CN111334467A - Photomagnetic bimodal indicator cell, preparation method thereof, detection method thereof and application thereof in-vivo tracing - Google Patents
Photomagnetic bimodal indicator cell, preparation method thereof, detection method thereof and application thereof in-vivo tracing Download PDFInfo
- Publication number
- CN111334467A CN111334467A CN201811548358.2A CN201811548358A CN111334467A CN 111334467 A CN111334467 A CN 111334467A CN 201811548358 A CN201811548358 A CN 201811548358A CN 111334467 A CN111334467 A CN 111334467A
- Authority
- CN
- China
- Prior art keywords
- bimodal
- cell
- photomagnetic
- indicator
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
-
- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
Abstract
The invention provides a preparation method of a photomagnetic bimodal indicator cell, which comprises the following steps: suspending erythrocytes in cell isotonic solution, and adding a fluorescent dye and a nuclear magnetic contrast agent to obtain a staining mixture; and incubating the staining mixture, washing the cells in the staining mixture by using cell isotonic solution, and finally harvesting the cells to obtain the photomagnetic bimodal indicator cells. The photomagnetic bimodal indicator cell prepared by the preparation method takes the cell as a photomagnetic bimodal tracer, can be used for fluorescence and nuclear magnetic resonance bimodal imaging, and can be used for reflecting the physiological condition of organisms from the cellular level to the integral level.
Description
Technical Field
The invention belongs to the field of biological imaging, and particularly relates to a photomagnetic bimodal indicator cell, a preparation method thereof, a detection method thereof and application thereof in-vivo tracing.
Background
In recent years, the development of biological imaging technology has enabled imaging objects to be organisms, tissues, cells, and even molecular levels, which reflect changes in molecular levels in a living state, and qualitatively and quantitatively analyze biological behaviors thereof, which has promoted the development of pharmaceutical research and clinical diagnosis.
The photomagnetic bimodal imaging technology combines the fluorescent imaging probe and the MRI imaging probe into a whole, so that the fluorescent imaging probe and the MRI imaging probe can be used for detection by two imaging technologies, and the inherent limitation of a single imaging technology is overcome. MRI is one of the most mature imaging techniques studied at present, and can provide high-resolution tissue information and three-dimensional structural imaging at the sub-millimeter level of the body depth, and is widely applied in scientific research and clinical. However, the imaging resolution is low, the sensitivity is low, and the micron level is difficult to achieve, so that the clinical application is greatly limited. The optical microscopic imaging technology has quite high sensitivity, and the detection limit can reach 0.1-0.2 micron, so that the combination of the MRI and the optical imaging technology can provide structural and histological information with high resolution, realize functional imaging of deep structures, and finally achieve perfect unification of the functional imaging and the structural tissue imaging.
Optical and magnetic imaging requires signal enhancement by means of magneto-optical molecular probes. The design of the current common photomagnetic bimodal probe is mainly based on two modes: the first is formed by directly or indirectly chelating nuclear magnetic signal elements by fluorescent dye molecules. The second method is to construct a functionalized nano bimodal system by utilizing a nano technology and modify fluorescent dye or magnetic particles on a nano material to form the nano bimodal system. However, the photomagnetic bimodal probe obtained by the two methods is in a solution state, the physiological condition of organisms is difficult to react from a cellular level, for example, the speed of red blood cells in blood flow is difficult to directly detect in a blood vessel imaging process, and the molecular probe is easy to permeate out of a blood vessel wall and distribute into tissues around the blood vessel wall.
Disclosure of Invention
The invention aims to provide a preparation method of a photomagnetic bimodal indicator cell, and the prepared photomagnetic bimodal indicator cell can be used for fluorescence imaging and nuclear magnetic resonance imaging.
It is another object of the present invention to provide a magneto-optical bimodal indicator cell that can be used for fluorescence imaging and magnetic resonance imaging, and can be used to observe red blood cell morphology.
Still another object of the present invention is to provide a method for detecting a photomagnetic bimodal indicator cell, which can perform fluorescence imaging and nuclear magnetic resonance imaging on the photomagnetic bimodal indicator cell.
