WO2016095572A1 - 双色荧光定位超分辨率生物显微方法及系统 - Google Patents

双色荧光定位超分辨率生物显微方法及系统 Download PDF

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WO2016095572A1
WO2016095572A1 PCT/CN2015/089047 CN2015089047W WO2016095572A1 WO 2016095572 A1 WO2016095572 A1 WO 2016095572A1 CN 2015089047 W CN2015089047 W CN 2015089047W WO 2016095572 A1 WO2016095572 A1 WO 2016095572A1
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fluorescent
image
super
fluorescent signal
resolution
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PCT/CN2015/089047
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English (en)
French (fr)
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赵腾
雷明德
杜胜望
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深圳市纳观生物有限公司
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Priority to US15/303,461 priority Critical patent/US10151697B2/en
Publication of WO2016095572A1 publication Critical patent/WO2016095572A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/061—Sources
    • G01N2201/06113—Coherent sources; lasers

Definitions

  • the invention relates to the field of biological microscopy, in particular to a two-color fluorescence localization super-resolution biological microscopic method and system.
  • fluorescence localization microscopy greatly simplifies sample preparation and enables live cell imaging. More importantly, fluorescence localization microscopy can easily achieve multi-channel co-localization imaging, thus obtaining the interaction between proteins, providing the most direct evidence for a large number of molecular biological research topics.
  • the performance of the prior art in multi-color super-resolution imaging still has deficiencies.
  • Nikon N-STORM is a representative of commercial fluorescence localization microscopy.
  • Its multi-channel imaging uses a "fluorescent switch" composed of two different excitation wavelength fluorescent molecules (for example, Alexa647-Alexa488 molecular pair and Alexa467-Alexa405 molecule). Yes) as a marker.
  • This method can achieve fast switching of different imaging channels, but the markers of such fluorescent switches are currently not commercialized, and laboratory preparation is complicated. More importantly, this multi-color imaging method produces more serious channel crosstalk, resulting in erroneous co-localization information.
  • the Leica SR GSD positioning microscope uses common fluorescent molecules of different excitation wavelengths as markers (eg, Alexa647 and Alexa532) and uses a filter system to achieve multi-channel imaging. The limitation of this method is that short-excitation wavelength fluorescent molecules are highly photo-bleaching, and shorter excitation wavelengths cause the autofluorescence background of the cells, affecting the image quality.
  • sample drift is a ubiquitous problem in super-resolution fluorescence localization microscopy. This means that due to environmental instability, such as airflow, temperature changes, noise, etc., the sample will move from tens to hundreds of nanometers during the shooting process. Although this drift phenomenon is ubiquitous in the microscopy system, Generally, the resolution of the microscope is less than 300 nm, and the phenomenon caused by drift is not obvious. But for super-resolution microscopes with resolutions of up to a dozen nanometers, drift can cause serious interference with imaging. Most of the existing solutions currently involve adding fluorescent particles to the sample and recording the displacement of these particles in the image, which is then subtracted from the resulting super-resolution image. The defect is that the preparation is troublesome; and the fluorescent particles occupy an imaging channel; in addition, due to photobleaching, the fluorescence of the fluorescent particles is attenuated with the imaging time, so the correction accuracy of the drift is also deteriorated with time.
  • the present invention provides a two-color fluorescence localization super-resolution biomicroscopic method, comprising:
  • Biological samples are subjected to two-color fluorescent labeling using Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules, and the biological samples are immersed in imaging buffer;
  • Irradiating the biological sample by laser respectively generating a first channel scintillation fluorescent signal corresponding to the Alexa647 or Cy5 fluorescent molecule, and a second channel scintillation fluorescent signal corresponding to the Alexa750 or Cy7 fluorescent molecule;
  • the first biological structure super-resolution image and the second biological structure super-resolution image are aligned to construct a third biological structure super-resolution image.
  • the step of irradiating the biological sample by laser includes:
  • the biological sample is illuminated by a predetermined activation laser to turn on the fluorescent signal, and the biological sample is illuminated by a predetermined excitation laser to turn off the fluorescent signal to generate a scintillation fluorescent signal.
  • the activating laser has a violet or ultraviolet wavelength; the excitation laser has an excitation wavelength of Alexa647 and Alexa750 fluorescent molecules, or the excitation laser has a excitation wavelength of Cy5 and Cy7 fluorescent molecules; using a predetermined excitation laser
  • the power and activation of the laser power cause most of the fluorescent molecules to turn off the fluorescent signal at the same time, and only a small fraction of the discrete fluorescent molecules turn on the fluorescent signal.
  • the imaging buffer comprises TCEP, cyclooctatetraene, oxygen scavenger, methyl viologen and/or ascorbic acid;
  • the imaging buffer comprises a sulfhydryl base, a cyclooctatetraene, an oxygen scavenger, methyl viologen and/or ascorbic blood acid.
  • the TCEP or ruthenium substrate is combined with a fluorescent molecule under irradiation of the excitation laser to form an adduct, and the adduct does not fluoresce under the irradiation of the excitation laser;
  • the adduct is re-decomposed into the TCEP or sulfhydryl substrate and fluorescent molecules under irradiation of the activating laser light, which fluoresces.
  • the cyclooctatetraene comprises a derivative of cyclooctatetraene
  • the oxygen scavenger comprises a combination of glucose oxidase, glucose and catalase, or the oxygen scavenger comprises A combination of pyranose oxidase, glucose, and catalase.
  • the step of constructing the first biological structure super-resolution image and the second biological structure super-resolution image respectively according to the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal include:
  • the location of the biological sample is locked in real time by a real-time locking system prior to data acquisition.
  • the step of real-time locking the position of the biological sample by the real-time locking system comprises:
  • the bright field image of the biological sample being imaged by a front imaging lens to a locking camera;
  • Offset compensation processing is performed on the current bright field image according to the offset.
  • the step of constructing the first biological structure super-resolution image and the second biological structure super-resolution image respectively according to the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal further comprises :
  • the two-color fluorescence localization super-resolution microscope is an inverted total Fluorescence microscope
  • the camera is an EMCCD camera; and/or
  • the beam splitting system includes a rectangular diaphragm, a two-color mirror, and two filters.
  • the invention also provides a two-color fluorescence localization super-resolution biological microscopy system, comprising:
  • a sample labeling module for performing two-color fluorescent labeling on a biological sample using Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules, and immersing the biological sample in an imaging buffer;
  • a signal generating module configured to irradiate the biological sample by laser to respectively generate a first channel scintillation fluorescent signal corresponding to the Alexa647 or Cy5 fluorescent molecule, and a second channel scintillation fluorescent signal corresponding to the Alexa750 or Cy7 fluorescent molecule ;
  • a first image construction module configured to respectively construct a first biological structure super-resolution image and a second biological structure super-resolution image according to the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal;
  • a second image construction module configured to perform alignment processing on the first biological structure super-resolution image and the second biological structure super-resolution image to construct a third biological structure super-resolution image.
  • the signal generating module is configured to illuminate the biological sample with a predetermined activation laser to turn on a fluorescent signal, and illuminate the biological sample with a predetermined excitation laser to turn off the fluorescent signal to generate a scintillation fluorescent light. signal.
  • the activating laser has a violet or ultraviolet wavelength
  • the excitation laser has an excitation wavelength of Alexa647 and Alexa750 fluorescent molecules, or the excitation laser has excitation wavelengths of Cy5 and Cy7 fluorescent molecules;
  • the signal generating module is configured to use a predetermined excitation laser power and an activation laser power such that most of the fluorescent molecules at the same time turn off the fluorescent signal, and only a small portion of the discrete fluorescent molecules turn on the fluorescent signal.
  • the imaging buffer comprises TCEP, cyclooctatetraene, oxygen scavenger, methyl viologen and/or ascorbic acid;
  • the imaging buffer comprises a sulfhydryl base, a cyclooctatetraene, an oxygen scavenger, methyl viologen, and/or ascorbic acid.
  • the TCEP or ruthenium substrate is combined with a fluorescent molecule to form an adduct under irradiation of the excitation laser, and the adduct does not fluoresce under the irradiation of the excitation laser;
  • the adduct is re-decomposed into the TCEP or sulfhydryl substrate and fluorescent molecules under irradiation of the activating laser light, which fluoresces.
  • the cyclooctatetraene comprises a derivative of cyclooctatetraene
  • the oxygen scavenger comprises a combination of glucose oxidase, glucose and catalase, or the oxygen scavenger comprises A combination of pyranose oxidase, glucose, and catalase.
  • the system according to the invention further comprises:
  • a real-time locking module for locking the position of the biological sample in real time by a real-time locking system prior to data acquisition.
  • the real-time locking module includes:
  • a bright field illumination sub-module for providing bright field illumination for the biological sample, the bright field image of the biological sample being imaged by the front imaging lens to the locking camera;
  • a module generating submodule configured to capture a bright field image of a biological sample or a reference body from the locking camera as a locking template
  • An image comparison sub-module configured to capture a current bright field image of the biological sample from the locked camera in real time, and compare the current bright field image with the locking template
  • An offset calculation submodule configured to calculate an offset between the current brightfield image and the locking template
  • an offset compensation submodule configured to perform offset compensation processing on the current bright field image according to the offset.
  • the first image building module further includes:
  • a signal acquisition sub-module for obtaining a first channel scintillation fluorescence signal and a second channel scintillation fluorescence signal by a two-color fluorescence localization super-resolution microscope
  • a signal separation sub-module for separating the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal by a two-color fluorescence localization super-resolution microscope, and respectively imaging the different regions of the photosensitive element of the camera;
  • a position recording sub-module for acquiring each image captured by the camera, determining a center coordinates of each of the fluorescent molecules in the image by Gaussian fitting and recording;
  • the image generation sub-module is configured to respectively construct a first biological structure super-resolution image corresponding to the first channel scintillation fluorescent signal and a second biological structure super-resolution image corresponding to the second channel scintillation fluorescent signal according to the recorded center coordinates of the Airy spot .
  • the two-color fluorescence localization super-resolution microscope is an inverted total reflection fluorescence microscope
  • the camera is an EMCCD camera; and/or
  • the beam splitting system includes a rectangular diaphragm, a two-color mirror, and two filters.
