A sample temperature control system for cryo-EM with sub-millisecond time resolution

Abstract

The present invention relates to the technical field of cryo-EM sample preparation, and in particular to a sub-millisecond time resolution cryo-EM sample temperature control system, the system comprising a heating module, a temperature sensing module, an adsorption quick freezing module and a computer control module, the heating module uses a high-frequency infrared pulse laser to heat the cryo-EM sample; the adsorption quick freezing module is used to control the amount of cryo-EM sample adsorbed by the grid, and drives the electron microscope grid adsorbed with the sample to move within a set area by a motor; the temperature sensing module is used to measure the temperature of the cryo-EM sample in real time to control the pulse frequency and / or output power of the infrared pulse laser in the heating module; the computer control module integrates the workflow of the other three modules to control the temperature control of rapid freezing and high time resolution synchronously. The present invention can realize real-time temperature control while realizing rapid temperature transition, and effectively realizes high-precision temperature control of cryo-EM samples.

Description

Technical Field

[0001] The present invention relates to the technical field of cryo-electron microscopy sample preparation, and in particular to a cryo-electron microscopy sample temperature control system with sub-millisecond time resolution. Background Art

[0002] Cryo-electron microscopy is an efficient method for determining molecular cell structures. Due to the rapid development of technology in recent years and the success of ALPHAFOLD2 / 3 in predicting the structure of biological macromolecules, single-particle cryo-electron microscopy can be used alongside X-ray crystallography to quickly obtain the structure of macromolecular complexes with near-atomic resolution. At present, there are more than 100 200 / 300KV cryo-electron microscopes installed in China, and there are more than 1,000 cryo-electron microscope systems worldwide. The use of each cryo-electron microscope requires the use of rapid freezing sample preparation technology to quickly cool the water in the biological sample to a glassy state to avoid damage to the molecular structure of the sample by tiny ice crystals.

[0003] There are two major categories of rapid freezing technologies: one is based on the adsorption rapid freezing method invented by Dubochet, whose time resolution can generally reach about 2-5 seconds; the other is the rapid injection or mixed spraying method, which can achieve a time resolution of about 10-15 milliseconds by rapidly spraying a trace amount of sample; however, many biochemical or physiological processes at the molecular level, such as the action potential discharge of the nervous system, often occur within about 1 millisecond. How to achieve sub-millisecond time resolution cryo-EM structure determination is still a major technical barrier. On the other hand, in the process of studying temperature-sensitive biological molecules, how to achieve sub-millisecond resolution temperature control has always been a huge technical problem. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a sub-millisecond time resolution cryo-EM sample temperature control system. The system includes a heating module, a temperature sensing module, an adsorption quick freezing module and a computer control module, wherein: the heating module uses a high-frequency infrared pulse laser to heat the cryo-EM sample; the adsorption quick freezing module is used to control the amount of cryo-EM sample adsorbed by the grid, and drives the electron microscope grid adsorbed with the cryo-EM sample to move within a set area through a motor to achieve rapid freezing; the temperature sensing module is used to measure the temperature of the cryo-EM sample in real time to control the pulse frequency or output power of the infrared pulse laser in the heating module. The present invention can perform real-time temperature control while achieving rapid temperature transition, effectively realizing high-precision control of the cryo-EM.

[0005] The present invention adopts the following technical scheme, a sub-millisecond time resolution cryo-electron microscope sample temperature control system, the system includes a heating module, an adsorption quick freezing module, a temperature sensing module and a computer control module, wherein:

[0006] The heating module uses a high-frequency infrared pulse laser to heat the cryo-EM sample;

[0007] The temperature sensing module is used to measure the temperature of the cryo-EM sample in real time to control the pulse frequency or output power of the infrared pulse laser in the heating module;

[0008] The adsorption quick freezing module is used to control the amount of cryo-EM samples adsorbed on the grid, and to drive the grid adsorbed with cryo-EM samples to move within a set area through a motor to achieve rapid freezing;

[0009] The computer control module has a user interface for receiving user instructions and input signals of various modules, so as to output corresponding control signals to control various modules in real time.

