CN109810167A - A method of control protein crystal growth is assisted using femtosecond laser - Google Patents
A method of control protein crystal growth is assisted using femtosecond laser Download PDFInfo
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Abstract
The present invention relates to a kind of methods for assisting control protein crystal growth using femtosecond laser, belong to femtosecond laser application field.The present invention by femto-second laser pulse sequence focus in protein solution established nucleating surface or near, change solute concentration and accumulation mode near protein crystal by laser capture effect or changes the mechanism such as protein surface growth pattern, realize the regulation for protein crystal growth process in the solution, the regulation of the speed of growth including the whole speed of growth or a certain crystal face.In addition, the invention also includes to femto-second laser pulse sequence carry out space-time shaping, can further decrease or avoid the thermal damage to protein, with realize to crystal structure destroy it is as small as possible in the case where Effective Regulation.
Description
Technical field
The present invention relates to a kind of methods for assisting control protein crystal growth using femtosecond laser, belong to femtosecond laser and answer
Use technical field.
Background technique
The crystallization of the larger molecular organics such as protein has important role in fields such as structural analysis, bio-pharmaceuticals,
In these applications, for the controllability of protein crystal size and shape, there is important meaning in scientific research and practical application.
Such as when analyzing protein structure, a kind of important method-X-ray diffraction needs to utilize large scale, high quality monocrystalline.
It mainly include two processes, the growth of protein forming core and protein nucleus generally for crystallization of protein.For nucleation process,
Conventional method mainly passes through control temperature humidity and realizes spontaneous nucleation, and there are also methods can pass through the methods of ultrasound, laser
Realize the forming core of some conventional method difficulty crystallization of protein.But for certain protein, due to its different crystal face speed of growth
Different reasons, even if realizing controllable forming core by existing method, can still be presented in crystal growing process strip or
Flat crystal face is not suitable for certain applications, such as noise is poor etc. in X-ray diffraction.For protein growth process
The control temperature and humidity by experience control protein crystal growth environment main at present, control its speed of growth or growth
Orientation, but these methods relatively depend on experience, and the effect for regulating and controlling realization is limited.
Femtosecond laser is since its pulse operating time is ultrashort, the high feature of peak power, is one non-with material effects
Linearly, nonequilibrium process is widely used in field of material processing.Especially because its high-precision, high-energy deposition efficiency,
The features such as smaller to material lesion size, Femtosecond-Laser Pulse Excitation can be avoided or reduced in biomaterial, such as when protein
Thermal damage has unique advantage, has obtained extensive research.Existing certain methods focus on protein by femtosecond laser
Supersaturated solution in regulate and control its formed nucleus quantity, such as the patent nuclei of crystallization manufacturing method and crystallization condition screening side
Described in method (application number 03820509.2) it is a kind of using pulsewidth be femtosecond to picosecond laser action in solution, by sharp
The phenomenon that solution confined explosion, generates nucleus at optical focus, but the method for this respect is mainly used for controlling the mistake of crystallization nucleation at present
Journey.It is that nucleus is formed by the Thermodynamic effect of laser and solution in its principle, although nucleus quantity when can control nucleation,
And finally influence to be formed the quantity and size of crystal, but because the growth of nucleus still uses foregoing experience control after forming core
System, in crystal growing process, is unable to control the speed and orientation of crystal growth, shape for finally obtained crystal and big
Small ability of regulation and control is limited.
In addition the dynamic control method of femtosecond laser has certain application in terms of with laser and biomaterial effect
Prospect.The dynamic control mechanism of femtosecond laser is the emerging technology in a kind of femtosecond laser minute manufacturing, in it is main
Rong Shi, by carrying out space-time shaping for femto-second laser pulse, i.e., energy, phase, polarization of change laser etc. are in time domain, airspace
On distribution, realize in laser action process for material surface local electronic excitation, free electron density develop etc. tune
Control, to realize that conventional laser processes the processing effect that cannot achieve, such as special surface micro-nano structure or reduce laser with
The heat-affected zone etc. of material.Such as three-dimensional periodic structure processing method (application number of the patent based on dynamic control
It 201310706949.9), is a kind of time domain shaping methods of femtosecond laser, this method is by the way that a femto-second laser pulse to be divided into
Multiple delays regulate and control Local Transient electronic Dynamic, realize traditional simple venation in solid material surface in the subpulse of 30-200fs
The coniform periodic structure of three-dimensional that punching laser processing cannot achieve.But it is special micro- that current this method is only used in the surface of solids
The realization of micro-nano structure, it can reduce material heat-affected zone etc. technical field of biological material have the characteristics of considerable advantage there are no
To sufficient application.