  • the two-color fluorescence localization super-resolution biomicroscopy technique of the present invention uses Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules to perform two-color fluorescent labeling on biological samples, and the emission spectrum of the fluorescent molecules used is completely separated, so theoretically there will be no Channel crosstalk is generated, and the excitation wavelength of the fluorescent molecules does not cause the cells to produce autofluorescence, thereby greatly reducing background noise, thereby improving image quality and being easy to operate.
  • the present invention employs an optimized imaging buffer to greatly reduce photobleaching of fluorescent molecules during imaging. More preferably, the present invention locks the sample through a real-time locking function Set to eliminate completely to eliminate any sample drift during imaging. The invention can be widely applied to the field of bio-optical super-resolution microscopy.
  • FIG. 1 is a schematic structural view of a two-color fluorescence localization super-resolution biological microscopy system of the present invention
  • FIG. 2 is a molecular structure diagram of the TCEP of the present invention.
  • Figure 3 is a schematic diagram showing the action of fluorescent molecules and TCEP components in imaging buffer in the embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing the action of a fluorescent molecule and a Thiol component in an imaging buffer in an embodiment of the present invention
  • Figure 5 is a schematic view showing the structure of a preferred two-color fluorescence localization super-resolution biological microscopy system of the present invention.
  • FIG. 6 is a flow chart of a two-color fluorescence localization super-resolution biomicroscopic method of the present invention.
  • FIG. 7 is a flow chart of a preferred two-color fluorescence localization super-resolution biomicroscopic method of the present invention.
  • FIG. 8 is a schematic structural view of a two-color fluorescence localization super-resolution biological microscopy system according to an embodiment of the present invention.
  • FIG. 1 is a schematic structural view of a two-color fluorescence localization super-resolution biological microscopy system of the present invention, the system 100 including a sample labeling module 10, a signal generating module 20, a first image building module 30, and a second image building module 40, wherein :
  • the sample labeling module 10 is for performing two-color fluorescent labeling on the biological sample using Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules, and immersing the labeled biological sample in the imaging buffer.
  • the Alexa647 and Alexa750 fluorescent molecules are respectively bound to different structural proteins of the biological sample by immunolabeling, the biological sample is made into a sealing piece, and the biological sample is to be immersed in the imaging buffer, and then the invading is performed.
  • An imaging buffer with biological samples was placed on the stage of a two-color fluorescence localization super-resolution microscope.
  • the fluorescent molecules of the present invention preferably employ Alexa647 and Alexa750 fluorescent molecules, but Alexa647 and Alexa750 fluorescent molecules can also be replaced by anthocyanin dye molecules of the same excitation wavelength such as Cy5 and Cy7.
  • the signal generating module 20 is configured to irradiate the biological sample by laser to respectively generate a first channel scintillation fluorescent signal corresponding to the Alexa647 or Cy5 fluorescent molecule, and a second channel scintillation fluorescent signal corresponding to the Alexa750 or Cy7 fluorescent molecule. If the Alexa647 and Alexa750 fluorescent molecules are used in the present invention, a first channel scintillation fluorescent signal corresponding to the Alexa647 fluorescent molecule and a second channel scintillation fluorescent signal corresponding to the Alexa750 fluorescent molecule are generated. The present invention generates a first channel scintillation fluorescent signal corresponding to the Cy5 fluorescent molecule and a corresponding Cy7 fluorescent molecule if the Cy5 and Cy7 fluorescent molecules are used. The second channel flashes the fluorescent signal.
  • the first image construction module 30 is configured to respectively construct a first biological structure super-resolution image and a second biological structure super-resolution image according to the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal.
  • the second image construction module 40 is configured to perform alignment processing on the first biological structure super-resolution image and the second biological structure super-resolution image to construct a third biological structure super-resolution image.
  • the specific component of the imaging buffer comprises TCEP (tris(2-carboxyethyl)phosphine), COT (cyclooctatetraene), oxygen scavenger, methyl viologen and/or ascorbic acid; or imaging
  • the specific components of the buffer include Thiol, COT, oxygen scavenger, methyl viologen and/or ascorbic acid.
  • the cost of TCEP or Thiol in the imaging buffer is combined with the fluorescent molecule to form an adduct under the irradiation of an excitation laser, the adduct does not fluoresce under the irradiation of the excitation laser, and the fluorescent molecule is thus turned off;
  • the compound re-decomposes into TCEP or Thiol and fluorescent molecules under the irradiation of an activated laser, and the fluorescent molecules emit fluorescence, and the fluorescent molecules are thus turned on.
  • FIG 2 shows the molecular structure of TCEP.
  • the Alexa647 fluorescent molecule interacts with the TCEP component in the imaging buffer to form an adduct as shown in Figure 3.
  • the Alexa647 fluorescent molecule in Figure 3 is only described as an example. It includes anthocyanin fluorescent molecules such as Alexa750, Cy5 and Cy7.
  • the Alexa647 fluorescent molecule interacts with the Thiol component in the imaging buffer to form an adduct as shown in Figure 4.
  • the Alexa647 fluorescent molecule in Figure 4 is only described as an example. This principle applies to cyanine including Alexa750, Cy5 and Cy7. Fluorescent molecules.
  • the Thiol includes ⁇ ME (2-mercaptoethanol), or MEA (ethanolamine), etc., but Thiol may reduce the photon number of the Alexa 750 molecule and increase the probability that the fluorescent molecule is photobleached, affecting the image quality.
  • the COT in the imaging buffer includes derivatives of COT.
  • the COT of the present invention is dissolved in DMSO (Dimethyl sulfoxide, dimethyl sulfoxide).
  • DMSO Dimethyl sulfoxide, dimethyl sulfoxide
  • the COT in the imaging buffer can inhibit the fluorescent molecules from entering the triplet state in the absence of oxygen molecules, thereby increasing the number of photons generated by the fluorescent molecules in the open state, improving the resolution, and reducing the probability of photo-bleaching of the fluorescent molecules, and improving imaging. quality.
  • the oxygen scavenger comprises a combination of glucose oxidase, glucose and catalase; or the oxygen scavenger comprises a combination of pyranose oxidase, glucose and catalase.
  • Glucose oxidase (or pyranose oxidase) in the imaging buffer, glucose and catalase combined as an oxygen scavenger can remove oxygen from the buffer, greatly reducing the probability of photobleaching of fluorescent molecules and improving image quality.
  • the present invention is not limited to the use of glucose oxidase (or pyranose oxidase), a combination of glucose and catalase as an oxygen scavenger. Other possible oxygen scavenging schemes can also be applied to the present invention.
  • Methyl viologen and ascorbic acid in the imaging buffer act as reducing agents to inhibit photobleaching of fluorescent molecules.
  • the present invention uses an optimized imaging buffer formulation to produce more than 2000 photons per fluorescent period in a fluorescent molecule, and the oxygen scavenger and COT components in the imaging buffer substantially reduce the molecular Photobleaching produces high quality two-color super-resolution images.
  • the lateral resolution of the Alexa647 channel is ideally 10 nanometers and the Alexa 750 channel is up to 20 nanometers.
  • the specific formulation of the imaging buffer in Example 1 is: 200 mM Tris phosphate buffer, pH 9.0; and contains 10% (w/v) glucose, 5 U/ml pyranose oxidase, 57 ⁇ g/ml catalase, 2 mM COT (dissolved in DMSO), 25 mM TCEP, 1 mM ascorbic acid and 1 mM methyl viologen.
  • the specific formulation of the imaging buffer in Example 2 is: 200 mM Tris phosphate buffer, pH 8.0; and contains 10% (w/v) glucose, 560 ⁇ g/ml glucose oxidase, 57 ⁇ g/ml catalase, 2 mM COT (dissolved in DMSO), 25 mM TCEP, 1 mM ascorbic acid and 1 mM methyl viologen.
  • the signal generating module 20 is configured to illuminate the biological sample with a predetermined activation laser to turn on the fluorescent signal, and illuminate the biological sample with a predetermined excitation laser to turn off the fluorescent signal to generate a scintillation fluorescent signal.
  • the activation laser has a violet or ultraviolet wavelength.
  • the excitation laser has an excitation wavelength of Alexa647 and Alexa750 fluorescent molecules, or the excitation laser has excitation wavelengths of Cy5 and Cy7 fluorescent molecules.
  • the signal generating module 20 is configured to use a predetermined excitation laser power and an activation laser power such that most of the fluorescent molecules at the same time turn off the fluorescent signal, and only a small portion of the discrete fluorescent molecules turn on the fluorescent signal.
  • the packaged biological sample is placed on a stage of an inverted microscope and simultaneously sampled in a total internal reflection mode using a 656.5 nm laser with a 300 mW output power and a 750 nm laser with a 300 mW output power. Irradiation.
  • the present invention does not theoretically have channel crosstalk due to the complete separation of the emission spectra of the Alexa647 and Alexa750 fluorescent molecules or the Cy5 and Cy7 fluorescent molecules. Moreover, the excitation wavelength of the fluorescent molecules used in the present invention does not cause autofluorescence of the cells, thereby greatly reducing background noise.
  • FIG. 5 is a schematic structural view of a preferred two-color fluorescence localization super-resolution biological microscopy system of the present invention, the system 100 including a sample labeling module 10, a signal generating module 20, a first image building module 30, a second image building module 40, and Real-time locking module 50, wherein:
  • the sample labeling module 10 is for performing two-color fluorescent labeling on the biological sample using Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules, and immersing the labeled biological sample in the imaging buffer.
  • the signal generating module 20 is configured to irradiate the biological sample by laser to respectively generate a first channel scintillation fluorescent signal corresponding to the Alexa647 or Cy5 fluorescent molecule, and a second channel scintillation fluorescent signal corresponding to the Alexa750 or Cy7 fluorescent molecule.
  • the biological sample is illuminated by a predetermined activation laser to turn on the fluorescent signal, and the biological sample is illuminated by a predetermined excitation laser to turn off the fluorescent signal to generate a scintillation fluorescent signal.
  • the biological sample was irradiated with an excitation laser of Alexa647 and Alexa750 to generate a fluorescent signal.