[0010] Furthermore, the heating module includes a pulsed laser, an optical fiber, a beam expander and a controller;

[0011] The pulse laser emits a 10-watt high-frequency infrared pulse laser through an optical fiber, and after being expanded by a beam expander, heats the cryo-EM sample within 0.5ms-4ms before the adsorption quick-freezing module cools it; the controller is used to control the pulse frequency or output power of the high-frequency infrared pulse laser.

[0012] Further, the adsorption quick freezing module includes a physical bracket, a motor, a motion sensor, an environmental controller, an ethane control device and an adsorption component;

[0013] A laser beam expander and a tweezers attachment rod are fixed on the physical support, and tweezers for clamping an electron microscope grid are arranged on the tweezers attachment rod;

[0014] The adsorption component comprises: a filter paper carrier, a rotating pushing device and a separating device; wherein the adsorption component comprises two filter paper carriers, filter paper is attached to the filter paper carriers, and after the filter paper is driven to contact and absorb the grid by the rotating pushing device, the grid is separated from the filter paper carrier by the separating device;

[0015] The motor-driven physical support drives the laser beam expander and the tweezers attachment rod to move up and down within a set range, so that the grid carrying the cryo-EM sample after being adsorbed by the filter paper is heated and inserted into liquid ethane during the downward movement to achieve rapid freezing;

[0016] The motion sensor divides the set range for controlling the movement of the physical support into a plurality of positions, and determines the current position through the environmental controller, so as to drive the physical support to obtain the corresponding acceleration and linear velocity when reaching the corresponding position through the motor.

[0017] Further, the temperature sensing module includes a temperature sensor;

[0018] The temperature sensor uses fluorescent dyes or fluorescent proteins to measure the temperature of the grid carrying the cryo-EM sample after being adsorbed on filter paper. When the heating temperature reaches the set value, the frequency or output power of the high-frequency infrared pulse laser is reduced to between 0.5% and 5% of the heating interval value through the controller.

[0019] Furthermore, the frequency of the high-frequency infrared pulse laser is adjusted down to 0.5%-5% of the heating interval frequency through the controller, specifically:

[0020] The controller includes laser modulation control and time course control;

[0021] The time course control sends a corresponding feedback signal according to the position and speed of the grid to drive the high-frequency infrared pulse laser to heat the grid;

[0022] When the heating temperature reaches the set value, the laser modulation control lowers the frequency of the high-frequency infrared pulse laser to between 500Hz-5KHz, or lowers the output power of the laser to between 0.05-0.5W.

[0023] Furthermore, the adsorption component adopts a 45-nanometer-thick gold grid and a 20-nanometer-thick porous carbon film.

[0024] The beneficial effects of the present invention are as follows: the present invention can realize instantaneous heating of the sample on the electron microscope grid within 1.0 millisecond at the fastest by using infrared laser, and can clamp the sample at a set temperature after heating by quickly modulating the frequency and / or output power of the laser pulse until it enters the liquid ethane; by adsorbing the position sensor in the quick freezing module, the linear velocity and acceleration of the grid within the set range can be measured in real time, the quick freezing rate can be estimated more accurately, and the frequency and / or output power (energy) of the pulsed laser can be modulated within a sub-millisecond time; the temperature sensing module can instantly detect the average temperature of the sample, and can output a feedback signal to the laser controller so as to adjust the energy output of the laser and realize high-precision control of the sample temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0026] Figure 1 This is a schematic structural diagram of a sub-millisecond time resolution cryo-electron microscope sample temperature control system according to an embodiment of the present invention;

[0027] Figure 2 A schematic diagram of a motor-driven bracket movement process according to an embodiment of the present invention;

[0028] Figure 3 This is an experimental principle diagram for realizing temperature transition and control in a rapid freezing sample preparation process according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the accompanying drawings in the embodiments of the present invention to provide a clearer and more complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] A schematic diagram of a sub-millisecond time resolution cryo-EM sample temperature control system according to an embodiment of the present invention is shown in FIG. Figure 1 As shown, it includes a heating module, a temperature sensing module, an adsorption quick freezing module and a computer control module, as follows:

[0031] The heating module uses high-frequency infrared pulse laser to heat the cryo-EM samples;

[0032] The heating module includes a pulse laser, an optical fiber, a beam expander and a controller; the pulse laser emits a 10-watt high-frequency infrared pulse laser through the optical fiber, and after expansion by the beam expander, heats the cryo-EM sample within 0.5ms-4ms before the adsorption quick-freezing module refrigerates the sample; the controller (not shown in the figure) is used to control the pulse frequency or output power of the high-frequency infrared pulse laser.

