CN109357975A - A method to measure the effective diffusion coefficient of biomolecules - Google Patents
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- 238000001215 fluorescent labelling Methods 0.000 claims 2
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- 238000002474 experimental method Methods 0.000 description 8
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- 238000004364 calculation method Methods 0.000 description 6
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- 238000004458 analytical method Methods 0.000 description 5
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- 238000006243 chemical reaction Methods 0.000 description 2
- 230000002121 endocytic effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000028023 exocytosis Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
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- 241000238631 Hexapoda Species 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000000571 coke Substances 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
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Abstract
The present invention proposes a kind of method for measuring biomolecule effective diffusion cofficient, includes the following steps；A1, planning need to measure the initial markers region ROI0 of the biomolecule of diffusion coefficient；Plan diffusion process collection of images region ROI1；A2, fluorescent marker or other optical markings are carried out to the biomolecule in the ROI0 of region；A3, the biomolecule in ROI0 is spread, and ensures that the diffusion zone of biomolecule is less than region ROI1, dynamic image collection is carried out to the biomolecule diffusion process in the ROI1 of region；Whether dynamic image collected by A4, verification is effective；A5, dimensionality reduction is carried out to image data, the plane coordinates data of biomolecule diffusion process is generated according to image data；A6, the plane coordinates data according to generation, the effective diffusion cofficient of biomolecule diffusion process is gone out with the derivation of equation；The present invention is few to the hypotheses of application, and application range is wider.
Description
Technical field
The present invention relates to experiment field of measuring technique, especially a kind of method for measuring biomolecule effective diffusion cofficient.
Background technique
Biosystem almost all carries out logistics management using Passive diffusion on all scales, and diffusion process is for lifes at different levels
Object process is most important, such as metabolism logistics, and enterprise schema forms and grows in growth course, lymph in immunoreaction process
The recruitment of cell and the vegetative coverage mode of ecosphere etc..Biomolecule usually moves in complicated vivo environment, and
It is not free diffusing, is influenced by multiple dimensioned factor, such as tortuosity, degradation, instantaneous combination and other dynamic process, because
This measure biomolecule in vivo environmental dissemination rate important parameter be effective diffusion cofficient (effective
Diffusioncoefficient), it is different from free diffusing coefficient (freediffusioncoefficient).
However, the effective diffusion cofficient for the biomolecule that precise measurement is spread in complicated biotic environment in vivo is very
It is challenging.Currently, the fluorescence after photobleaching restores (FRAP) or mirror image FRAP (iFRAP) is commonly used to measure effective expansion
The rate of dissipating.However, FRAP or iFRAP method is due to needing stringent hypothesis and there is serious applied defects, because these are false
If not being able to satisfy in biosystem usually.Firstly, current FRAP or iFRAP analysis is strictly dependent on entire target area
The distribution of diffusing molecules does not require nothing more than it and is in stable state, and requiring target area is the distribution of homogeneity or diffusing molecules
Meet certain specific function.Therefore, FRAP or iFRAP is unrealistic for most of highly dynamic biological process
, and may cause misleading conclusion or explanation.Secondly, FRAP or iFRAP analysis method is by involved by biomolecule
Various kineticses strong influence, such as with acceptor interaction, Reversible binding, cell endocytic and exocytosis, drop
Solution.Ignoring these influence factors may cause quite inaccurate measurement result.Third, in the current analysis side FRAP/iFRAP
In method, sample geography geometry excessively simplifies, thus causes very big error.Therefore, it is resulted at present using traditional technology
Many misleading scientific conclusions or explanation and application, need the appearance of new technology.
Summary of the invention
The present invention proposes a kind of method for measuring biomolecule effective diffusion cofficient, few to the hypotheses of application, and answers
It is wider with range.
The present invention uses following technical scheme.
A method of measurement biomolecule effective diffusion cofficient, the measurement method include the following steps；
A1, planning need to measure the biomolecule of diffusion coefficient the initial markers region region ROI0, ROI0 be label to
The distributed areas put between surveying biomolecule at the beginning；Plan diffusion process collection of images the region ROI1, ROI1 of biomolecule
Range is greater than biomolecule diffusion process to be measured and carries out biomolecule diffusion rear region in collection of images time window；The ROI1
Range be greater than ROI0；
A2, fluorescent marker or other optical markings are carried out to the biomolecule in the ROI0 of region；
A3, the biomolecule in ROI0 is spread, and ensures that the diffusion zone of biomolecule is less than region ROI1, to region
Biomolecule diffusion process in ROI1 carries out dynamic image collection；
Whether dynamic image collected by A4, verification is effective, such as expansion of the labeled biomolecule in collection of images process
Scattered region is always positioned in the areas imaging of dynamic image, then determines that collected dynamic image is effective dynamic image；
A5, dimensionality reduction is carried out to image data, the plane coordinates data of biomolecule diffusion process is generated according to image data；
A6, the plane coordinates data according to generation, the effective diffusion cofficient of biomolecule diffusion process is gone out with the derivation of equation.