  • the fluorescent molecules are randomly turned on (fluorescent) or turned off (no fluorescence) under the excitation of a strong excitation laser to produce flicker.
  • the opening speed of the fluorescent molecules of the present invention can be controlled by an activation laser of the order of milliwatts, and the opening speed is proportional to the activation laser power.
  • the closing speed of the fluorescent molecule can be excited by the laser
  • the closing speed is proportional to the intensity of the excitation laser.
  • the excitation laser is generated by a laser having an excitation wavelength of Alexa 647 molecules, and a laser having an excitation wavelength of Alexa 750 molecules; the activation laser is generated by a laser having a violet or ultraviolet wavelength.
  • the present invention uses appropriate excitation laser power and activating laser power to cause most of the fluorescent molecules to be in a closed state at the same time, with only a small portion of the discrete molecules being turned on. There is no coincidence between the imaging of these open fluorescent molecules.
  • the real-time locking module 50 is used to lock the position of the biological sample in real time through the real-time locking system before data acquisition to completely remove the sample drift during imaging.
  • the real-time locking module 50 includes:
  • the brightfield illumination sub-module 51 is configured to provide brightfield illumination for the biological sample, and the brightfield image of the biological sample is imaged by the front imaging lens to the locking camera.
  • the module generation sub-module 52 is configured to grab a bright field image of a biological sample or a reference body from the locking camera as a locking template.
  • the image comparison sub-module 53 is configured to capture the current bright field image of the biological sample from the locked camera in real time, and compare the current bright field image with the locking template.
  • the offset calculation sub-module 54 is configured to calculate an offset between the current brightfield image and the lock template. Specifically, the correlation calculation, or the normalized cross-correlation operation, can be used to obtain the offset of the biological sample.
  • the offset compensation sub-module 55 is configured to perform offset compensation processing on the current bright field image according to the offset, and the process loops to the end of shooting.
  • the real-time sample locking method used in the invention can lock the position of the biological sample with nanometer precision without adding any fluorescent label, remove the sample drift during the shooting process, and further improve the imaging resolution of the system. Since the bright field image of the biological sample itself is used as the locking reference, unlike the fluorescent particles, photobleaching does not occur during long-term data acquisition, thereby reducing the locking precision.
  • the real-time sample locking method used in the present invention locks cells for a long time. When the biological sample taken is too small or too thin to produce a bright field image with a high signal-to-noise ratio, a reference object such as a 1.2 micron diameter polystyrene microsphere can be attached to the slide to produce a bright field image instead of the biological sample. As a target of locking.
  • the first image construction module 30 is configured to respectively construct a first biological structure super-resolution image and a second biological structure super-resolution image according to the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal.
  • the second image construction module 40 is configured to perform alignment processing on the first biological structure super-resolution image and the second biological structure super-resolution image to construct a third biological structure super-resolution image.
  • the first image building module 30 further includes:
  • the signal acquisition sub-module 31 is configured to acquire the first channel scintillation fluorescence signal and the second channel scintillation fluorescence signal by a two-color fluorescence positioning super-resolution microscope.
  • the two-color fluorescence localization super-resolution microscope is an inverted internal reflection fluorescence microscope (TIRFM). That is, the scintillation fluorescent signal is received by an inverted total reflection fluorescence microscope.
  • Signal separation sub-module 32 for splitting system for positioning a super-resolution microscope through two-color fluorescence
  • the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal are separated and imaged respectively in different regions of the camera's photosensitive element.
  • the camera is an EMCCD (Electron-Multiplying Charge-coupled Devic) camera, the spectroscopic system comprising a rectangular aperture, a two-color mirror and two filters. Fluorescent signals from the dye molecules are received by the microscope and separated by a spectroscopic system and imaged by lenses into different regions of the photosensitive element of the EMCCD camera. Using the appropriate excitation laser power and activating the laser power, most of the fluorescent molecules are off at the same time, and only a small fraction of the discrete molecules are on. There is no coincidence between the imaging of these open fluorescent molecules.
  • EMCCD Electro-Multiplying Charge-coupled Devic
  • the position recording sub-module 33 is configured to acquire each image captured by the camera, determine the center coordinates of each of the fluorescent molecules in the image by Gaussian fitting and record. Due to the optical resolution limit, each fluorescent molecule forms an image of an Airy Disk.
  • the positioning software obtains the image on the EMCCD camera and determines the center coordinates of each Airy spot by Gaussian fitting. This coordinate more accurately represents the actual position of the fluorescent molecules and is recorded by the program.
  • the EMCCD camera records the flickering fluorescent signal at high speed until almost all of the fluorescent molecules are located and recorded.
  • the image generating sub-module 34 is configured to respectively construct a first biological structure super-resolution image corresponding to the first channel scintillation fluorescent signal and a second biological structure super-resolution corresponding to the second channel scintillation fluorescent signal according to the recorded Elysian center coordinates image.
  • the program reconstructs the super-resolution biological structure of each channel by Gaussian-fitted center coordinates of the Airy spot.
  • the present invention provides a super-resolution two-color fluorescence localization microscopy system based on Alexa647 and Alexa750 fluorescent molecular labeling. At the same time, this microscopy system has a cell locking function to eliminate sample drift.
  • the microscope system is simple and easy to operate.
  • the application of optimized imaging buffers greatly reduces the photobleaching of fluorescent molecules during imaging and eliminates channel crosstalk.
  • the sample is directly locked using the brightfield image of the cell to eliminate any sample drift during imaging.
  • the invention can be widely applied to the field of bio-optical super-resolution microscopy.
  • FIG. 6 is a flow chart of a two-color fluorescence localization super-resolution biomicroscopic method of the present invention, which can be implemented by the system 100 as shown in FIG. 1 or FIG. 4, including:
  • step S601 the biological sample is subjected to two-color fluorescent labeling using Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules, and the biological sample is immersed in the imaging buffer.
  • the Alexa647 and Alexa750 fluorescent molecules are respectively bound to different structural proteins of the biological sample by immunolabeling, the biological sample is made into a sealing piece, and the biological sample is to be immersed in the imaging buffer, and then the invading is performed.
  • An imaging buffer with biological samples was placed on the stage of a two-color fluorescence localization super-resolution microscope.
  • the fluorescent molecules of the present invention preferably employ Alexa647 and Alexa750 fluorescent molecules, but Alexa647 and Alexa750 fluorescent molecules can also be replaced by anthocyanin dye molecules of the same excitation wavelength such as Cy5 and Cy7.
  • Step S602 irradiating a biological sample with a laser to respectively generate a fluorescent molecule with Alexa647 or Cy5 Corresponding first channel scintillation fluorescent signal, and second channel scintillation fluorescent signal corresponding to Alexa750 or Cy7 fluorescent molecule.
  • Alexa647 and Alexa750 fluorescent molecules are used in the present invention, a first channel scintillation fluorescent signal corresponding to the Alexa647 fluorescent molecule and a second channel scintillation fluorescent signal corresponding to the Alexa750 fluorescent molecule are generated. If the Cy5 and Cy7 fluorescent molecules are used in the present invention, a first channel scintillation fluorescent signal corresponding to the Cy5 fluorescent molecule and a second channel scintillation fluorescent signal corresponding to the Cy7 fluorescent molecule are generated.
  • the biological sample is illuminated by a predetermined activation laser to turn on the fluorescent signal, and the biological sample is irradiated by a predetermined excitation laser to turn off the fluorescent signal to generate a scintillation fluorescent signal.
  • the activation laser has a violet or ultraviolet wavelength.
  • the excitation laser has an excitation wavelength of Alexa647 and Alexa750 fluorescent molecules, or the excitation laser has excitation wavelengths of Cy5 and Cy7 fluorescent molecules.
  • the signal generating module 20 is configured to use a predetermined excitation laser power and an activation laser power such that most of the fluorescent molecules at the same time turn off the fluorescent signal, and only a small portion of the discrete fluorescent molecules turn on the fluorescent signal.
  • Step S603 respectively constructing a first biological structure super-resolution image and a second biological structure super-resolution image according to the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal.
  • Step S604 performing alignment processing on the first biological structure super-resolution image and the second biological structure super-resolution image to construct a third biological structure super-resolution image.
  • the specific component of the imaging buffer comprises TCEP (tris(2-carboxyethyl)phosphine), COT (cyclooctatetraene), oxygen scavenger, methyl viologen and/or ascorbic acid; or imaging
  • the specific components of the buffer include Thiol, COT, oxygen scavenger, methyl viologen and/or ascorbic acid.
  • the cost of TCEP or Thiol in the imaging buffer is combined with the fluorescent molecule to form an adduct under the irradiation of an excitation laser, the adduct does not fluoresce under the irradiation of the excitation laser, and the fluorescent molecule is thus turned off;
  • the compound re-decomposes into TCEP or Thiol and fluorescent molecules under the irradiation of an activated laser, and the fluorescent molecules emit fluorescence, and the fluorescent molecules are thus turned on.
  • FIG 2 shows the molecular structure of TCEP.
  • the Alexa647 fluorescent molecule interacts with the TCEP component in the imaging buffer to form an adduct as shown in Figure 3.
  • the Alexa647 fluorescent molecule in Figure 3 is only described as an example. It includes anthocyanin fluorescent molecules such as Alexa750, Cy5 and Cy7.
  • the Alexa647 fluorescent molecule interacts with the Thiol component in the imaging buffer to form an adduct as shown in Figure 4.
  • the Alexa647 fluorescent molecule in Figure 4 is only described as an example. This principle applies to cyanine including Alexa750, Cy5 and Cy7. Fluorescent molecules.
  • the Thiol includes ⁇ ME (2-mercaptoethanol), or MEA (ethanolamine), etc., but Thiol may reduce the photon number of the Alexa 750 molecule and increase the probability that the fluorescent molecule is photobleached, affecting the image quality.
  • the COT in the imaging buffer includes derivatives of COT.
  • the COT of the present invention is dissolved in DMSO (Dimethyl sulfoxide, dimethyl sulfoxide).
  • DMSO Dimethyl sulfoxide, dimethyl sulfoxide
  • the COT in the imaging buffer can inhibit the fluorescent molecules from entering the triplet state in the environment lacking oxygen molecules, thereby increasing the photons generated when the fluorescent molecules are in an open state. Number, improve resolution, while reducing the probability of fluorescent molecules being photobleached, improving image quality.