The invention also aims to provide application of the photomagnetic bimodal indicator cell in-vivo tracing, which can realize in-vivo tracing of the photomagnetic bimodal indicator cell under fluorescence imaging and nuclear magnetic resonance imaging.
The invention provides a preparation method of a photomagnetic bimodal indicator cell, which comprises the following steps: suspending erythrocytes in cell isotonic solution, and adding a fluorescent dye and a nuclear magnetic contrast agent to obtain a staining mixture; and incubating the staining mixture, washing the cells in the staining mixture by using cell isotonic solution, and finally harvesting the cells to obtain the photomagnetic bimodal indicator cells.
The photomagnetic bimodal indicator cell prepared by the preparation method can be used for fluorescence and nuclear magnetic resonance bimodal imaging and can be used for reflecting the physiological condition of organisms from a cellular level to an integral level.
In another illustrative embodiment of the method for producing a photomagnetic bimodal indicator cell, the fluorescent dye is selected from the group consisting of a combination of one or more of fluoric CH dipotassium salt, fluorescein isothiocyanate, rhodamine B, and Alexa Fluor 594; the nuclear magnetic contrast agent is gadopentetate dimeglumine. Therefore, better fluorescence imaging and nuclear magnetic resonance imaging effects can be realized.
In yet another illustrative embodiment of the method for preparing a photomagnetic bimodal indicator cell, the final concentration of fluoric CH dipotassium salt is not less than 0.001mM, the final concentration of fluorescein isothiocyanate is not less than 0.001mM, the final concentration of rhodamine B is not less than 0.005mM, and the final concentration of Alexa Fluor594 is not less than 0.005 mM; the final concentration of the gadopentetate dimeglumine is not less than 0.1 mM. Therefore, better fluorescence imaging and nuclear magnetic resonance imaging effects can be realized. More preferably, the final concentration of the fluorite CH dipotassium salt is not less than 0.1mM, the final concentration of fluorescein isothiocyanate is not less than 0.1mM, the final concentration of rhodamine B is not less than 5 mu M, and the final concentration of Alexa Fluor594 is not less than 0.5 mM; the final concentration of the gadopentetate dimeglumine is not less than 7.5mM, so that the fluorescence imaging and nuclear magnetic resonance imaging effects are further improved.
In yet another illustrative embodiment of the method for preparing a photomagnetic bimodal indicator cell, the red blood cells are immobilized red blood cells prepared by placing live red blood cells in a cell isotonic solution containing 4% paraformaldehyde, and incubating at 20 to 25 ℃ for not less than 30 minutes. Therefore, better fluorescence imaging and nuclear magnetic resonance imaging effects are realized.
In still another illustrative embodiment of the method for preparing a magneto-optical bimodal indicator cell, the method for obtaining viable red blood cells is: anticoagulating blood with anticoagulant, centrifuging to remove serum, washing cells in blood with cell isotonic solution, and centrifuging to obtain viable erythrocyte. Therefore, better fluorescence imaging and nuclear magnetic resonance imaging effects are realized.
In a further exemplary embodiment of the method for the preparation of a magneto-optical bimodal indicator cell, the incubation of the staining mixture is performed at 20 to 25 ℃ for 0.5 to 72 hours, preferably 6 to 72 hours.
In yet another illustrative embodiment of the method for preparing a photomagnetic bimodal indicator cell, the cellular isotonic fluid is physiological saline. Therefore, the interference can be reduced, and better fluorescence imaging and nuclear magnetic resonance imaging effects can be realized.
In yet another illustrative embodiment of the method for preparing a magneto-optical bimodal indicator cell, the method for harvesting the cell is centrifugation.
The invention also provides a photomagnetic bimodal indicator cell which is prepared by adopting the preparation method. The photomagnetic bimodal indicator cell can be used for fluorescence and nuclear magnetic resonance bimodal imaging and can be used for reflecting the physiological condition of organisms from a cellular level to an integral level.