[0033] In another specific embodiment of the present invention, the heating module uses a high-frequency infrared laser to output 20-50 pulses between 0.5-1.0 milliseconds, and the output power can be adjusted within 0.05-10W, so that the cryo-EM sample can be heated in a short time, and the temperature can be increased in steps of 2 degrees between ambient temperature and 60 degrees Celsius. After the heating is completed, the frequency and / or output power of the laser pulse can be reduced to between 0.5%-5% of the heating period, thereby achieving stable temperature clamping.

[0034] The controller in the heating module includes modulation control and time control, wherein the modulation control specifically includes frequency control and output power control. During the heating period, the pulse frequency is up to 100KHz, and during the temperature clamping period, the frequency can be reduced to between 500Hz-5.0KHz. Combined with the adjustment of the output power, the temperature control accuracy can be achieved within 0.5 degrees Celsius; time control requires synchronization of the physical movement of the tweezers / electron microscope grid, temperature sensing and laser heating temperature control. After being soaked with filter paper, when the grid reaches the set speed and vertical position, the feedback signal can activate the laser beam (reaction time is less than 1 microsecond), and the output laser pulse heats the solution on the grid surface in a short time; when the temperature sensor detects that the temperature is close to the preset value, it will output a feedback signal to drive the laser controller to reduce the frequency and / or energy output (reaction time is less than 1 microsecond) so that the sample temperature can be clamped at the set value.

[0035] The adsorption quick freezing module is used to control the filter paper adsorption of cryo-EM samples in the grid, and to drive the grid adsorbed with cryo-EM samples to move within a set area through a motor, so as to achieve high-speed freezing after the sample is quickly inserted into liquid ethane.

[0036] The adsorption quick freezing module includes a physical support, a motor, a motion sensor, an environmental controller, an ethane control device and an adsorption component; wherein, a laser beam expander and a tweezer attachment rod are fixed on the physical support, and tweezers for clamping the grid are provided on the tweezer attachment rod; in the embodiment of the present invention, tweezers with a heat-insulating tip are selected for operation so as to minimize or eliminate the interference of the tweezer tip on the grid temperature control process; the adsorption component includes a filter paper carrier, a rotating pushing device and a separating device, wherein the adsorption component includes two filter paper carriers, the filter paper carriers are attached with filter paper, and the filter paper carriers are connected to the ethane control device by the rotating pushing device. After the filter paper is driven to be in close contact with and adsorbed on the grid, the grid is separated from the filter paper carrier by a separation device; the motion sensor divides the set range for controlling the movement of the physical bracket into multiple positions, and the environmental controller determines the current position, so that the motor drives the physical bracket to obtain the corresponding acceleration and linear velocity when reaching a specific position; the motor drives the physical bracket to drive the laser beam expander and the tweezers attachment rod to move up and down within the set range, so that the electron microscope grid adsorbed with the cryo-EM sample is quickly inserted into liquid ethane after being heated by the laser pulse during the downward movement to achieve rapid freezing.

[0037] In another specific embodiment of the present invention, the motor drives the physical support to drive the laser beam expander and the tweezers attachment rod to move up and down within a set physical distance. The laser beam is introduced into the beam expander by the optical fiber and then irradiated to the surface of the grid, so as to realize heating control of the electron microscope grid in a short time after the filter paper is adsorbed with the sample. The downward movement of the tweezers attachment rod can accelerate the electron microscope grid carrying the sample from a static state after the filter paper is adsorbed, and then insert it into the liquid ethane cup to achieve rapid freezing. After the filter paper carrier is separated from the grid, the adsorption component does not move up and down; the motion sensor will provide feedback information to the stepper motor, thereby detecting and controlling the vertical movement of the electron microscope grid attached to the physical support at multiple positions within a set range, and can also drive the physical support and the grid to reach the corresponding acceleration and set linear speed.