  • the oxygen scavenger comprises a combination of glucose oxidase, glucose and catalase; or the oxygen scavenger comprises a combination of pyranose oxidase, glucose and catalase.
  • Glucose oxidase (or pyranose oxidase) in the imaging buffer, glucose and catalase combined as an oxygen scavenger can remove oxygen from the buffer, greatly reducing the probability of photobleaching of fluorescent molecules and improving image quality.
  • the present invention is not limited to the use of glucose oxidase (or pyranose oxidase), a combination of glucose and catalase as an oxygen scavenger. Other possible oxygen scavenging schemes can also be applied to the present invention.
  • Methyl viologen and ascorbic acid in the imaging buffer act as reducing agents to inhibit photobleaching of fluorescent molecules.
  • the packaged biological sample is placed on a stage of an inverted microscope and simultaneously sampled in a total internal reflection mode using a 656.5 nm laser with a 300 mW output power and a 750 nm laser with a 300 mW output power. Irradiation.
  • FIG. 7 is a flow diagram of a preferred two-color fluorescence localization super-resolution biomicroscopy method of the present invention, which may be implemented by system 100 as shown in FIG. 4, including:
  • step S701 the biological sample is subjected to two-color fluorescent labeling using Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules, and the biological sample is immersed in the imaging buffer.
  • the two-color fluorescent super-resolution microscopy method of the present invention uses Alexa647 and Alexa750 fluorescent molecules to label a specific structure of a biological sample by immunological reaction, and soaks the biological sample in an imaging buffer containing TCEP.
  • Step S702 irradiating the biological sample with a laser to respectively generate a first channel scintillation fluorescent signal corresponding to the Alexa647 or Cy5 fluorescent molecule, and a second channel scintillation fluorescent signal corresponding to the Alexa750 or Cy7 fluorescent molecule.
  • the biological sample is illuminated by a predetermined activation laser to turn on the fluorescent signal, and the biological sample is illuminated by a predetermined excitation laser to turn off the fluorescent signal to generate a scintillation fluorescent signal.
  • the opening speed of the fluorescent molecules of the present invention can be controlled by an activation laser of the order of milliwatts, and the opening speed is proportional to the activation laser power.
  • the closing speed of the fluorescent molecule can be controlled by its excitation laser intensity, which is proportional to the intensity of the excitation laser.
  • the excitation laser is generated by a laser having an excitation wavelength of Alexa 647 molecules, and a laser having an excitation wavelength of Alexa 750 molecules; the activation laser is generated by a laser having a violet or ultraviolet wavelength.
  • the present invention uses appropriate excitation laser power and activating laser power to cause most of the fluorescent molecules to be in a closed state at the same time, with only a small portion of the discrete molecules being turned on. There is no coincidence between the imaging of these open fluorescent molecules.
  • step S703 the position of the biological sample is locked in real time by the real-time locking system before data acquisition.
  • This step further includes:
  • Brightfield illumination is provided for the biological sample, and the brightfield image of the biological sample is imaged by the front imaging lens to the locked camera.
  • a bright field image of a biological sample or reference body is captured from the locked camera as a locking template.
  • the current brightfield image of the biological sample is captured in real time from the locked camera, and the current brightfield image is compared with the locked template.
  • the current brightfield image is subjected to offset compensation processing according to the offset, and the process loops to the end of shooting.
  • Step S704 obtaining a first channel scintillation fluorescence signal and a second channel scintillation fluorescence signal by a two-color fluorescence localization super-resolution microscope.
  • the two-color fluorescence localization super-resolution microscope is an inverted total reflection fluorescence microscope.
  • Step S705 separating the first channel scintillation fluorescent signal and the second channel scintillation fluorescent signal by a spectroscopic system of a two-color fluorescence positioning super-resolution microscope, and respectively imaging different regions of the photosensitive element of the camera.
  • the camera is an EMCCD (Electron-Multiplying Charge-coupled Devic) camera, the spectroscopic system comprising a rectangular aperture, a two-color mirror and two filters. Fluorescent signals from the dye molecules are received by the microscope and separated by a spectroscopic system and imaged by lenses into different regions of the photosensitive element of the EMCCD camera. Using the appropriate excitation laser power and activating the laser power, most of the fluorescent molecules are off at the same time, and only a small fraction of the discrete molecules are on. There is no coincidence between the imaging of these open fluorescent molecules.
  • EMCCD Electro-Multiplying Charge-coupled Devic
  • Step S706 acquiring each image captured by the camera, determining the center coordinates of each of the fluorescent molecules in the image by Gaussian fitting and recording.
  • each fluorescent molecule forms an image of an Airy Disk.
  • the positioning software obtains the image on the EMCCD camera and determines the center coordinates of each Airy spot by Gaussian fitting. This coordinate more accurately represents the actual position of the fluorescent molecules and is recorded by the program.
  • the EMCCD camera records the flickering fluorescent signal at high speed until almost all of the fluorescent molecules are located and recorded.
  • Step S707 respectively constructing a first biological structure super-resolution image corresponding to the first channel scintillation fluorescent signal and a second biological structure super-resolution image corresponding to the second channel scintillation fluorescent signal according to the recorded Elysian center coordinates.
  • the program reconstructs the super-resolution biological structure of each channel by Gaussian-fitted center coordinates of the Airy spot.
  • Step S708 performing alignment processing on the first biological structure super-resolution image and the second biological structure super-resolution image to construct a third biological structure super-resolution image.
  • FIG. 8 is a schematic view showing the structure of a two-color fluorescence localization super-resolution biological microscopy system according to an embodiment of the present invention, including an optical structure diagram of a real-time cell locking system.
  • the present invention uses a real-time cell locking system to completely remove sample drift during imaging.
  • the principle is as follows: 1) Using the light-emitting diode of FIG. 8 and the filter 1 to generate blue light to provide bright field illumination for the sample. 2) The bright field image of the sample is imaged by the front imaging lens to the locking camera. 4)
  • the program will first grab a template image of the sample from the locked camera as a locking template. 5) The locking program then grabs the brightfield image of the sample in real time from the locked camera and compares it with the locking template through the associated algorithm. 6) The correlation algorithm will get the offset of the current image relative to the locked template. 7) The locking program then drives the piezoceramic platform through the drive circuit to compensate for the offset. 8) This process loops to the end of shooting.
  • the detection optical path used in the present invention is as shown in FIG.
  • the scintillation fluorescent signal emitted by the fluorescent label on the sample is received by a 100x objective lens.
  • Fluorescent signals include: channel 1: fluorescence from the Alexa 647 molecule; and channel 2: fluorescence from the Alexa 758 molecule.
  • the signals of the two channels are separated using a spectroscopic system consisting of a rectangular aperture, a two-color mirror 2 and a filter 3, and a filter 4, and imaged separately in different regions of the EMCCD camera photosensitive element.
  • the EMCCD camera simultaneously records the flashing fluorescent dot signals from both channels in a recording mode.
  • the positioning software acquires each image acquired by the EMCCD and determines the center coordinates of each Airy spot on each channel in each image by Gaussian fitting and records.
  • the program then plots the recorded center coordinates in the form of points to reconstruct the super-resolution biostructural patterns of the Alexa647 and Alexa750 channels, respectively. Finally, the images of the two channels are precisely aligned by the algorithm to obtain a two-color fluorescence super-resolution micrograph of the labeled sample.
  • the two-color fluorescence localization super-resolution biomicroscopy technique of the present invention uses Alexa647 and Alexa750 fluorescent molecules, or Cy5 and Cy7 fluorescent molecules to perform two-color fluorescent labeling on biological samples, and the emission spectrum of the fluorescent molecules used is completely separated.
  • the present invention employs an optimized imaging buffer to greatly reduce photobleaching of fluorescent molecules during imaging.
  • the present invention locks the sample with a real-time locking function to eliminate completely to eliminate any sample drift during imaging.
  • the invention can be widely applied to the field of bio-optical super-resolution microscopy.