The invention also provides a detection method of the photomagnetic bimodal indicator cell, which comprises the following steps: suspending the photomagnetic bimodal indicator cells in cell isotonic solution, then placing the cell isotonic solution in a laser scanning confocal microscope for detection, and imaging through a fluorescence channel and an optical lens; wherein the parameters of the fluorescent channel are determined according to the excitation and emission wavelengths of the fluorescent dye; and placing the photomagnetic bimodal indicator cells into an EP tube, and diluting the indicator cells by using cell isotonic solution until the number of the cells is 104To 108The parameters of MRI imaging are that the wrist coil is used and scanning is carried out by adopting a T13D MP-RAGE sequence, the imaging parameters are that the echo Time (TE) is 3.7ms, the repetition Time (TR) is 1500ms, the inversion angle (FA) is 90, the inversion Time (TI) is 900ms, the layer thickness (SL) is 1mm, the visual Field (FOV) is 267mm, the matrix 512 × 96 and the resolution is 0.5mm × 0.5mm × 0.5.5 mm.
The invention also provides an application of the photomagnetic bimodal indicator cell in-vivo tracing, which comprises the following steps: introducing photomagnetic bimodal indicator cells into an animal body; placing the animal in a magnetic resonance imaging system for MRI imaging; and placing the animal, the tissue section of the animal or the cell of the animal in a laser scanning confocal microscope for detection, wherein the parameters of the fluorescence channel are determined according to the excitation and emission wavelengths of the fluorescent dye. The application can realize the in-vivo tracing of the photomagnetic bimodal indicator cells under the fluorescent imaging and the nuclear magnetic resonance imaging so as to reflect the physiological condition of organisms from the cellular level.
In another illustrative embodiment of the use of photomagnetic bimodal indicator cells for in vivo tracking, MRI imaging is performed using a wrist coil and scanning with a T13D MP-RAGE sequence with echo Time (TE) of 3.7ms, repetition Time (TR) of 1500ms, Flip Angle (FA) of 90, flip Time (TI) of 900ms, layer thickness (SL) of 1mm, field of view (FOV) of 267mm, matrix 512 × 96, resolution of 0.5mm × 0.5mm × 0.5.5 mm.
In another illustrative embodiment of the use of a magneto-optical bimodal indicator cell for in vivo tracking, the magneto-optical bimodal indicator cell is introduced into a cisterna magna of an animal and the tissue section is brain tissue of the animal. The application can utilize fluorescence imaging and nuclear magnetic resonance imaging to reflect the physiological condition of the animal brain from the cellular level.
Drawings
The following drawings are only schematic illustrations and explanations of the present invention, and do not limit the scope of the present invention:
FIG. 1 is a 488nm excitation fluorescence image (A), a light mirror image (B) and a fluorescence light mirror superposition image (C) of a photomagnetic bimodal indicator cell corresponding to the serial number 6;
FIG. 2 is a 488nm excitation fluorescence image (A), a light mirror image (B) and a fluorescence light mirror overlay image (C) of the magneto-optical bimodal indicator cell corresponding to the reference number 13;
FIG. 3 is a 488nm excitation light fluorescence image (A), a 633nm excitation light fluorescence image (B), a light mirror image (C) and a fluorescence light mirror overlay image (D) of the photomagnetic bimodal indicator cell corresponding to the reference number 19;
FIG. 4 is an MRI image of the subarachnoid space region after laxation;
fig. 5 is an image obtained by laser scanning confocal microscope examination of the subarachnoid space region after drug administration.
Detailed Description
In order to more clearly understand the technical features, objects and effects of the present invention, specific embodiments of the present invention will be described with reference to the following examples.
Reagents and apparatus used in the following examples:
laser Scanning Confocal Microscope (LSCM) Leica TCS SP 8;
magnetic resonance imager (3.0T, Trio) is Siemens, germany;
the small animal experiment brain stereotaxic instrument is purchased from Stoelting of the United states;
rats were purchased from department of medicine, Beijing university;
fluorescent yellow CH dipotassium salt fluorescent probe was purchased from Sigma-Aldrich co.llc;
fluorescein isothiocyanate was purchased from life technology, usa;
rhodamine B is available from sahn chemical technology (shanghai) ltd;
alexa fluor594 is available from life technology, USA;
the brain slice mold is purchased from Riword Life technologies, Inc. of Shenzhen;
high speed centrifuges were purchased from Beckman, usa;
physiological saline (500ml) was purchased from Beijing Solaibao Tech Co., Ltd;
4% tissue cell fixative (4% paraformaldehyde) was purchased from Beijing Solaibao Tech Co., Ltd;
confocal microscopy glass-bottom culture dishes were purchased from mercy ltd, zhongkohamhen (beijing);
microsyringes were purchased from Hamilton, usa;
the gadopentetate meglumine injection is purchased from Beijing Hotan pharmaceutical industry Co.