[0038] The temperature sensing module is used to measure the temperature of the cryo-EM sample in real time to control the pulse frequency and / or output power of the infrared pulse laser in the heating module; the temperature sensing module includes a temperature sensor, which uses temperature-sensitive fluorescent dyes or fluorescent proteins to measure the temperature of the grid adsorbed with the cryo-EM sample. The feedback signal output by the temperature sensor can drive the laser controller through the computer control module to reduce the frequency or output power of the high-frequency infrared pulse laser to 0.5%-5% of the heating range frequency or power.

[0039] The computer control module is used to receive input instructions from operators and input signals from other hardware modules, and output control signals to other modules after signal processing to achieve real-time control of each module, and can also display system status, integrate and coordinate operations of different modules, achieve synchronous control and coordination of heating process and rapid freezing process, and achieve temperature control with sub-millisecond time resolution. The user interface of the computer control module in the present invention can allow different operators to adjust and record system status parameters for repeated use and parameter optimization.

[0040] In a specific embodiment of the present invention, Figure 2As shown, the laser beam expander and the tweezers attachment rod are fixed on the horizontal bracket. The motor drive can accelerate the vertical speed from 0 to 10m / s (can also be adjusted to 20m / s) within 2.5cm. After reaching the H2 position in the figure, it moves at a constant speed. In the remaining 4.0cm, the laser switch time can be controlled to 0.5-4.0 milliseconds, thereby realizing the time course of timed heating and temperature clamping. The specific operation process is: after the filter paper carrier of the adsorption component uses filter paper to adsorb the excess liquid on the grid at the height of H0, about 1.0-2.0 nanoliters of solution will remain; then, the motor drives the physical bracket to start the laser beam expander and the grid together, and within a distance of 2.5cm, the speed is accelerated from 0 to 10m / s (can also be adjusted to 20m / s); in the remaining distance, the time of infrared laser irradiation on the grid surface can be controlled to 0.5-4.0 milliseconds. At the same time, the environmental detector is implemented by a fixed laser, which can be placed at any height of H2, H3, H4, or H5. The signal input of the laser can accurately control the time of turning on the laser pulse in the heating module through the computer control module.

[0041] In another specific embodiment of the present invention, the spatial position and relative orientation of the beam expander and the electron microscope grid are adjustable, thereby avoiding a small distance above the liquid ethane surface where the laser cannot heat or control the temperature.

[0042] It should be noted that if the time course is longer than 4.0 milliseconds, the grid can be moved to the H1 height after the filter paper adsorption is completed, and then the grid is accelerated and quickly frozen after heating and clamping. The time length of temperature clamping can be controlled between 4.0–500 milliseconds and can be adjusted as needed during specific implementation.

[0043] like Figure 3 As shown in the figure, the frequency-adjustable high-frequency infrared pulse laser is introduced by optical fiber and expanded by a beam expander, which can quickly heat the solution in the grid, and the output energy is sufficient to achieve a heating temperature change of up to 50 degrees Celsius within 0.3 milliseconds. At the same time, after reaching the preset sample temperature, the temperature can be clamped by lowering the pulse frequency or output power; the optical fiber and beam expander run synchronously with the electron microscope grid and can control the length of the heating time. Figure 3 S1 represents the end zone of laser heating; S2 represents the last position of the grid in the liquid ethane cup. Between these two positions, the fastest travel time of the electron microscope grid is about 0.3 milliseconds. Thus, the highest time resolution that can be achieved in the embodiment of the present invention is limited to about 0.3 milliseconds.

[0044] The present invention heats a trace solution (~50-200 nanometers thick) on the surface of the grid within 0.3 milliseconds, and uses a temperature detector to measure the sample temperature for negative feedback control and clamps the sample temperature after transient heating; during the heating process, the present invention uses an ultra-thin gold grid (45 nanometers thick) and a 20 nanometer thick porous carbon film to achieve rapid temperature transition; a 10-watt infrared high-frequency (0.1-100KHz) frequency-adjustable pulse laser can achieve a temperature change of 50 degrees Celsius within 0.30 milliseconds, and its specific energy is shown in Table 1.