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Abstract

一种双色荧光定位超分辨率生物显微方法和系统（100），所述方法包括有：使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将生物样品浸泡在成像缓冲液中（S601）；通过激光照射生物样品，分别产生与Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号(S602)；根据第一、第二通道闪烁荧光信号，分别构建第一、第二生物结构超分辨率图像(S603)；将第一、第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像(S604)。借此，该超分辨率生物显微成像技术不会产生通道串扰，并且可大幅降低背景噪音，从而提高了成像质量。

Description

双色荧光定位超分辨率生物显微方法及系统 技术领域
本发明涉及生物显微技术领域，尤其涉及一种双色荧光定位超分辨率生物显微方法及系统。
背景技术
自恩斯特·阿贝(Ernst Abbe)于十八世纪七十年代提出光学成像分辨率极限的理论以来，人们一直在寻找各种各样的方法方式以求突破这一分辨率极限。目前，通过运用现代尖端技术，庄小威，埃里克·贝齐格(Eric Betzig)分别于2006年先后分别提出了随机光学重构显微技术(Stochastic Optical Reconstruction Microscopy，SORM)和荧光定位显微技术(Photoactivation Laser Microscopy，PLM)，均实现了突破光学分辨率极限十倍的超分辨率成像。埃里克·贝齐格更是通过这一技术获得了2014年诺贝尔化学奖。目前荧光定位显微技术已经实现部分商业化，并且开始被用于基础生命科学，尤其是分子生物学以及生物化学的研究当中。通过这一技术，研究人员可以以10到20纳米的横向分辨率和50纳米的纵向分辨率研究生物样品的细节结构。与其他已有的超分辨率技术，如电子显微技术相比，荧光定位显微技术大大简化了样品的制备环节，并且可以实现活体细胞成像。更重要的是，荧光定位显微技术可以轻松的实现多通道共定位成像，从而得到蛋白质之间的相互作用关系，为大量分子生物研究课题提供最直接的证据。然而，现有技术在多色超分辨率成像方面的表现仍有不足之处。尼康(Nikon)N-STORM作为商业化荧光定位显微镜的代表，其多通道成像使用由两种不同激发波长荧光分子结合而成的“荧光开关”(例如，Alexa647-Alexa488分子对以及Alexa467-Alexa405分子对)作为标记物。这种方法可以实现不同成像通道的快速切换，但是这类荧光开关的标记物目前并无商业化，且实验室制备较为复杂。更重要的是，这种多色成像的方法会产生较严重的通道串扰(Channel Crosstalk)，产生错误的共定位信息。徕卡(Leica)SR GSD定位显微镜使用不同激发波长的普通荧光分子作为标记物(例如，Alexa647和Alexa532)，并使用滤镜系统来实现多通道成像。这一方法的局限性在于，短激发波长荧光分子极易被光漂白(Photo-bleach)，并且较短的激发波长会导致细胞的自发荧光背景，影响成像质量。
另外，样品漂移是一种普遍存在于超分辨率荧光定位显微镜的问题。这是指由于环境的不稳定，如气流、温度变化、噪音等等，会使样品在拍摄过程中发生几十至几百纳米的移动。尽管这一漂移现象普遍存在于显微系统中，由于 一般显微镜的分辨率不足300纳米，漂移造成的现象并不明显。但是对于分辨率可达十几纳米的超分辨率显微镜而言，漂移会对成像造成严重的干扰。目前已有的解决方法大多为向样品中加入荧光颗粒，并在成像中记录这些颗粒的位移，随后从所得超分辨率图像中将这一位移减去。其缺陷是制备麻烦；而且荧光颗粒会占用一个成像通道；另外，由于光漂白，荧光颗粒的荧光会随成像时间衰减，因此漂移的修正精度也会随时间变差。
综上可知，现有技术在实际使用上显然存在不便与缺陷，所以有必要加以改进。
发明内容
针对上述的缺陷，本发明的目的在于提供一种双色荧光定位超分辨率生物显微方法及系统，其不会产生通道串扰，并且可大幅降低背景噪音，从而提高了成像质量。
为了实现上述目的，本发明提供一种双色荧光定位超分辨率生物显微方法，包括有：
使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将所述生物样品浸泡在成像缓冲液中；
通过激光照射所述生物样品，分别产生与所述Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与所述Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号；
根据所述第一通道闪烁荧光信号和所述第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像；
将所述第一生物结构超分辨率图像和所述第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
根据本发明所述的方法，所述通过激光照射所述生物样品的步骤包括：
通过预定的激活激光照射所述生物样品以开启荧光信号，并通过预定的激发激光照射所述生物样品以关闭荧光信号，以产生闪烁荧光信号。
根据本发明所述的方法，所述激活激光具有紫色或紫外波长；所述激发激光具有Alexa647和Alexa750荧光分子激发波长，或者所述激发激光具有Cy5和Cy7荧光分子激发波长；使用预定的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子关闭荧光信号，仅有小部分离散的荧光分子开启荧光信号。
根据本发明所述的方法，所述成像缓冲液包含有TCEP、环辛四烯、除氧剂、甲基紫精和/或抗坏血酸；或者
所述成像缓冲液包含有巯基物、环辛四烯、除氧剂、甲基紫精和/或抗坏血 酸。
根据本发明所述的方法，所述TCEP或巯基物与荧光分子在所述激发激光的照射下结合形成加合物，所述加合物不会在所述激发激光的照射下发出荧光；所述加合物在所述激活激光的照射下重新分解为所述TCEP或巯基物和荧光分子，所述荧光分子发出荧光。
根据本发明所述的方法，所述环辛四烯包括环辛四烯的衍生物；所述除氧剂包括葡萄糖氧化酶、葡萄糖和过氧化氢酶的组合，或者，所述除氧剂包括吡喃糖氧化酶、葡萄糖和过氧化氢酶的组合。
根据本发明所述的方法，所述根据第一通道闪烁荧光信号和所述第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像的步骤之前还包括：
在数据采集之前，通过实时锁定系统将所述生物样品的位置实时锁定。
根据本发明所述的方法，所述实时锁定系统将所述生物样品的位置实时锁定的步骤包括：
为所述生物样品提供明场照明，所述生物样品的明场图像经过前成像透镜成像于锁定相机；
从所述锁定相机中抓取一张生物样品或参照体的明场图像作为锁定模板；
从所述锁定相机中实时抓取所述生物样品的当前明场图像，将所述当前明场图像与所述锁定模板进行对比；
计算所述当前明场图像与所述锁定模板之间的偏移量；
根据所述偏移量对所述当前明场图像进行偏移补偿处理。
根据本发明所述的方法，所述根据第一通道闪烁荧光信号和所述第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像的步骤还包括：
通过双色荧光定位超分辨率显微镜获取所述第一通道闪烁荧光信号和所述第二通道闪烁荧光信号；
通过所述双色荧光定位超分辨率显微镜的分光系统将所述第一通道闪烁荧光信号和所述第二通道闪烁荧光信号分离，并分别成像于相机的感光元件的不同区域；
获取所述相机采集的每一张图像，通过高斯拟合确定所述图像中每个荧光分子的爱里斑中心坐标并记录；
根据记录的所述爱里斑中心坐标分别构建所述第一通道闪烁荧光信号对应的所述第一生物结构超分辨率图像和所述第二通道闪烁荧光信号对应的所述第二生物结构超分辨率图像。
根据本发明所述的方法，所述双色荧光定位超分辨率显微镜为倒置的全反 射荧光显微镜；
所述相机为EMCCD相机；和/或
所述分光系统包括矩形光阑、双色反射镜和两个滤镜。
本发明还提供一种双色荧光定位超分辨率生物显微系统，包括有：
样品标记模块，用于使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将所述生物样品浸泡在成像缓冲液中；
信号产生模块，用于通过激光照射所述生物样品，分别产生与所述Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与所述Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号；
第一图像构建模块，用于根据所述第一通道闪烁荧光信号和所述第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像；
第二图像构建模块，用于将所述第一生物结构超分辨率图像和所述第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
根据本发明所述的系统，所述信号产生模块用于通过预定的激活激光照射所述生物样品以开启荧光信号，并通过预定的激发激光照射所述生物样品以关闭荧光信号，以产生闪烁荧光信号。
根据本发明所述的系统，所述激活激光具有紫色或紫外波长；所述激发激光具有Alexa647和Alexa750荧光分子激发波长，或者所述激发激光具有Cy5和Cy7荧光分子激发波长；
所述信号产生模块用于使用预定的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子关闭荧光信号，仅有小部分离散的荧光分子开启荧光信号。
根据本发明所述的系统，所述成像缓冲液包含有TCEP、环辛四烯、除氧剂、甲基紫精和/或抗坏血酸；或者
所述成像缓冲液包含有巯基物、环辛四烯、除氧剂、甲基紫精和/或抗坏血酸。
根据本发明所述的系统，所述TCEP或巯基物与荧光分子在所述激发激光的照射下结合形成加合物，所述加合物不会在所述激发激光的照射下发出荧光；所述加合物在所述激活激光的照射下重新分解为所述TCEP或巯基物和荧光分子，所述荧光分子发出荧光。
根据本发明所述的系统，所述环辛四烯包括环辛四烯的衍生物；所述除氧剂包括葡萄糖氧化酶、葡萄糖和过氧化氢酶的组合，或者，所述除氧剂包括吡喃糖氧化酶、葡萄糖和过氧化氢酶的组合。
根据本发明所述的系统，还包括：
实时锁定模块，用于在数据采集之前，通过实时锁定系统将所述生物样品的位置实时锁定。
根据本发明所述的系统，所述实时锁定模块包括：
明场照明子模块，用于为所述生物样品提供明场照明，所述生物样品的明场图像经过前成像透镜成像于锁定相机；
模块生成子模块，用于从所述锁定相机中抓取一张生物样品或参照体的明场图像作为锁定模板；
图像对比子模块，用于从所述锁定相机中实时抓取所述生物样品的当前明场图像，将所述当前明场图像与所述锁定模板进行对比；