Example 1: and preparing and detecting the photomagnetic bimodal indicator cell.
1. Preparing a photomagnetic bimodal indicator cell:
1.1, collecting blood from the heart of an SD rat, anticoagulating the blood by EDTA, centrifuging to remove serum, cleaning cells in the blood by normal saline for 3-4 times, and finally centrifuging at 2000r/min for 10min to obtain living erythrocytes (generally, leukocytes are mixed in the erythrocytes and are ignored because the proportions of the erythrocytes and the leukocytes are very different);
1.2, taking live red blood cells, putting the live red blood cells into physiological saline (namely 4% tissue cell fixing solution) containing 4% paraformaldehyde, and incubating for 30 minutes at 20-25 ℃ (the time can be prolonged according to practical conditions as long as the cells are not cracked) to obtain immobilized red blood cells, wherein the volume ratio of the immobilized red blood cells to the physiological saline containing 4% paraformaldehyde is 1: 5;
1.3, suspending the immobilized red blood cells in physiological saline, and adding a fluorescent dye and a nuclear magnetic contrast agent to obtain a dyeing mixture, wherein the types of the fluorescent dye and the nuclear magnetic contrast agent and the final concentration in the dyeing mixture are shown in table 1;
1.4, incubating the staining mixture at 20-25 ℃ for 48 hours, washing the cells in the staining mixture by using physiological saline, and finally harvesting the cells by centrifugation to obtain the magneto-optical bimodal indicator cells.
2. Detecting the photomagnetic bimodal indicator cell:
2.1, suspending the photomagnetic bimodal indicator cells in physiological saline, dripping the physiological saline in a confocal glass cuvette, then placing the confocal glass cuvette in a laser scanning confocal microscope for detection, and imaging through a fluorescence channel and an optical lens to obtain the average single cell fluorescence intensity shown in the following table 1; wherein, the parameters of the fluorescence channel are determined according to the corresponding excitation and emission wavelengths of the disclosed fluorescent dye, which is not described herein again; the magneto-optical bimodal indicating cell is the magneto-optical bimodal indicating cell which is just prepared;
2.2, placing the magneto-optical bimodal indicator cells in an EP tube, and diluting the indicator cells with physiological saline until the cell number is 106cell/mL (cell concentration can be adjusted to 10 according to actual conditions)4To 108cell/mL), the EP tube was then placed in a 3.0T magnetic resonance imaging system for MRI imaging, and a scan was performed using a T13D MP-RAGE sequence using a wrist coil with imaging parameters of echo Time (TE) of 3.7ms, repetition Time (TR) of 1500ms, Flip Angle (FA) of 90, inversion Time (TI) of 900ms, layer thickness (SL) of 1mm, field of view (FOV) of 267mm, matrix 512 × 96, resolution of 0.5mm × 0.5.5 mm × 0.5.5 mm, and measured nuclear magnetic signal intensities are shown in table 1 below.
Table 1:
FIG. 1 shows the fluorescence image (A) of 488nm excitation light, the optical mirror image (B) and the fluorescence mirror overlay image (C) of the magneto-optical bimodal indicator cell corresponding to the number 6 in the above Table 1.
FIG. 2 shows the fluorescence image (A) of the 488nm excitation light, the light mirror image (B) and the fluorescence light mirror overlay image (C) of the magneto-optical bimodal indicator cell corresponding to the number 13 in the above Table 1.
FIG. 3 shows the 488nm excitation fluorescence image (A), the 633nm excitation fluorescence image (B), the light mirror image (C) and the fluorescence mirror overlay image (D) of the magneto-optical bimodal indicator cell corresponding to the number 19 in Table 1 above.
As can be seen from Table 1 above and FIGS. 1-3, the magneto-optical bimodal indicator cells prepared in the above numbers 1-19 can be used for fluorescence-MRI bimodal imaging.
The saline used in this embodiment may be replaced with other cell isotonic solutions.