Summary of the invention
The purpose of the present invention is realizing the regulation of the nucleation process for protein, propose that a kind of utilization femtosecond laser assists
The method of protein crystal growth focuses on the nucleating surface formed in protein solution using femto-second laser pulse sequence
Near or, in the case where avoiding or reducing to material thermal damage, changed by laser capture effect molten near protein crystal
Matter concentration and accumulation mode change the mechanism such as protein surface growth pattern, realize for protein crystal in the solution
The regulation of growth course, the regulation of the speed of growth including the whole speed of growth or a certain crystal face.Wherein dynamic control master
If, can be with by suitably selected dynamic control method by the modulation for carrying out time domain and airspace for femtosecond laser
In femtosecond laser and material effects, ablation heat affected area is smaller, realizes damage smaller for protein.
The purpose of the present invention is achieved through the following technical solutions:
A kind of method assisting control protein crystal growth using femtosecond laser proposed by the present invention, which is characterized in that
The following steps are included:
S1, it prepares protein supersaturated solution and crystallizes required pond liquid, by protein supersaturated solution in the pendular ring border of pond
Constant temperature is stood until generating the primary crystalline that some sizes are 10-100 μm by spontaneous crystallization；
S2, object judgement: if be used for the regulation protein crystal entirety speed of growth, step S3 is executed；If for regulating and controlling
When the speed of growth of a certain plane of crystal of protein crystal, step S4 is executed；
S3, select laser parameter for the femtosecond laser of single pulse energy 0.5-2.5nJ, pulse recurrence frequency 10-100MHz
Pulse train；Selected femto-second laser pulse sequence is focused on into the satiety of the protein near the primary crystalline that step S1 is obtained
In solution, continuous action 1-12 hours, until obtaining required crystal size, and protein is kept in laser action process
Supersaturated solution sealing and standing in the pond pendular ring border of room temperature；
S4, femtosecond of the laser parameter for single pulse energy equal to the ablation threshold on 1-1.1 times of protein crystal surface is selected
Laser pulse sequence；Selected femto-second laser pulse sequence is focused on to the primary crystalline surface for needing the speed of growth,
Continuous action is until the plane of crystal generates ablation point；Then by the protein with crystal in the liquid of pond sealing and standing at room temperature
1-7 days, make crystal autonomous growth to being sized.
Further, in step S3, by selected femto-second laser pulse sequence focus on step S1 obtain it is initial
It is further comprising the steps of before in protein supersaturated solution near crystal:
Space-time shaping is carried out to selected femto-second laser pulse sequence: unfocused preceding light is made by spatial light modulator
Beam obtain have certain space be distributed phase, regulate and control femto-second laser pulse sequence after object lens focus focal zone in space
Distribution to realize that the femto-second laser pulse sequence near multiple protein crystals of selected areas focuses, while regulating and controlling more
A protein crystal growth speed.
Further, in step S3, by selected femto-second laser pulse sequence focus on step S1 obtain it is initial
It is further comprising the steps of before in protein supersaturated solution near crystal:
Space-time shaping is carried out to selected femto-second laser pulse sequence: selected femto-second laser pulse sequence is carried out
Space-time shaping: time domain shaping methods are used, pulse shaping is modulated to the series of sub-pulses being made of 2~5 subpulses, phase
Time interval between adjacent subpulse is 100fs-10ps.
Further, in step S4, need the speed of growth focusing on selected femto-second laser pulse sequence
Plane of crystal before, it is further comprising the steps of:
Space-time shaping is carried out to selected femto-second laser pulse sequence: time domain shaping methods are used, by pulse shaping
It is modulated to the series of sub-pulses being made of 2~5 subpulses, the time interval between adjacent subpulse is 100fs-10ps.
Further, in step S4, need the speed of growth focusing on selected femto-second laser pulse sequence
Initial crystal face on before, it is further comprising the steps of:
Space-time shaping is carried out to selected femto-second laser pulse sequence: unfocused preceding light is made by spatial light modulator
Beam obtain have certain space be distributed phase, regulate and control femto-second laser pulse sequence after object lens focus focal zone in space
Distribution realizes simultaneous processing in multiple spot.
The features of the present invention and the utility model has the advantages that
1, relative to traditional protein crystallization mode, a kind of new control in addition to the parameters such as control temperature humidity is realized
The method of the speed and the crystal face orientation of growth of protein crystalline substance crystal growing process；
2, it by the control for the crystal entirety speed of growth or a certain crystal face speed of growth, can further influence final
Obtain the properties such as the quality, size, shape of protein crystal；
3, due to using femtosecond laser to handle protein, further pass through the optimization of dynamic control method, generation
Heat-affected zone is small, may be implemented to destroy crystal structure it is as small as possible in the case where Effective Regulation.