偏移计算子模块，用于计算所述当前明场图像与所述锁定模板之间的偏移量；
偏移补偿子模块，用于根据所述偏移量对所述当前明场图像进行偏移补偿处理。
根据本发明所述的系统，所述第一图像构建模块还包括：
信号获取子模块，用于通过双色荧光定位超分辨率显微镜获取第一通道闪烁荧光信号和第二通道闪烁荧光信号；
信号分离子模块，用于通过双色荧光定位超分辨率显微镜的分光系统将第一通道闪烁荧光信号和第二通道闪烁荧光信号分离，并分别成像于相机的感光元件的不同区域；
位置记录子模块，用于获取相机采集的每一张图像，通过高斯拟合确定图像中每个荧光分子的爱里斑中心坐标并记录；
图像生成子模块，用于根据记录的爱里斑中心坐标分别构建第一通道闪烁荧光信号对应的第一生物结构超分辨率图像和第二通道闪烁荧光信号对应的第二生物结构超分辨率图像。
根据本发明所述的系统，所述双色荧光定位超分辨率显微镜为倒置的全反射荧光显微镜；
所述相机为EMCCD相机；和/或
所述分光系统包括矩形光阑、双色反射镜和两个滤镜。
本发明双色荧光定位超分辨率生物显微技术采用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，所采用的荧光分子的发射光谱完全分离，因此理论上不会有通道串扰产生，并且荧光分子的激发波长不会使细胞产生自发荧光，从而大幅降低了背景噪音，从而提高了成像质量，且易于操作。优选的是，本发明采用优化的成像缓冲液使荧光分子在成像过程中的光漂白大大减少。更好的是，本发明通过实时锁定功能对样品进行锁 定，以彻底消除以消除成像过程中的任何样品漂移。本发明可以广泛应用于生物光学超分辨率显微领域。
附图说明
图1是本发明双色荧光定位超分辨率生物显微系统的结构示意图；
图2是本发明TCEP的分子结构图；
图3是本发明实施例中荧光分子与成像缓冲液中TCEP成分作用原理图
图4是本发明实施例中荧光分子与成像缓冲液中Thiol成分作用原理图；
图5是本发明优选双色荧光定位超分辨率生物显微系统的结构示意图；
图6是本发明双色荧光定位超分辨率生物显微方法的流程图；
图7是本发明优选双色荧光定位超分辨率生物显微方法的流程图；
图8是本发明一实施例中双色荧光定位超分辨率生物显微系统的结构示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
图1是本发明双色荧光定位超分辨率生物显微系统的结构示意图，所述系统100包括有样品标记模块10、信号产生模块20、第一图像构建模块30以及第二图像构建模块40，其中：
样品标记模块10，用于使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将标记后的生物样品浸泡在成像缓冲液中。在一个实施例中，将Alexa647和Alexa750荧光分子通过免疫标记法分别结合在生物样品的不同结构蛋白上，将生物样品制作成封片，并且生物样品需浸泡在成像缓冲液中，再将侵泡有生物样品的成像缓冲液放在双色荧光定位超分辨率显微镜的载物台上。本发明的荧光分子优选采用Alexa647和Alexa750荧光分子，但Alexa647和Alexa750荧光分子亦可由相同激发波长的花青素染料分子如Cy5和Cy7来替代。
信号产生模块20，用于通过激光照射生物样品，分别产生与Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号。本发明如果使用Alexa647和Alexa750荧光分子，则产生与Alexa647荧光分子对应的第一通道闪烁荧光信号和与Alexa750荧光分子对应的第二通道闪烁荧光信号。本发明如果使用Cy5和Cy7荧光分子，则产生与Cy5荧光分子对应的第一通道闪烁荧光信号和与Cy7荧光分子对应的 第二通道闪烁荧光信号。
第一图像构建模块30，用于根据第一通道闪烁荧光信号和第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像。
第二图像构建模块40，用于将第一生物结构超分辨率图像和第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
优选的是，所述成像缓冲液的具体成分包含有TCEP(三(2-羧乙基)膦)、COT(环辛四烯)、除氧剂、甲基紫精和/或抗坏血酸；或者成像缓冲液的具体成分包含有巯基物(Thiol)、COT、除氧剂、甲基紫精和/或抗坏血酸。
所述成像缓冲液中的TCEP或Thiol成本与荧光分子在激发激光的照射下结合形成加合物，该加合物不会在激发激光的照射下发出荧光，荧光分子因此被关闭；且该加合物在激活激光的照射下重新分解为TCEP或Thiol和荧光分子，荧光分子发出荧光，荧光分子因此被开启。
图2示出了TCEP的分子结构，Alexa647荧光分子与成像缓冲液中TCEP成分作用后生成如图3所示的加合物，图3中的Alexa647荧光分子仅作为实施例具体说明，此原理适用于包括Alexa750，Cy5和Cy7等花青素荧光分子。
Alexa647荧光分子与成像缓冲液中Thiol成分作用后生成如图4所示的加合物，图4中的Alexa647荧光分子仅作为实施例具体说明，此原理适用于包括Alexa750，Cy5和Cy7等花青素荧光分子。
所述Thiol包括βME(2-巯基乙醇)，或MEA(乙醇胺)等，但是Thiol可能会减少Alexa750分子的光子数，且增加荧光分子被光漂白的概率，影响图像质量。
成像缓冲液中的COT包括COT的衍生物。本发明COT溶解于DMSO(Dimethyl sulfoxide，二甲基亚砜)。成像缓冲液中的COT可抑制荧光分子在缺少氧分子的环境中进入三重态，从而提高荧光分子在打开状态时产生的光子数，提高分辨率，同时减少荧光分子被光漂白的概率，提高成像质量。
所述除氧剂包括葡萄糖氧化酶、葡萄糖和过氧化氢酶的组合；或者，除氧剂包括吡喃糖氧化酶、葡萄糖和过氧化氢酶的组合。成像缓冲液中的葡萄糖氧化酶(或吡喃糖氧化酶)，葡萄糖和过氧化氢酶组合作为除氧剂可去除缓冲液中的氧气，大幅减少荧光分子被光漂白的概率，提高成像质量。本发明并不局限于使用葡萄糖氧化酶(或吡喃糖氧化酶)，葡萄糖和过氧化氢酶组合作为除氧剂。其他可行的除氧方案亦可应用于本发明。
成像缓冲液中的甲基紫精和抗坏血酸作为还原剂可抑止荧光分子的光漂白。
本发明使用经过优化的成像缓冲液配方使荧光分子在每次发光周期都会产生超过2000个光子，并且成像缓冲液中的除氧剂和COT成分大幅减少分子的 光漂白，从而产生高质量的双色超分辨率图像。Alexa647通道的横向分辨率在理想条件下可达10纳米，Alexa750通道可达20纳米。
实施例1中成像缓冲液的具体配方为：200mM Tris磷酸缓冲液，pH9.0；并含有10％(w/v)葡萄糖,5U/ml吡喃糖氧化酶,57μg/ml过氧化氢酶,2mM COT(溶于DMSO),25mM TCEP，1mM抗坏血酸和1mM甲基紫精。
实施例2中成像缓冲液的具体配方为：200mM Tris磷酸缓冲液，pH8.0；并含有10％(w/v)葡萄糖,560μg/ml葡萄糖氧化酶,57μg/ml过氧化氢酶,2mM COT(溶于DMSO),25mM TCEP,1mM抗坏血酸和1mM甲基紫精。
优选的是，信号产生模块20用于通过预定的激活激光照射生物样品以开启荧光信号，并通过预定的激发激光照射生物样品以关闭荧光信号，以产生闪烁荧光信号。所述激活激光具有紫色或紫外波长。所述激发激光具有Alexa647和Alexa750荧光分子激发波长，或者激发激光具有Cy5和Cy7荧光分子激发波长。信号产生模块20用于使用预定的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子关闭荧光信号，仅有小部分离散的荧光分子开启荧光信号。
在一个实施例中，将封装的生物样品放置在倒置显微镜的载物台上，并使用300mW输出功率的656.5纳米激光，和300mW输出功率的750纳米激光，以全内反射的模式对样品同时进行照射。
本发明由于使用的Alexa647和Alexa750荧光分子，或者Cy5和Cy7荧光分子的发射光谱完全分离，因此理论上不会有通道串扰产生。而且，本发明所用荧光分子的激发波长不会使细胞产生自发荧光，从而大幅降低了背景噪音。
图5是本发明优选双色荧光定位超分辨率生物显微系统的结构示意图，所述系统100包括有样品标记模块10、信号产生模块20、第一图像构建模块30、第二图像构建模块40以及实时锁定模块50，其中：
样品标记模块10，用于使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将标记后的生物样品浸泡在成像缓冲液中。
信号产生模块20，用于通过激光照射生物样品，分别产生与Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号。
通过预定的激活激光照射生物样品以开启荧光信号，并通过预定的激发激光照射生物样品以关闭荧光信号，以产生闪烁荧光信号。同时使用Alexa647和Alexa750的激发激光照射生物样品以产生荧光信号。在成像缓冲液的作用下，荧光分子在强激发激光的照射下被随机的开启(发出荧光)或关闭(不发出荧光)，产生闪烁。本发明荧光分子的开启速度可由一束毫瓦量级的激活激光控制，开启速度和激活激光功率成正比。荧光分子的关闭速度可由其激发激光强 度来控制，关闭速度和激发激光强度成正比。所述激发激光为具有Alexa647分子激发波长的激光器生成，和具有Alexa750分子激发波长的激光器生成；所述激活激光为具有紫色或紫外波长的激光器生成。本发明使用适当的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子处于关闭状态，仅有小部分离散的分子处于开启状态。这些开启的荧光分子所成像之间没有重合。
实时锁定模块50，用于在数据采集之前，通过实时锁定系统将生物样品的位置实时锁定，以彻底去除成像时的样品漂移。优选的是，实时锁定模块50包括：
明场照明子模块51，用于为生物样品提供明场照明，生物样品的明场图像经过前成像透镜成像于锁定相机。
模块生成子模块52，用于从锁定相机中抓取一张生物样品或参照体的明场图像作为锁定模板。
图像对比子模块53，用于从锁定相机中实时抓取生物样品的当前明场图像，将当前明场图像与锁定模板进行对比。
偏移计算子模块54，用于计算当前明场图像与锁定模板之间的偏移量。具体可使用相关度运算，或归一化互相关运算获取生物样品的偏移量。
偏移补偿子模块55，用于根据偏移量对当前明场图像进行偏移补偿处理，此过程循环至拍摄结束。
本发明所使用的实时样品锁定方法无需添加任何荧光标记物便可以纳米精度锁定生物样品位置，去除拍摄过程中的样品漂移，进一步提高系统的成像分辨率。由于使用生物样品本身的明场图像作为锁定参照，因此不同于荧光微粒，不会在长时间的数据采集过程发生光漂白，从而降低锁定精度。本发明所使用的实时样品锁定方法可将细胞长时间锁定。当所拍摄的生物样品过小或过薄无法产生较高信噪比的明场图像时，可向载玻片附着少许1.2微米直径的聚苯乙烯微球等参照体以产生明场图像代替生物样品作为锁定目标。