Formulation process in step A6 is as follows；
Region ROI1 is the target area that dynamic image is collected；
If a is that target area testing molecule is distributed in extracellular ratio；When a value is known or is surveyed by other methods
When the value obtained；
Derivation process A is；
Some pixel position x subscript in observation visual field when if I (t) represents observation time point t when dynamic image is collected
The corresponding light intensity of the diffusing molecules of note, the light intensity are proportional to molecular number；X is located at the area of the big visual field ROI1 [a, b] of imaging observation
Interior corresponding position coordinate, the origin of coordinate are typically chosen in the geometric center of ROI1；x2For square of location coordinate；Adduction
It number indicates to sum to the product of the light intensity of all positions and location coordinate square, is approximately integral if data point is continuous enough；t
For the time point of sampling, take the moment of optical markings as starting point t0；The fluorescence intensity of each position in target area is multiplied by position
That square summation set obtains is F1 (t)；
Function performance graph then is obtained by biomolecule diffusion experiment data, formula 1 is derived from by function performance graph:
The denominator of the formula is the total fluorescence intensity in ROI1, as the correction of optical signalling, referring to attached drawing 1；
This experimental data is fitted by the function that formula 2 is stated again；
Formula 2:
K is obtained by fitting0And D α/k0.Wherein K0And a is constant, D is effective diffusion cofficient.
Preferably, when a value is unknown,
Derivation process B is；
If s be observe imaging time window [0, t] in some time point, [- l, l] be initial markers region ROI0 or
It is that any one includes initial markers area but the region sufficiently small relative to ROI1, which is denoted as ROI2；λ is constant, referring to
Attached drawing 1,
Function performance graph then is obtained by biomolecule diffusion experiment data, formula 3 is derived from by function performance graph:
The function curve stated by formula 3 can the Fitting Calculation go out a value；Again via K obtained by derivation process A0And D α/k0
Numerical value, D can be calculated.
Preferably, derivation process B does not carry out direct the Fitting Calculation to D with formula 3.
Reason is that the D of formula 3 is appeared in exponential function, therefore if deviation will be non-by the D that the formula fitting comes out
Chang great, therefore formula 3 should not carry out direct the Fitting Calculation to D
In step A2, the method for fluorescent marker include use can light turn or can light activated fluorophor biology is divided
Son is marked；The fluorophor includes that light turning egg(s) is white.
In step A5, when collection of images region ROI1 has symmetrical spatial symmetry, according to image data
The 2-D data of biomolecule diffusion process is projected to formation one-dimensional data at symmetry axis.
In step A3, when carrying out dynamic image collection, optical signal is collected using detector, and cut using the light of superposition
Cylinder carries out light signal collection and is reached with eliminating the movement of fluorescent tag molecule in the Z-axis direction influence caused by dynamic data
The dimensionality reduction effect acquired to data.
In step A4, whether keep permanent at any time by label optical signalling total in ROI1 during calculating imaging
It is fixed, the imaging model of dynamic image whether is always positioned to determine labeled biomolecule in the diffusion zone of collection of images process
In enclosing, if total label optical signalling is kept constant at any time, illustrate that diffusing molecules were not both sent out in time of measuring window
Raw degradation, also without diffusion out-of-bounds.
During the dynamic image of step A3 is collected, need to guaranteeing sample belonging to biomolecule, there is no whole shiftings
Dynamic or deformation, to prevent the biomolecule DYNAMIC DISTRIBUTION data obtained from deviation occur.
Compared with prior art, present invention has an advantage that
A. few using hypotheses, it does not require the distribution of diffusing molecules to be in stable state, dynamic process can be analyzed；Also it does not require
Target area is that the distribution of homogeneity or diffusing molecules meets certain specific function.
B. it is contemplated that the influence of various kineticses, for example, with acceptor interaction, Reversible binding, cell endocytic and
The processes such as exocytosis, degradation, the real time different scale that can be occurred according to various dynamic processes adjust reality when measurement
Test time scale.
C. the case where being applicable not only to stable state is equally applicable to the measurement of highly dynamic cell, tissue, organ.
D. measurement method has a wide range of application, and is applicable not only in contrast more inert diffusing molecules, is also applied for
Biological diffusing molecules in complex biological vivo environment.