Detailed description of the invention
Fig. 1 is that femtosecond laser used by the embodiment of the present invention 1 and embodiment 3 processes index path.
Fig. 2 is the Femtosecond-Laser Pulse Excitation method and schematic illustration of the embodiment of the present invention 1.
Fig. 3 is the crystal growth result obtained of the embodiment of the present invention 1 and traditional method for crystallising comparative result figure.Wherein
(a), (c), (e) be respectively the obtained crystal growth of traditional method for crystallising 20 minutes, 40 minutes, 60 minutes front and back comparison diagrams；
(b) during (d) (f) is respectively femtosecond laser continuous action, crystal growth 20 minutes, 40 minutes, front and back comparison in 60 minutes
Figure.
Fig. 4 is that femtosecond laser used by the embodiment of the present invention 2 and embodiment 4 processes index path.
Fig. 5 is the Femtosecond-Laser Pulse Excitation method and schematic illustration of the embodiment of the present invention 2.
Fig. 6 is the crystal growth result figure obtained of the embodiment of the present invention 2, wherein (a) is after femtosecond laser is just finished
Obtained microscope figure；It (b) is 96 hours after laser action microscope figures.
Specific embodiment
Technical solution of the present invention is further described below in conjunction with the drawings and specific embodiments.
A kind of method assisting control protein crystal growth using femtosecond laser proposed by the present invention, including following step
It is rapid:
S1, it prepares protein supersaturated solution and crystallizes required pond liquid, by protein supersaturated solution in the pendular ring border of pond
Constant temperature is stood until generating the primary crystalline that some sizes are 10-100 μm by spontaneous crystallization.
S2, object judgement: if be used for the regulation protein crystal entirety speed of growth, step S3 is executed；If for regulating and controlling
When the speed of growth of a certain plane of crystal of protein crystal, step S4 is executed.
S3, select laser parameter for the femtosecond laser of single pulse energy 0.5-2.5nJ, pulse recurrence frequency 10-100MHz
Pulse train；Selected femto-second laser pulse sequence is focused on into the satiety of the protein near the primary crystalline that step S1 is obtained
In solution, continuous action 1-12 hours, until obtaining required crystal size, and protein is kept in laser action process
Supersaturated solution sealing and standing in the pond pendular ring border of room temperature.
S4, the ablation threshold for selecting laser parameter to be equal to 1-1.1 times of protein crystal surface for single pulse energy are (specific to burn
Erosion threshold value can be measured or be consulted pertinent literature by laser ablation plane of crystal and obtains) femto-second laser pulse sequence；It will be selected
The femto-second laser pulse sequence selected focuses on the primary crystalline surface for needing the speed of growth, and continuous action is until the crystal table
Face generates ablation point；Then by the protein with crystal in the liquid of pond sealing and standing 1-7 days at room temperature, make crystal autonomous growth
To being sized.
Further, in step S3, by selected femto-second laser pulse sequence focus on step S1 obtain it is initial
It is further comprising the steps of before in protein supersaturated solution near crystal:
Space-time shaping is carried out to selected femto-second laser pulse sequence: regulation Proteins In Aqueous Solutions crystal is integrally given birth to
The demand of long speed then uses airspace shaping methods if necessary to regulate and control multiple protein crystals of selection area simultaneously, is flying
Before second laser pulse sequence focuses on inside protein supersaturated solution, by spatial light modulator (SLM) make it is unfocused before
Light beam obtain have certain space be distributed phase, to regulate and control femto-second laser pulse sequence focal zone after object lens focus
Distribution in space, to realize that the femto-second laser pulse sequence near multiple protein crystals of selected areas focuses, together
When regulate and control multiple protein crystal growth speed.In this step, the spatial distribution and space light modulation of specific laser focal zone
Phase relation added by device can be obtained by conventional diffraction theory reverse.
Further, in step S3, by selected femto-second laser pulse sequence focus on step S1 obtain it is initial
It is further comprising the steps of before in protein supersaturated solution near crystal:
Space-time shaping is carried out to selected femto-second laser pulse sequence: selected femto-second laser pulse sequence is carried out
Space-time shaping: time domain shaping methods are used, pulse shaping is modulated to the series of sub-pulses being made of 2~5 subpulses, phase
Time interval between adjacent subpulse is 100fs-10ps.