第一图像构建模块30，用于根据第一通道闪烁荧光信号和第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像。
第二图像构建模块40，用于将第一生物结构超分辨率图像和第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
优选的是，第一图像构建模块30还包括：
信号获取子模块31，用于通过双色荧光定位超分辨率显微镜获取第一通道闪烁荧光信号和第二通道闪烁荧光信号。优选的，双色荧光定位超分辨率显微镜为倒置的全反射荧光显微镜(Total internal Reflection Fluorescence Microscope，TIRFM)。即通过倒置的全反射荧光显微镜接收闪烁荧光信号。
信号分离子模块32，用于通过双色荧光定位超分辨率显微镜的分光系统将 第一通道闪烁荧光信号和第二通道闪烁荧光信号分离，并分别成像于相机的感光元件的不同区域。优选的，所述相机为EMCCD(Electron-Multiplying Charge-coupled Devic，电子倍增电荷耦合元件)相机，所述分光系统包括矩形光阑、双色反射镜和两个滤镜。来自染料分子的荧光信号被显微镜接收后通过分光系统分离，并分别通过透镜成像于EMCCD相机的感光元件上的不同区域。使用适当的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子处于关闭状态，仅有小部分离散的分子处于开启状态。这些开启的荧光分子所成像之间没有重合。
位置记录子模块33，用于获取相机采集的每一张图像，通过高斯拟合确定图像中每个荧光分子的爱里斑中心坐标并记录。由于光学分辨率极限，每个荧光分子所成的像都为一个爱里斑(Airy Disk)。定位软件获取EMCCD相机上的图像，通过高斯拟合确定每个爱里斑的中心坐标，这一坐标更精确的代表了荧光分子的实际位置，并被程序记录下来。EMCCD相机高速记录闪烁的荧光信号直至几乎所有荧光分子都被定位且记录。
图像生成子模块34，用于根据记录的爱里斑中心坐标分别构建第一通道闪烁荧光信号对应的第一生物结构超分辨率图像和第二通道闪烁荧光信号对应的第二生物结构超分辨率图像。程序通过高斯拟合出的爱里斑中心坐标分别重构出每个通道的超分辨率生物结构图样。
本发明提供了一种基于Alexa647和Alexa750荧光分子标记的超分辨率双色荧光定位显微系统。同时此显微系统具有实施细胞锁定功能以消除样品漂移。本显微系统结构简单易于操作。应用优化的成像缓冲液使荧光分子在成像过程中的光漂白大大减少，并且消除了通道串扰。利用细胞的明场图像对样品直接进行锁定以消除成像过程中的任何样品漂移。本发明可以广泛应用于生物光学超分辨率显微领域。
图6是本发明双色荧光定位超分辨率生物显微方法的流程图，其可通过如图1或图4所示的系统100是实现，包括有：
步骤S601，使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将生物样品浸泡在成像缓冲液中。
在一个实施例中，将Alexa647和Alexa750荧光分子通过免疫标记法分别结合在生物样品的不同结构蛋白上，将生物样品制作成封片，并且生物样品需浸泡在成像缓冲液中，再将侵泡有生物样品的成像缓冲液放在双色荧光定位超分辨率显微镜的载物台上。本发明的荧光分子优选采用Alexa647和Alexa750荧光分子，但Alexa647和Alexa750荧光分子亦可由相同激发波长的花青素染料分子如Cy5和Cy7来替代。
步骤S602，通过激光照射生物样品，分别产生与Alexa647或Cy5荧光分子 对应的第一通道闪烁荧光信号，以及与Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号。
本发明如果使用Alexa647和Alexa750荧光分子，则产生与Alexa647荧光分子对应的第一通道闪烁荧光信号和与Alexa750荧光分子对应的第二通道闪烁荧光信号。本发明如果使用Cy5和Cy7荧光分子，则产生与Cy5荧光分子对应的第一通道闪烁荧光信号和与Cy7荧光分子对应的第二通道闪烁荧光信号。
优选的是，通过预定的激活激光照射生物样品以开启荧光信号，并通过预定的激发激光照射生物样品以关闭荧光信号，以产生闪烁荧光信号。所述激活激光具有紫色或紫外波长。所述激发激光具有Alexa647和Alexa750荧光分子激发波长，或者激发激光具有Cy5和Cy7荧光分子激发波长。信号产生模块20用于使用预定的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子关闭荧光信号，仅有小部分离散的荧光分子开启荧光信号。
步骤S603，根据第一通道闪烁荧光信号和第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像。
步骤S604，将第一生物结构超分辨率图像和第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
优选的是，所述成像缓冲液的具体成分包含有TCEP(三(2-羧乙基)膦)、COT(环辛四烯)、除氧剂、甲基紫精和/或抗坏血酸；或者成像缓冲液的具体成分包含有巯基物(Thiol)、COT、除氧剂、甲基紫精和/或抗坏血酸。
所述成像缓冲液中的TCEP或Thiol成本与荧光分子在激发激光的照射下结合形成加合物，该加合物不会在激发激光的照射下发出荧光，荧光分子因此被关闭；且该加合物在激活激光的照射下重新分解为TCEP或Thiol和荧光分子，荧光分子发出荧光，荧光分子因此被开启。
图2示出了TCEP的分子结构，Alexa647荧光分子与成像缓冲液中TCEP成分作用后生成如图3所示的加合物，图3中的Alexa647荧光分子仅作为实施例具体说明，此原理适用于包括Alexa750，Cy5和Cy7等花青素荧光分子。
Alexa647荧光分子与成像缓冲液中Thiol成分作用后生成如图4所示的加合物，图4中的Alexa647荧光分子仅作为实施例具体说明，此原理适用于包括Alexa750，Cy5和Cy7等花青素荧光分子。
所述Thiol包括βME(2-巯基乙醇)，或MEA(乙醇胺)等，但是Thiol可能会减少Alexa750分子的光子数，且增加荧光分子被光漂白的概率，影响图像质量。
成像缓冲液中的COT包括COT的衍生物。本发明COT溶解于DMSO(Dimethyl sulfoxide，二甲基亚砜)。成像缓冲液中的COT可抑制荧光分子在缺少氧分子的环境中进入三重态，从而提高荧光分子在打开状态时产生的光子 数，提高分辨率，同时减少荧光分子被光漂白的概率，提高成像质量。
所述除氧剂包括葡萄糖氧化酶、葡萄糖和过氧化氢酶的组合；或者，除氧剂包括吡喃糖氧化酶、葡萄糖和过氧化氢酶的组合。成像缓冲液中的葡萄糖氧化酶(或吡喃糖氧化酶)，葡萄糖和过氧化氢酶组合作为除氧剂可去除缓冲液中的氧气，大幅减少荧光分子被光漂白的概率，提高成像质量。本发明并不局限于使用葡萄糖氧化酶(或吡喃糖氧化酶)，葡萄糖和过氧化氢酶组合作为除氧剂。其他可行的除氧方案亦可应用于本发明。
成像缓冲液中的甲基紫精和抗坏血酸作为还原剂可抑止荧光分子的光漂白。
在一个实施例中，将封装的生物样品放置在倒置显微镜的载物台上，并使用300mW输出功率的656.5纳米激光，和300mW输出功率的750纳米激光，以全内反射的模式对样品同时进行照射。
图7是本发明优选双色荧光定位超分辨率生物显微方法的流程图，其可通过如图4所示的系统100是实现，包括有：
步骤S701，使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将生物样品浸泡在成像缓冲液中。
优选的是，本发明涉及的双色荧光超分辨率显微方法使用Alexa647和Alexa750荧光分子通过免疫反应标记生物样品的特定结构，并且将生物样品浸泡在含有TCEP的成像缓冲液。
步骤S702，通过激光照射生物样品，分别产生与Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号。
通过预定的激活激光照射生物样品以开启荧光信号，并通过预定的激发激光照射生物样品以关闭荧光信号，以产生闪烁荧光信号。本发明荧光分子的开启速度可由一束毫瓦量级的激活激光控制，开启速度和激活激光功率成正比。荧光分子的关闭速度可由其激发激光强度来控制，关闭速度和激发激光强度成正比。所述激发激光为具有Alexa647分子激发波长的激光器生成，和具有Alexa750分子激发波长的激光器生成；所述激活激光为具有紫色或紫外波长的激光器生成。本发明使用适当的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子处于关闭状态，仅有小部分离散的分子处于开启状态。这些开启的荧光分子所成像之间没有重合。
步骤S703，在数据采集之前，通过实时锁定系统将生物样品的位置实时锁定。本步骤进一步包括：
为生物样品提供明场照明，生物样品的明场图像经过前成像透镜成像于锁定相机。
从锁定相机中抓取一张生物样品或参照体的明场图像作为锁定模板。
从锁定相机中实时抓取生物样品的当前明场图像，将当前明场图像与锁定模板进行对比。
计算当前明场图像与锁定模板之间的偏移量。
根据偏移量对当前明场图像进行偏移补偿处理，此过程循环至拍摄结束。
步骤S704，通过双色荧光定位超分辨率显微镜获取第一通道闪烁荧光信号和第二通道闪烁荧光信号。
优选的是，双色荧光定位超分辨率显微镜为倒置的全反射荧光显微镜。
步骤S705，通过双色荧光定位超分辨率显微镜的分光系统将第一通道闪烁荧光信号和第二通道闪烁荧光信号分离，并分别成像于相机的感光元件的不同区域。
优选的，所述相机为EMCCD(Electron-Multiplying Charge-coupled Devic，电子倍增电荷耦合元件)相机，所述分光系统包括矩形光阑、双色反射镜和两个滤镜。来自染料分子的荧光信号被显微镜接收后通过分光系统分离，并分别通过透镜成像于EMCCD相机的感光元件上的不同区域。使用适当的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子处于关闭状态，仅有小部分离散的分子处于开启状态。这些开启的荧光分子所成像之间没有重合。
步骤S706，获取相机采集的每一张图像，通过高斯拟合确定图像中每个荧光分子的爱里斑中心坐标并记录。
由于光学分辨率极限，每个荧光分子所成的像都为一个爱里斑(Airy Disk)。定位软件获取EMCCD相机上的图像，通过高斯拟合确定每个爱里斑的中心坐标，这一坐标更精确的代表了荧光分子的实际位置，并被程序记录下来。EMCCD相机高速记录闪烁的荧光信号直至几乎所有荧光分子都被定位且记录。
步骤S707，根据记录的爱里斑中心坐标分别构建第一通道闪烁荧光信号对应的第一生物结构超分辨率图像和第二通道闪烁荧光信号对应的第二生物结构超分辨率图像。
程序通过高斯拟合出的爱里斑中心坐标分别重构出每个通道的超分辨率生物结构图样。
步骤S708，将第一生物结构超分辨率图像和第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