E. can comprehensively consider the geometry of irregular organism, object under test, the specific imaging of regulation experiment and
Stakeout & Homicide Preservation Strategy.
It is also an advantage of the present invention that:
A. the precision of measurement biomolecule effective diffusion cofficient is improved；
B. measurement method has a wide range of application, and is applicable not only in contrast more inert diffusing molecules, is also applied for
Biological diffusing molecules in complex biological vivo environment；The case where being applicable not only to stable state is equally applicable to highly dynamic
Cell, tissue, organ measurement；
C. this method can be made as application module, to be directly embedded into the operating system of current all high-end microscope imagings
In, there is stronger practicability and preferable social benefit.
Detailed description of the invention
The present invention is described in more detail with reference to the accompanying drawings and detailed description:
Attached drawing 1 is in embodiment, and dispersion ability propagates schematic diagram after biomolecule light turns label
Attached drawing 2 is in embodiment, and disk Dpp::Dendra2 occurs for drosophila larvae eye before light turns after (left side) and light turn
The distribution schematic diagram on (right side)；
Attached drawing 3 is red fluorescence dynamic communication dynamic image data after Dpp::Dendra2 is turned by light in analysis drosophila eye disk
Schematic diagram；
Attached drawing 4 is the F that Dpp::Dendra2 is turned rear red fluorescence dynamic communication dynamic data by light in drosophila eye disk1(t)、
F2(t) it is fitted legend；
Specific embodiment
As shown in the picture, a method of measurement biomolecule effective diffusion cofficient, it is characterised in that: the measurement method
Include the following steps；
A1, planning need to measure the biomolecule of diffusion coefficient the initial markers region region ROI0, ROI0 be label to
The distributed areas put between surveying biomolecule at the beginning；Plan diffusion process collection of images the region ROI1, ROI1 of biomolecule
Range is greater than biomolecule diffusion process to be measured and carries out biomolecule diffusion rear region in collection of images time window；The ROI1
Range be greater than ROI0；
A2, fluorescent marker or other optical markings are carried out to the biomolecule in the ROI0 of region；
A3, the biomolecule in ROI0 is spread, and ensures that the diffusion zone of biomolecule is less than region ROI1, to region
Biomolecule diffusion process in ROI1 carries out dynamic image collection；
Whether dynamic image collected by A4, verification is effective, such as expansion of the labeled biomolecule in collection of images process
Scattered region is always positioned in the areas imaging of dynamic image, then determines that collected dynamic image is effective dynamic image；
A5, dimensionality reduction is carried out to image data, the plane coordinates data of biomolecule diffusion process is generated according to image data；
A6, the plane coordinates data according to generation, the effective diffusion cofficient of biomolecule diffusion process is gone out with the derivation of equation.
Formulation process in step A6 is as follows；
Region ROI1 is the target area that dynamic image is collected；
If a is that target area testing molecule is distributed in extracellular ratio；When a value is known or is surveyed by other methods
When the value obtained；
Derivation process A is；
Some pixel position x subscript in observation visual field when if I (t) represents observation time point t when dynamic image is collected
The corresponding light intensity of the diffusing molecules of note, the light intensity are proportional to molecular number；X is located at the area of the big visual field ROI1 [a, b] of imaging observation
Interior corresponding position coordinate, the origin of coordinate are typically chosen in the geometric center of ROI1；x2For square of location coordinate；Adduction
It number indicates to sum to the product of the light intensity of all positions and location coordinate square, is approximately integral if data point is continuous enough；t
For the time point of sampling, take the moment of optical markings as starting point t0；The fluorescence intensity of each position in target area is multiplied by position
That square summation set obtains is F1 (t)；
Function performance graph then is obtained by biomolecule diffusion experiment data, formula 1 is derived from by function performance graph:
The denominator of the formula is the total fluorescence intensity in ROI1, as the correction of optical signalling, referring to attached drawing 1；
This experimental data is fitted by the function that formula 2 is stated again；
Formula 2:
K is obtained by fitting0And D α/k0.Wherein k0And a is constant, D is effective diffusion cofficient.
Preferably, when a value is unknown,
Derivation process B is；
If s be observe imaging time window [0, t] in some time point, [- l, l] be initial markers region ROI0 or
It is that any one includes initial markers area but the region sufficiently small relative to ROI1, which is denoted as ROI2；λ is constant, referring to
Attached drawing 1,
Function performance graph then is obtained by biomolecule diffusion experiment data, formula 3 is derived from by function performance graph:
The function curve stated by formula 3 can the Fitting Calculation go out a value；Again via k obtained by derivation process A0And D α/k0
Numerical value, D can be calculated.