Further, in step S4, need the speed of growth focusing on selected femto-second laser pulse sequence
Plane of crystal before, it is further comprising the steps of:
Space-time shaping is carried out to selected femto-second laser pulse sequence: the life of setting crystal face a certain for regulation protein
The demand of long speed improves final crystalline quality, then if necessary to reduce laser action to the greatest extent to the thermal damage of protein simultaneously
Using time domain shaping methods, pulse shaping is modulated to the series of sub-pulses being made of 2~5 subpulses, adjacent subpulse
Between time interval be 100fs-10ps, the femtosecond laser by time domain shaping can regulate and control femtosecond laser and when material effects
Material surface local electronic density realizes in the identical situation of ablation effect that heat-affected zone is small as far as possible.In this step, specifically
The parameters such as time interval are different for different protein between the subpulse number of selection and pulse, can be by obtaining in experiment
Crystallization effect best parameter determines.
Further, in step S4, need the speed of growth focusing on selected femto-second laser pulse sequence
Initial crystal face on before, it is further comprising the steps of:
Space-time shaping is carried out to selected femto-second laser pulse sequence: unfocused preceding light is made by spatial light modulator
Beam obtain have certain space be distributed phase, regulate and control femto-second laser pulse sequence after object lens focus focal zone in space
Distribution realizes simultaneous processing in multiple spot.
With reference to the accompanying drawing and example is further introduced the present invention, assists control Egg-white molten with femtosecond laser
For the growth of bacterium enzyme crystal.
Femto-second laser uses central wavelength for the titanium sapphire femto-second laser of 800nm.Other femto-second lasers, including
The pulsewidths such as titanium sapphire laser device, optical fiber laser should also have similar auxiliary control egg in the femto-second laser of 35-400fs
White matter crystal growth effects.
The raw material of crystallization is Hen egg-white lysozyme powder of the purchase from Sigma-Ardrich.Other oroteins substance, is answered
Also there is similar crystallization nucleation effect.
Embodiment 1: the method for assisting control protein crystal growth speed using femtosecond laser, comprising the following steps:
S1, it prepares protein supersaturated solution and crystallizes required pond liquid, wherein supersaturated solution uses Hen egg-white lysozyme
Powder 40mg/ml, sodium chloride crystal 2.5%wt, acetic acid sodium crystal 0.1M (molar concentration)；It crystallizes pond liquid used and uses 10%wt
Sodium chloride solution takes 100 μ L protein supersaturated solutions to be placed in the aperture of 96 orifice plates, is placed in same closed appearance with pond liquid
Device is placed in 19 degrees Celsius of isoperibols, produces 10-20 μm or so of primary crystalline after 1 hour in observing protein solution.
S2, object judgement: the present embodiment is used for the speed of growth of regulation protein crystal entirety, executes step S3.
S3, laser parameter is determined, focuses in the protein supersaturated solution near the primary crystalline that step S1 is obtained have
Body realizes that process is as follows:
S31, selection laser pulse sequence parameter are as follows: single pulse energy 2.5nJ (mean power 200mW), pulse pulse weight
The femto-second laser pulse sequence of frequency 80MHz.
S32, it builds fs-laser system and carries out light path calibration, the present embodiment uses femtosecond laser system as shown in Figure 1
System, which includes femto-second laser 1, neutral density attenuator 2 and the mechanical shutter 3 that common optical axis is set gradually,
Reflecting mirror 4,5,6 is passed sequentially through by the femtosecond laser that mechanical shutter 3 exports, it is incident vertically downward after optical path is lifted, by position
10 times of object lens 7 in 6 side of reflecting mirror focus on the protein supersaturated solution 9 equipped with primary crystalline placed on translation stage 8,
And the image side faces unit 13 by being located at 8 side of translation stage is imaged, 6 other side of reflecting mirror, which is equipped with, is total to light with 10 times of object lens 7
The front imaging unit 14 of axis；Wherein femto-second laser 1, for generating femtosecond laser；Neutral density attenuator 2, for adjusting
Laser energy；Mechanical shutter 3, for controlling pulse operating time；Reflecting mirror 4~6 is used to guidance optical path and adjusts optical path standard
Directly；10 times of object lens 9 are used for focused laser beam；Front imaging unit 14, the first charge coupled cell being arranged by common optical axis
(CCD) 12 and imaging len 11 constitute；Image side faces unit 13, using the second charge coupled cell with camera lens and lighting source
(CCD)10；Solid line shown in Fig. 1 is femtosecond laser light, and chain-dotted line is imaging unit light.Fs-laser system builds completion
After carry out light path calibration, there is air breakdown phenomenon by 10 times of focusing of object lens 7 in single pulse energy 20 μ J, repetition rate 1kHz,
In the visual field that image side faces unit 13 and front imaging unit 14 are observed, air breakdown point is found respectively, is labeled as laser
Focal position, in subsequent step by the way that laser spot is focused in solution in imaging optical path.