[0045] Table 1 Estimation of the energy required to achieve temperature transition in the gold grid

[0046]

[0047] In the embodiment of the present invention, a temperature-sensitive fluorescent dye or fluorescent protein is further used to measure the sample temperature and provide negative feedback, and the maximum Celsius temperature error that can be expected is ~2.5%; after the temperature is stabilized, the frequency and / or output power of the laser pulse can be reduced to 0.5%-5% of the heating interval frequency and / or power according to the temperature clamping level achieved, thereby achieving stable temperature clamping.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the specific implementation of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sub-millisecond time resolution cryo-electron microscope sample temperature control system, comprising a heating module, a temperature sensing module, an adsorption quick freezing module and a computer control module; characterized in that: The heating module uses a high-frequency infrared pulse laser to heat the cryo-EM sample; The temperature sensing module is used to measure the temperature of the cryo-EM sample in real time to control the pulse frequency or output power of the infrared pulse laser in the heating module; The adsorption quick freezing module is used to control the amount of cryo-EM samples adsorbed by the grid, and to drive the grid adsorbed with cryo-EM samples to move within a set area through a motor to achieve rapid freezing; The computer control module has a user interface for receiving user instructions and input signals of various modules, and outputting corresponding control signals to control various modules in real time; The adsorption quick freezing module includes a physical bracket, a motor, a motion sensor, an environmental controller, an ethane control device and an adsorption component; A laser beam expander and a tweezers attachment rod are fixed on the physical support, and tweezers for clamping an electron microscope grid are arranged on the tweezers attachment rod; The adsorption component comprises: a filter paper carrier, a rotating pushing device and a separating device; wherein the adsorption component comprises two filter paper carriers, filter paper is attached to the filter paper carriers, and after the filter paper is driven to contact and absorb the grid by the rotating pushing device, the grid is separated from the filter paper carrier by the separating device; The motor-driven physical support drives the laser beam expander and the tweezers attachment rod to move up and down within a set range, so that the grid carrying the cryo-EM sample after being adsorbed by the filter paper is heated and inserted into liquid ethane during the downward movement to achieve rapid freezing; The motion sensor divides the set range for controlling the movement of the physical support into a plurality of positions, and determines the current position through the environmental controller, so as to drive the physical support to obtain the corresponding acceleration and linear velocity when reaching the corresponding position through the motor.

2. A sub-millisecond time resolution cryo-electron microscopy sample temperature control system according to claim 1, characterized in that: The heating module includes a pulse laser, an optical fiber, a beam expander and a controller; The pulse laser emits a 10-watt high-frequency infrared pulse laser through an optical fiber, and after being expanded by a beam expander, heats the cryo-EM sample within 0.5ms-4ms before the adsorption quick-freezing module cools it; the controller is used to control the pulse frequency or output power of the high-frequency infrared pulse laser.

3. A sub-millisecond time resolution cryo-electron microscopy sample temperature control system according to claim 1, characterized in that: The temperature sensing module includes a temperature sensor; The temperature sensor uses fluorescent dyes or fluorescent proteins to measure the temperature of the grid carrying the cryo-EM sample after being adsorbed on filter paper. When the heating temperature reaches the set value, the frequency or output power of the high-frequency infrared pulse laser is reduced to between 0.5% and 5% of the heating interval value through the controller.

4. A sub-millisecond time resolution cryo-electron microscopy sample temperature control system according to claim 3, characterized in that: The frequency of the high-frequency infrared pulse laser is adjusted down to 0.5%-5% of the heating interval frequency through the controller, specifically: The controller includes laser modulation control and time course control; The time course control sends a corresponding feedback signal according to the position and speed of the grid to drive the high-frequency infrared pulse laser to heat the grid; When the heating temperature reaches the set value, the laser modulation control lowers the frequency of the high-frequency infrared pulse laser to between 500Hz-5KHz, or lowers the output power of the laser to between 0.05-0.5W.

5. The sub-millisecond time resolution cryo-electron microscopy sample temperature control system according to claim 1, characterized in that: The adsorption component adopts a 45-nanometer-thick gold grid and a 20-nanometer-thick porous carbon film.

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