry of the cryo sample, which is supported by a holey gold film. (b)–(e) Experimental concept. (b) Apoferritin particles are irradiated with the electron beam, which alters their structure and thus serves as a stimulus for dynamics to occur. (c) The cryo sample is melted in situ by heating with a laser, (d) allowing the structure of the particles to freely evolve. (e) Once the laser is switched off, the sample rapidly revitrifies and particles are arrested in their transient configurations, which can be subsequently imaged with conventional cryo-EM techniques. Cartoon of apoferritin adapted from Ref. . (f)–(j) Proof-of-principle demonstration. (f) Composite micrograph of a cryo sample of apoferritin in which only the two circular areas in the top left and bottom right have been exposed with a dose of 5 and 10 electrons/Å2, respectively. Scale bar, 200 nm. (g) The sample is melted in situ with a 15 μs laser pulse and revitrifies. Particles that were illuminated with the electron beam prior to melting have unraveled during the short period when the sample was liquid, while those in the unexposed areas remain intact. (h)–(j) Details of the square areas marked in (f) and (g). Scale bar, 50 nm.
FIG. 2.
FIG. 2.
Phase behavior of cryo samples heated with laser pulses of increasing power. (a)–(g) Micrographs of a cryo sample under exposure to laser pulses of increasing power. (a) The sample (b) remains vitreous after heating with a 10 μs laser pulse of 14 mW power, but (c) crystallizes at a power of 19 mW. (d)–(f) The crystal morphology changes as the power is increased in steps to 35 mW. (g) A single pulse of 46 mW power melts the sample, causing it to revitrify when it cools after the end of the laser pulse. (h)–(m) Diffraction patterns of a second identical cryo sample under exposure to laser pulses of the same powers as in (a)–(f). (n) Diffraction pattern of the revitrified sample in (g). Scale bars, 500 nm and 5 nm−1.
FIG. 3.
FIG. 3.
Phase behavior of a cryo sample in a melting and revitrification experiment. (a) Micrograph of a cryo sample after irradiation with a 10 μs laser pulse (46 mW). The green circle indicates the laser position and spot size (24 μm FWHM). Scale bar, 10 μm. (b) Enlarged view of the region marked with the white square in (a). Melting and revitrification have occurred in all but one of the holes in the area marked with the small solid circle, while the sample has crystallized within the bounds of the large dashed circle. The rest of the sample has remained vitreous. Scale bar, 5 μm. (c)–(h) Micrographs and (i)–(n) diffraction patterns of the regions marked with colored squares in (b). Scale bars, 500 nm and 5 nm−1.
FIG. 4.
FIG. 4.
Time–temperature–transformation diagram of supercooled water. The black curve indicates the crystallization time of supercooled water, which is estimated from experimental nucleation and growth rates (see Note S2). It exhibits a minimum of 5 μs at about 225 K, in a region of the phase diagram of water commonly referred to as “no man's land,” where rapid crystallization hinders the characterization of supercooled water. Crystallization is dominated by rapid crystal growth above 225 K, while nucleation dominates at lower temperatures. The solid blue lines indicate transition temperatures, while the dashed red lines denote the boundaries of “no man's land.”
FIG. 5.
FIG. 5.
Heat transfer simulations of a melting and revitrification experiment. (a,b) Temperature distribution of a cryo sample after irradiation with a 10 μs laser pulse (46 mW, 24 μm spot size, indicated with a green circle). Scale bar, 10 μm. The white line in (b) indicates the isotherm at 273 K, and the dashed circle represents the boundary of the crystalline region from the experiment in Fig. 3(b), at which the simulation predicts a temperature of 170 K. (c) Plateau temperature of the sample [at the position of the blue dot in (b)] as a function of laser power for different sample geometries. (d) Diameter of area that melts as a function of the laser power [probed at the position of the blue dot in (b)]. The curve serves as a guide for the eye.
FIG. 6.
FIG. 6.
The size of the revitrified area increases with laser power. (a)–(c) Micrographs of cryo samples irradiated by a 15 μs laser pulse of increasing power. Areas that melted and revitrified are highlighted in red. Scale bar, 4 μm.
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