S33, above-mentioned femto-second laser pulse sequence is focused in the protein supersaturated solution of step S1 configuration: takes step
The pond liquid for having grown the supersaturated solution of protein primary crystalline obtained in S1 and its having been placed, whole container is placed in flat
In moving stage 8.By the imaging optical path of front imaging unit 14 and image side faces unit 13,8 position of translation stage is adjusted, step is made
The laser spot marked in imaging systems in S32 is in the supersaturated solution near the protein crystal for needing to regulate and control, distance
At about 10 μm of protein crystal, change the laser energy 200mW (arteries and veins of 10 times of object lens 7 of arrival by adjusting neutral density attenuator 2
Rush repetition 80MHz, single pulse energy 2.5nJ), focus on laser inside solution.
S34, solution are still placed in the pond pendular ring border of S1 configuration, and laser continuous action after sixty minutes, is regulated growth
Protein crystal afterwards；Keep protein supersaturated solution close in the pond pendular ring border of room temperature (such as 19 DEG C) in laser action process
Envelope is stood.
The schematic illustration of embodiment 1 is needing as shown in Fig. 2, laser focuses in the protein solution being nucleated
Near the protein crystal of regulation；The electromagnetic field of laser in solution protein molecule and cluster have capture effect, this reality
It applies in example, the effect of laser mainly will lead to molecules in solution and the regular aggregation of cluster at laser action, itself has reached energy
Measure lower state, it is not easy to grow in plane of crystal, to inhibit the speed of growth of crystal entirety, finally obtain required shape
Or the preferable crystal of quality.
The experimental result picture of embodiment 1 obtains as shown in figure 3, wherein scheming the respectively traditional method for crystallising of (a), (c), (e)
Crystal growth 20 minutes, 40 minutes, 60 minutes front and back comparison diagrams, figure (b), (d), (f) are respectively that femtosecond laser is persistently made
With in the process, crystal growth 20 minutes, 40 minutes, 60 minutes front and back comparison diagrams, wherein cross is Femtosecond-Laser Pulse Excitation focus,
As described in above-mentioned principle, Femtosecond-Laser Pulse Excitation is mainly the speed of growth for inhibiting crystal in this experiment.This figure work is said as follows
It is bright: for the ease of local observation, to have selected different number of samples, the protein crystal number taken in right column figure in two column figures of left and right
Amount distribution is fewer, part is easy observation；Protein crystal quantity is not both due to chosen area at the beginning in two column figures of left and right
It is different and cause, and the influence of non-laser；The scattering generated when strip is laser action in right column figure, figure unrelated with this method
As not can be removed when processing.
Embodiment 2: the method for the femtosecond laser auxiliary control protein crystal growth speed of airspace shaping, this implementation are utilized
The difference of example and embodiment 1 embodies the relevant operation for increasing airspace shaping in step s3, the step S1 of the present embodiment
Identical with embodiment 1 with step S2, details are not described herein again, and now only the step S3 of the present embodiment is described in detail:
S3, laser parameter and dynamic control parameter are determined, focuses on the egg near the primary crystalline that step S1 is obtained
In white matter supersaturated solution, the specific implementation process is as follows:
S31, selection laser pulse sequence parameter are as follows: single pulse energy 2.5nJ (mean power 200mW), pulse pulse weight
The femto-second laser pulse sequence of frequency 80MHz；Selection dynamic control parameter: the present embodiment is in order to realize in an elongated area
The control of neighbouring protein crystal growth speed selects the femtosecond laser of airspace shaping, by femto-second laser pulse sequence
Progress spacing shaping is concentrated in an elongated area self-energy (by conventional diffraction theory it is found that can be in object lens after focusing it
Before focusing plus an axicon lens phase realizes the purpose, will be described in detail in step s 32).