图8是本发明一具体实施例中双色荧光定位超分辨率生物显微系统的结构示意图，包括实时细胞锁定系统的光学结构图。
首先，本发明使用了实时细胞锁定系统以彻底去除成像时的样品漂移。其原理为：1)利用图8中的发光二极管和滤镜1产生蓝光为样品提供明场照明。2)样品的明场图像经过前成像透镜成像于锁定相机。4)当计算机的锁定程序 启动，程序会先从锁定相机中抓取一张样品的模板图像作为锁定模板。5)锁定程序随后从锁定相机中实时抓取样品的明场图像，并通过相关算法与锁定模板进行对比。6)相关算法会得到目前图像相对于锁定模板的偏移量。7)锁定程序随后通过驱动电路驱动压电陶瓷平台以补偿偏移。8)此过程循环至拍摄结束。
本发明采用的探测光路如图8所示。通过100倍物镜接收样品上荧光标记物发出的闪烁荧光信号。荧光信号包括：通道1：Alexa647分子发出的荧光；和通道2：Alexa758分子发出的荧光。使用由包含矩形光阑，双色反射镜2和滤镜3、滤镜4组成的分光系统将两个通道的信号分离，并分别成像于EMCCD相机感光元件的不同区域。EMCCD相机以录像模式同时记录来自两个通道的闪烁荧光点信号。定位软件获取EMCCD采集的每一副图像，并通过高斯拟合确定每幅图像中，每个通道上的每个爱里斑的中心坐标并记录。随后，程序将所记录的中心坐标以点的形式绘出，以此分别重构出Alexa647和Alexa750通道的超分辨率生物结构图样。最后通过算法将两个通道的图像精确对准，得到所标记样品的双色荧光超分辨率显微图。
综上所述，本发明双色荧光定位超分辨率生物显微技术采用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，所采用的荧光分子的发射光谱完全分离，因此理论上不会有通道串扰产生，并且荧光分子的激发波长不会使细胞产生自发荧光，从而大幅降低了背景噪音，从而提高了成像质量，且易于操作。优选的是，本发明采用优化的成像缓冲液使荧光分子在成像过程中的光漂白大大减少。更好的是，本发明通过实时锁定功能对样品进行锁定，以彻底消除以消除成像过程中的任何样品漂移。本发明可以广泛应用于生物光学超分辨率显微领域。
当然，本发明还可有其它多种实施例，在不背离本发明精神及其实质的情况下，熟悉本领域的技术人员当可根据本发明作出各种相应的改变和变形，但这些相应的改变和变形都应属于本发明所附的权利要求的保护范围。

Claims (20)

  1. 一种双色荧光定位超分辨率生物显微方法，其特征在于，包括有：
    使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将所述生物样品浸泡在成像缓冲液中；
    通过激光照射所述生物样品，分别产生与所述Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与所述Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号；
    根据所述第一通道闪烁荧光信号和所述第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像；
    将所述第一生物结构超分辨率图像和所述第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
  2. 根据权利要求1所述的方法，其特征在于，所述通过激光照射所述生物样品的步骤包括：
    通过预定的激活激光照射所述生物样品以开启荧光信号，并通过预定的激发激光照射所述生物样品以关闭荧光信号，以产生闪烁荧光信号。
  3. 根据权利要求2所述的方法，其特征在于，所述激活激光具有紫色或紫外波长；所述激发激光具有Alexa647和Alexa750荧光分子激发波长，或者所述激发激光具有Cy5和Cy7荧光分子激发波长；使用预定的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子关闭荧光信号，仅有小部分离散的荧光分子开启荧光信号。
  4. 根据权利要求1所述的方法，其特征在于，所述成像缓冲液包含有TCEP、环辛四烯、除氧剂、甲基紫精和/或抗坏血酸；或者
    所述成像缓冲液包含有巯基物、环辛四烯、除氧剂、甲基紫精和/或抗坏血酸。
  5. 根据权利要求4所述的方法，其特征在于，所述TCEP或巯基物与荧光分子在所述激发激光的照射下结合形成加合物，所述加合物不会在所述激发激光的照射下发出荧光；所述加合物在所述激活激光的照射下重新分解为所述TCEP或巯基物和荧光分子，所述荧光分子发出荧光。
  6. 根据权利要求4所述的方法，其特征在于，所述环辛四烯包括环辛四烯的衍生物；所述除氧剂包括葡萄糖氧化酶、葡萄糖和过氧化氢酶的组合，或者，所述除氧剂包括吡喃糖氧化酶、葡萄糖和过氧化氢酶的组合。
  7. 根据权利要求1所述的方法，其特征在于，所述根据第一通道闪烁荧光信号和所述第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像的步骤之前还包括：
    在数据采集之前，通过实时锁定系统将所述生物样品的位置实时锁定。
  8. 根据权利要求7所述的方法，其特征在于，所述实时锁定系统将所述生物样品的位置实时锁定的步骤包括：
    为所述生物样品提供明场照明，所述生物样品的明场图像经过前成像透镜成像于锁定相机；
    从所述锁定相机中抓取一张生物样品或参照体的明场图像作为锁定模板；
    从所述锁定相机中实时抓取所述生物样品的当前明场图像，将所述当前明场图像与所述锁定模板进行对比；
    计算所述当前明场图像与所述锁定模板之间的偏移量；
    根据所述偏移量对所述当前明场图像进行偏移补偿处理。
  9. 根据权利要求1～8任一项所述的方法，其特征在于，所述根据第一通道闪烁荧光信号和所述第二通道闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像的步骤还包括：
    通过双色荧光定位超分辨率显微镜获取所述第一通道闪烁荧光信号和所述第二通道闪烁荧光信号；
    通过所述双色荧光定位超分辨率显微镜的分光系统将所述第一通道闪烁荧光信号和所述第二通道闪烁荧光信号分离，并分别成像于相机的感光元件的不同区域；
    获取所述相机采集的每一张图像，通过高斯拟合确定所述图像中每个荧光分子的爱里斑中心坐标并记录；
    根据记录的所述爱里斑中心坐标分别构建所述第一通道闪烁荧光信号对应的所述第一生物结构超分辨率图像和所述第二通道闪烁荧光信号对应的所述第二生物结构超分辨率图像。
  10. 根据权利要求9所述的方法，其特征在于，所述双色荧光定位超分辨率显微镜为倒置的全反射荧光显微镜；
    所述相机为EMCCD相机；和/或
    所述分光系统包括矩形光阑、双色反射镜和两个滤镜。
  11. 一种双色荧光定位超分辨率生物显微系统，其特征在于，包括有：
    样品标记模块，用于使用Alexa647和Alexa750荧光分子，或Cy5和Cy7荧光分子对生物样品进行双色荧光标记，并将所述生物样品浸泡在成像缓冲液中；
    信号产生模块，用于通过激光照射所述生物样品，分别产生与所述Alexa647或Cy5荧光分子对应的第一通道闪烁荧光信号，以及与所述Alexa750或Cy7荧光分子对应的第二通道闪烁荧光信号；
    第一图像构建模块，用于根据所述第一通道闪烁荧光信号和所述第二通道 闪烁荧光信号，分别构建第一生物结构超分辨率图像和第二生物结构超分辨率图像；
    第二图像构建模块，用于将所述第一生物结构超分辨率图像和所述第二生物结构超分辨率图像进行对准处理以构建出第三生物结构超分辨率图像。
  12. 根据权利要求11所述的系统，其特征在于，所述信号产生模块用于通过预定的激活激光照射所述生物样品以开启荧光信号，并通过预定的激发激光照射所述生物样品以关闭荧光信号，以产生闪烁荧光信号。
  13. 根据权利要求12所述的系统，其特征在于，所述激活激光具有紫色或紫外波长；所述激发激光具有Alexa647和Alexa750荧光分子激发波长，或者所述激发激光具有Cy5和Cy7荧光分子激发波长；
    所述信号产生模块用于使用预定的激发激光功率和激活激光功率，使同一时刻大部分的荧光分子关闭荧光信号，仅有小部分离散的荧光分子开启荧光信号。
  14. 根据权利要求11所述的系统，其特征在于，所述成像缓冲液包含有TCEP、环辛四烯、除氧剂、甲基紫精和/或抗坏血酸；或者
    所述成像缓冲液包含有巯基物、环辛四烯、除氧剂、甲基紫精和/或抗坏血酸。
  15. 根据权利要求14所述的系统，其特征在于，所述TCEP或巯基物与荧光分子在所述激发激光的照射下结合形成加合物，所述加合物不会在所述激发激光的照射下发出荧光；所述加合物在所述激活激光的照射下重新分解为所述TCEP或巯基物和荧光分子，所述荧光分子发出荧光。
  16. 根据权利要求14所述的系统，其特征在于，所述环辛四烯包括环辛四烯的衍生物；所述除氧剂包括葡萄糖氧化酶、葡萄糖和过氧化氢酶的组合，或者，所述除氧剂包括吡喃糖氧化酶、葡萄糖和过氧化氢酶的组合。
  17. 根据权利要求11所述的系统，其特征在于，还包括：
    实时锁定模块，用于在数据采集之前，通过实时锁定系统将所述生物样品的位置实时锁定。
  18. 根据权利要求17所述的系统，其特征在于，所述实时锁定模块包括：
    明场照明子模块，用于为所述生物样品提供明场照明，所述生物样品的明场图像经过前成像透镜成像于锁定相机；
    模块生成子模块，用于从所述锁定相机中抓取一张生物样品或参照体的明场图像作为锁定模板；
    图像对比子模块，用于从所述锁定相机中实时抓取所述生物样品的当前明场图像，将所述当前明场图像与所述锁定模板进行对比；
    偏移计算子模块，用于计算所述当前明场图像与所述锁定模板之间的偏移 量；
    偏移补偿子模块，用于根据所述偏移量对所述当前明场图像进行偏移补偿处理。
  19. 根据权利要求11～18任一项所述的系统，其特征在于，所述第一图像构建模块还包括：
    信号获取子模块，用于通过双色荧光定位超分辨率显微镜获取第一通道闪烁荧光信号和第二通道闪烁荧光信号；
    信号分离子模块，用于通过双色荧光定位超分辨率显微镜的分光系统将第一通道闪烁荧光信号和第二通道闪烁荧光信号分离，并分别成像于相机的感光元件的不同区域；
    位置记录子模块，用于获取相机采集的每一张图像，通过高斯拟合确定图像中每个荧光分子的爱里斑中心坐标并记录；
    图像生成子模块，用于根据记录的爱里斑中心坐标分别构建第一通道闪烁荧光信号对应的第一生物结构超分辨率图像和第二通道闪烁荧光信号对应的第二生物结构超分辨率图像。
  20. 根据权利要求19所述的系统，其特征在于，所述双色荧光定位超分辨率显微镜为倒置的全反射荧光显微镜；
    所述相机为EMCCD相机；和/或
    所述分光系统包括矩形光阑、双色反射镜和两个滤镜。
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