Preferably, derivation process B does not carry out direct the Fitting Calculation to D with formula 3.
Reason is that the D of formula 3 is appeared in exponential function, therefore if deviation will be non-by the D that the formula fitting comes out
Chang great, therefore formula 3 should not carry out direct the Fitting Calculation to D
In step A2, the method for fluorescent marker include use can light turn or can light activated fluorophor biology is divided
Son is marked；The fluorophor includes that light turning egg(s) is white.
In step A5, when collection of images region ROI1 has symmetrical spatial symmetry, according to image data
The 2-D data of biomolecule diffusion process is projected to formation one-dimensional data at symmetry axis.
In step A3, when carrying out dynamic image collection, optical signal is collected using detector, and cut using the light of superposition
Cylinder carries out light signal collection and is reached with eliminating the movement of fluorescent tag molecule in the Z-axis direction influence caused by dynamic data
The dimensionality reduction effect acquired to data.
In step A4, whether keep permanent at any time by label optical signalling total in ROI1 during calculating imaging
It is fixed, the imaging model of dynamic image whether is always positioned to determine labeled biomolecule in the diffusion zone of collection of images process
In enclosing, if total label optical signalling is kept constant at any time, illustrate that diffusing molecules were not both sent out in time of measuring window
Raw degradation, also without diffusion out-of-bounds.
During the dynamic image of step A3 is collected, need to guaranteeing sample belonging to biomolecule, there is no whole shiftings
Dynamic or deformation, to prevent the biomolecule DYNAMIC DISTRIBUTION data obtained from deviation occur.
Embodiment:
As shown in attached drawing 1-4, the eyes organ (eyes formation disk) of Dpp::Dendra2 drosophila larvae is dissected, is carried out external
Culture.
In vitro culture is carried out according to normal process, and culture medium is Life company Schneider ' s Insect culture medium, is added
1% calf serum of Hyclone, culture vessel are that Nest brand is copolymerized burnt microscopy special culture dish.
It carries out light and turns-dynamic imaging experiment:
Leica Laser Scanning Confocal Microscope, which is inverted, using SP5 carries out real time imagery experiment.Pass through (the discoloration of two-photon pulse laser
Dragon) at about 800nm to Dendra2 carry out light conversion: laser power 25%-30%, MP gain 100%, MP deviate 12%-
60%, scanning excitation 2-3 times within 2 seconds.Dendra2 can emit red fluorescence after light turns, and (drosophila larvae eye occurs referring to fig. 2
Distribution map of the disk Dpp::Dendra2 before light turns and after light turn).Light turns region ROI0 and is selected as 10 × 10 μ in tissue plane
M2 square.Collect image using 40 × 1.25 oil immersion lens: the output of laser rays 543nm 15% (is imaged) for red fluorescence
With the output of 488nm laser lower than 0.5% (if desired, green fluorescence is imaged before light conversion), every 0.30 μ m of pixel
0.30 μm of progress confocal scanning imaging.Copolymerization coke Z lamination is sliced (about 12 μ m-thick in total) by five confocal optics and forms, with 20
The interval real time imagery of second obtains 5~10 minutes.
Dynamic imaging data is analyzed, the effective diffusion cofficient of Dpp is calculated:
During image real time transfer, specifying ROI1 is 30-40 × 30-40 μm2Square, light turns region ROI0
In its center.Select ROI1 sufficiently large to guarantee total red fluorescence in the time window of monitoring in ROI1 in two directions
Signal strength is kept constant, this quality guarantee link can guarantee that the Dpp::Dendra2 molecule in ROI1 does not spread out-of-bounds,
It is not degraded in observation time window.ROI2 may be selected it is Chong Die with ROI0 or with ROI0 with length wide, with the same length of ROI1
It is rectangular.Referring to Fig. 3, (Dpp::Dendra2 turns rear red fluorescence dynamic communication dynamic image data by light in analysis drosophila eye disk
Schematic diagram).After light turns, image is obtained according to same time interval.Two specific ROI are chosen on each image
(ROI1 and ROI2).By the variation of the fluorescence distribution in ROI1 from two dimension drop at one-dimensional, then to fluorescence intensity on each position
The as F obtained multiplied by square summation of position1(t).F is fitted using the variation of fluorescence intensity in ROI2 in different time2(t)。
Go out D according to two formula fittingsαEffective diffusion cofficient D can be calculated by being divided by with α.Representative fitting result is referring to fig. 4.According to
Such process measures 8 samples, is averaged, and obtaining Dpp molecule in the effective diffusion cofficient of drosophila larvae eye disk is 0.48 μ
m2/s。
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