S32, it builds fs-laser system and carries out light path calibration, the present embodiment uses femtosecond laser system as shown in Figure 4
System, which includes femto-second laser 1, neutral density attenuator 2 and the mechanical shutter 3 that common optical axis is set gradually,
Enter space-time shaping unit 17 after passing sequentially through reflecting mirror 4, beam splitter 5 by the femtosecond laser that mechanical shutter 3 exports, for winged
Second laser pulse sequence carries out space-time shaping, incident vertically downward after optical path is lifted using reflecting mirror 6, anti-by being located at
10 times of object lens 7 for penetrating 6 side of mirror focus on the protein supersaturated solution 9 equipped with primary crystalline placed on translation stage 8, and by
Image side faces unit 13 positioned at 8 side of translation stage is imaged, and 6 other side of reflecting mirror is equipped with and 10 times of 7 common optical axis of object lens
Front imaging unit 14；Wherein femto-second laser 1, for generating femtosecond laser；Neutral density attenuator 2, for adjusting laser
Energy；Mechanical shutter 3, for controlling pulse operating time；Reflecting mirror 4,6 is used to guidance optical path and adjusts beam path alignment, point
Beam mirror 5 is for guiding optical path, optical path beam splitting and adjusting beam path alignment；10 times of object lens 9 are used for focused laser beam；Entering object
Before mirror；Front imaging unit 14 is made of the first charge coupled cell (CCD) 12 of common optical axis setting and imaging len 11；Side
Face imaging unit 13, using the second charge coupled cell (CCD) 10 with camera lens and lighting source；The space-time shaping of the present embodiment
Unit 17 is airspace shaping unit, including spatial light modulator 14 and two lens 15,16, and spatial light modulator 14 is used for entering
It penetrates light and increases the phase back reflection of a spatially Arbitrary distribution and enter subsequent optical path, lens 15,16 constitute 4f system, for will
10 times of object lens 7 are transferred to light field salt free ligands, wherein the focal length f=40cm of lens 15,16, spatial light modulator 14 arrives lens 15
Distance d1=f=40cm, lens 15 arrive lens 16 distance d2+d3=2f=80cm, the distance of 15 to 10 times of object lens of lens 7
D4+d5=f=40cm；Solid line shown in Fig. 4 optical path is femtosecond laser light, and chain-dotted line is imaging unit light.Femtosecond laser
Light path calibration is carried out after the completion of system building, single pulse energy 20 μ J, repetition rate 1kHz are focused by 10 times of object lens 7 and occurred
Air breakdown phenomenon finds air breakdown in the visual field that image side faces unit 13 and front imaging unit 14 are observed respectively
Point, be labeled as focal position of laser, in subsequent step by the way that laser spot is focused in solution in imaging optical path.
S33, above-mentioned femto-second laser pulse sequence is focused in the protein supersaturated solution of step S1 configuration: takes step
The pond liquid for having grown the supersaturated solution of protein primary crystalline obtained in S1 and its having been placed, whole container is placed in flat
In moving stage 8.By the imaging optical path of front imaging unit 14 and image side faces unit 13,8 position of translation stage is adjusted, step is made
The laser spot marked in imaging systems in S32 is in the supersaturated solution near the protein crystal for needing to regulate and control, distance
At about 10 μm of protein crystal, change the laser energy 200mW (arteries and veins of 10 times of object lens 7 of arrival by adjusting neutral density attenuator 2
Rush repetition 80MHz, single pulse energy 2.5nJ), focus on laser inside solution；Then airspace shaping is carried out to laser,
Increase by one 2 degree of the angle of wedge of axicon lens phase in spatial light modulator 14.
S34, solution are still placed in the pond pendular ring border of S1 configuration, and laser continuous action after sixty minutes, is regulated growth
Protein crystal afterwards；Keep protein supersaturated solution close in the pond pendular ring border of room temperature (such as 19 DEG C) in laser action process
Envelope is stood.
Relative in embodiment 1 as a result, the present embodiment is because be a strip region, Neng Goushi by laser focusing range
The growth for now regulating and controlling multiple protein crystals in certain area simultaneously, for needing the case where preparing multiple protein crystals,
Regulation efficiency can be improved.
Embodiment 3: the method for assisting a certain setting crystal face speed of growth of control protein using femtosecond laser, including it is following
Step:
S1, it prepares protein supersaturated solution and crystallizes required pond liquid, wherein supersaturated solution uses Hen egg-white lysozyme
Powder 40mg/ml, sodium chloride crystal 2.5%wt, acetic acid sodium crystal 0.1M (molar concentration)；It crystallizes pond liquid used and uses 10%wt
Sodium chloride solution takes 100 μ l protein solutions to be placed in the aperture of 96 orifice plates, is placed in same closed container with pond liquid, is placed in
In 19 degrees Celsius of isoperibols, 12 as a child produced primary crystalline of the size at 100 μm or so in observing protein solution.
S2, object judgement: the present embodiment is used for the speed of growth of a certain crystal face of regulation protein crystal, executes step S4.
S4, paraffin fluid-tight is carried out to 96 orifice plates of the protein supersaturated solution of the primary crystalline obtained equipped with step S1,
Guarantee the water not re-evaporation in solution, a period of time (the present embodiment stands 48 hours) is being stood, until primary crystalline does not regenerate
It is long；It determines laser parameter, focuses on the primary crystalline surface for needing the speed of growth, the specific implementation process is as follows:
S41, design femto-second laser pulse sequence: protein used by the present embodiment is Hen egg-white lysozyme, and selection is single
Pulse energy is slightly above the femtosecond of Hen egg-white lysozyme ablation threshold (consulting pertinent literature single pulse ablation threshold value is about 0.2 μ J)
Laser pulse sequence, pulse repetition 1kHz, single pulse energy 0.2 μ J, action time 0.1s.
S42, it builds fs-laser system and carries out light path calibration, it is identical as step S32 in embodiment 1, it is no longer superfluous herein
It states.
S43, above-mentioned femto-second laser pulse sequence is focused on a certain crystal face of protein crystal being nucleated: will
The solution for growing protein crystal is placed on translation stage 8, passes through the imaging of front imaging unit 14 and image side faces unit 13
Optical path adjusts 8 position of translation stage, makes the laser spot marked in step S42 in imaging system in one needs of protein crystal
On the crystal face for promoting growth, so that so that laser is focused on crystal needs the surface of regulation, changed by adjusting neutral density decaying wheel 2
Listed by changing to up in the laser energy such as step S41 of object lens, controlled in laser action time such as step S41 using mechanical shutter 3
It is listed, rhegmalypt is observed that on protein crystal surface；
S44, by the protein crystal after femto-second laser pulse series processing in protein supersaturated solution, it is Celsius 19
Continue sealing and standing 96 hours under the conditions of degree, the protein crystal after being regulated growth.
The schematic illustration of embodiment 3 is as shown in figure 5, laser focuses on a certain crystal face of the protein crystal being nucleated
On, slightly above ablation threshold generates rhegmalypt on surface；Laser treatment forms helical dislocation at ablation in the present embodiment, from
And the crystal growth mode on the surface is changed, become the growth of surface helix formula from the growth of stratiform, such as figure circular arrows institute
Show.Since the speed of growth of crystal spiral growth will be much higher than layer growth, it improves the speeds of growth of the crystal face.
The experimental result picture of embodiment 3 is as shown in fig. 6, wherein scheme to obtain after (a) is just finished for femtosecond laser micro-
Mirror figure is wherein the surface in laser ablation hole at spider, burns in the mark plane of two, left side protein crystal by laser
Erosion, the right crystal is without laser ablation；Scheme 96 after (b) illustrates laser action with dotted line frame on the basis of figure (a)
The crystal profile of hour, for comparing each crystal face growing state.As described in above-mentioned principle, Femtosecond-Laser Pulse Excitation is main in this experiment
It is the speed of growth for improving the plane of crystal of laser action, realizes the regulation of crystalline orientation.
Embodiment 4: the side of a certain setting crystal face speed of growth of femtosecond laser auxiliary control protein of time domain shaping is utilized
The difference of method, the present embodiment and embodiment 3 embodies the relevant operation for increasing time domain shaping in step s 4, the present embodiment
Step S1 it is identical with embodiment 3 with step S2, details are not described herein again, now only carries out to the step S4 of the present embodiment detailed
Illustrate:
S4, paraffin fluid-tight is carried out to 96 orifice plates of the protein supersaturated solution of the primary crystalline obtained equipped with step S1,
Guarantee the water not re-evaporation in solution, a period of time (the present embodiment stands 48 hours) is being stood, until primary crystalline does not regenerate
It is long；It determines laser parameter and dynamic control parameter, focuses on the primary crystalline surface for needing the speed of growth, it is specific real
Existing process is as follows:
S41, design femto-second laser pulse sequence: protein used by the present embodiment is Hen egg-white lysozyme, and selection is single
Pulse energy is slightly above the femtosecond laser arteries and veins of protein ablation threshold (consulting pertinent literature single pulse ablation threshold value is about 0.2 μ J)
Rush sequence, pulse repetition 1kHz, single pulse energy 0.2 μ J, action time 0.1s；Dynamic control parameter: selection space
Each pulse in above-mentioned femto-second laser pulse sequence is shaped as 2 energy sizes as original by the femtosecond pulse sequence of shaping
The half of energy, time interval are the subpulse of 1ps.
S42, it builds fs-laser system and carries out light path calibration, the fs-laser system and implementation that the present embodiment is built
The difference of fs-laser system is embodied in example 2, and spatial light modulator 14 therein is adjusted to pulse shaper, is used for
Single femto-second laser pulse is shaped as on the time to the multiple series of sub-pulses for there are certain intervals；The femtosecond that the present embodiment is built
Remaining component in laser system is same as Example 2, and details are not described herein again.
S43, the femto-second laser pulse sequence is focused on a certain crystal face of protein crystal being nucleated: will be raw
The solution for growing protein crystal is placed on translation stage 8, passes through the imaging of front imaging unit 14 and image side faces unit 13
Road adjusts 8 position of translation stage, the laser spot marked in step S42 in imaging system is made to need to promote at protein crystal one
Into on the crystal face of growth, so that so that laser is focused on crystal needs the surface of regulation.Changed by adjusting neutral density decaying wheel 2
Listed by reaching in the laser energy such as step S41 of object lens, then to pulse shaper, each femtosecond is swashed with pulse shaper
The shaping of light pulse is the half that 2 energy sizes are proper energy amount, and time interval is the subpulse of 1ps.Utilize mechanical shutter 3
Listed by controlling in laser action time such as step S41, rhegmalypt is observed that on protein crystal surface.
S44, identical with the step S44 of embodiment 3, details are not described herein again.
Relative in embodiment 3 as a result, due to having used the femto-second laser pulse ablation of temporal shaping to need in embodiment 4
The protein crystal surface to be regulated and controled can reduce heat-affected zone around to the greatest extent in the case where reaching identical ablation effect,
Finally obtained protein crystal quality be better than do not do space-time shaping as a result, structural analysis of protein etc. application in can obtain
Better signal.
The present invention demand different with the setting speed of growth of crystal face for the whole speed of growth for regulating and controlling crystal, except above-mentioned
Outside listed embodiment, the different combinations that femtosecond laser, time domain shaping and airspace shaping can be also carried out, to realize mesh of the present invention
's.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. a kind of method for assisting control protein crystal growth using femtosecond laser, which comprises the following steps:
S1, prepare protein supersaturated solution and crystallize needed for pond liquid, by protein supersaturated solution in the pendular ring border of pond constant temperature
It stands until generating the primary crystalline that some sizes are 10-100 μm by spontaneous crystallization；
S2, object judgement: if be used for the regulation protein crystal entirety speed of growth, step S3 is executed；If being used for modulin
When the speed of growth of a certain plane of crystal of matter crystal, step S4 is executed；
S3, select laser parameter for the femto-second laser pulse of single pulse energy 0.5-2.5nJ, pulse recurrence frequency 10-100MHz
Sequence；It is molten that selected femto-second laser pulse sequence is focused on into the protein supersaturation near the primary crystalline that step S1 is obtained
In liquid, continuous action 1-12 hours, until obtaining required crystal size, and protein satiety is kept in laser action process
With solution in the pond pendular ring border of room temperature sealing and standing；
S4, femtosecond laser of the laser parameter for single pulse energy equal to the ablation threshold on 1-1.1 times of protein crystal surface is selected
Pulse train；Selected femto-second laser pulse sequence is focused on to the primary crystalline surface for needing the speed of growth, is continued
Effect is until the plane of crystal generates ablation point；Then by the protein with crystal in the liquid of pond sealing and standing 1-7 at room temperature
It, makes crystal autonomous growth to being sized.
2. utilizing the method for femtosecond laser auxiliary control protein crystal growth as described in claim 1, which is characterized in that step
In rapid S3, selected femto-second laser pulse sequence is being focused on into the satiety of the protein near the primary crystalline that step S1 is obtained
It is further comprising the steps of before in solution:
Space-time shaping is carried out to selected femto-second laser pulse sequence: obtaining unfocused preceding light beam by spatial light modulator
The phase that must have certain space to be distributed regulates and controls femto-second laser pulse sequence point of focal zone in space after object lens focus
Cloth to realize that the femto-second laser pulse sequence near multiple protein crystals of selected areas focuses, while regulating and controlling multiple
Protein crystal growth speed.
3. utilizing the method for femtosecond laser auxiliary control protein crystal growth as described in claim 1, which is characterized in that step
In rapid S3, selected femto-second laser pulse sequence is being focused on into the satiety of the protein near the primary crystalline that step S1 is obtained
It is further comprising the steps of before in solution:
Space-time shaping is carried out to selected femto-second laser pulse sequence: space-time is carried out to selected femto-second laser pulse sequence
Shaping: time domain shaping methods are used, pulse shaping is modulated to the series of sub-pulses being made of 2~5 subpulses, adjacent son
Time interval between pulse is 100fs-10ps.
4. utilizing the method for femtosecond laser auxiliary control protein crystal growth as described in claim 1, which is characterized in that step
In rapid S4, before being focused on selected femto-second laser pulse sequence and needing the plane of crystal of the speed of growth, also wrap
Include following steps:
Space-time shaping is carried out to selected femto-second laser pulse sequence: using time domain shaping methods, pulse shaping is modulated
For the series of sub-pulses being made of 2~5 subpulses, the time interval between adjacent subpulse is 100fs-10ps.
5. utilizing the method for femtosecond laser auxiliary control protein crystal growth as described in claim 1, which is characterized in that step
In rapid S4, before focusing on selected femto-second laser pulse sequence on the initial crystal face for need the speed of growth, also
The following steps are included:
Space-time shaping is carried out to selected femto-second laser pulse sequence: obtaining unfocused preceding light beam by spatial light modulator
The phase that must have certain space to be distributed regulates and controls femto-second laser pulse sequence point of focal zone in space after object lens focus
Cloth realizes simultaneous processing in multiple spot.
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