Although only red blood cells are used in the above examples, it is not limited thereto, and other types of cells may be used in other exemplary embodiments.
Example 2 (incubation time).
The incubation time in step 1.4 of example 1 was set to 6 hours, 48 hours and 72 hours, respectively, and the types of fluorescent dye and nuclear magnetic contrast agent and the final concentration in the staining mixture were given number 13 in table 1, with the remaining parameters being unchanged. The average single cell fluorescence intensity and nuclear magnetic signal intensity of the prepared photomagnetic bimodal indicator cells are shown in the following table 2.
Table 2:
| fluorescence intensity of single cell | Nuclear magnetic signal intensity | |
| Incubation time: 6 hours | 178.212±7.62 | 489.03±9.91 |
| Incubation time: 48 hours | 252.316±3.54 | 877.34±12.54 |
| Incubation time: 72 hours | 252.623±2.32 | 834.45±13.62 |
As shown in Table 2, when the incubation time is 6 hours, the fluorescence intensity and the nuclear magnetic signal intensity of a single cell reach higher contrast, and fluorescence imaging and nuclear magnetic imaging can be realized. When the incubation time reaches 48 hours, the photomagnetic cytosphere reaches the optimal single cell fluorescence intensity and nuclear magnetic signal intensity. And the incubation time is continuously increased to 72 hours, and the fluorescence intensity and nuclear magnetic signal intensity of the single cells are not increased when reaching a plateau.
Example 3 (stability).
The photomagnetic bimodal indicator cell corresponding to the serial number 13 in the table 1 of the example 1 is placed in the dark for 1 day and 7 days at room temperature (20-25 ℃) after the preparation is finished, and the average single cell fluorescence intensity and the nuclear magnetic signal intensity are detected, and the detection result is shown in the following table 3.
Table 3:
| fluorescence intensity of single cell | Nuclear magnetic signal intensity | |
| Detection time: 1 day | 252.316±3.54 | 877.34±12.54 |
| Detection time: 7 days | 220.293±2.37 | 690.21±11.67 |
。
As can be seen from Table 3 above, the fluorescence signal and nuclear magnetic signal of the photomagnetic bimodal indicator cells can be kept stable within 7 days of storage.
Example 4: photomagnetic bimodal indicates the use of cells for in vivo tracking.
The method comprises the following steps:
1. the rat is anesthetized by 250g, preserved skin is marked, the rat is fixed by a brain stereotaxic apparatus, 50 mu L of photomagnetic bimodal indicator cells corresponding to the serial number 13 in the table 1 of the embodiment 1 are injected into the occipital cisterna, the rat is placed in a 3.0T magnetic resonance imaging system for MRI imaging after 15 min, 30 min and 60min respectively, a wrist coil is used for scanning by adopting a T13D MP-RAGE sequence, the imaging parameters are that echo Time (TE) is 3.7ms, repetition Time (TR) is 1500ms, inversion angle (FA) is 90, inversion Time (TI) is 900ms, layer thickness (SL) is 1mm, visual Field (FOV) is 267mm, matrix 512 × 96, resolution is 0.5mm × 0.5mm × 0.5mm, the result is shown in figure 4, wherein row A is an MRI image of 0, 15, 30 and 60min, row B is an MRI image of 0, 15, 30 and 60min, row C is an MRI image of 0, a coronary signal ratio is 0, and a visible subarachnoid space is 0-MRI is a visible area after 0-time of a scanning is shown in a visible area;
2. anesthetizing a rat, perfusing the heart, taking the brain, and placing the brain in 4% paraformaldehyde for fixation for 2 hours; and (3) placing the fixed rat brain in a rat brain mould, slicing the rat brain at the coronal position, wherein the thickness of each slice is about 2mm, and placing the obtained slices in a laser scanning confocal microscope for detection, wherein the parameters of a fluorescence channel are determined according to the excitation wavelength and the emission wavelength of the fluorescent dye. The detection result is shown in FIG. 5, wherein A is a fluorescence image, B is a light mirror image, C is a reflected light image, D is a fluorescence light mirror image, and E is a partially enlarged view of A; from the figure, it can be seen that the fluorescence distribution is in the subarachnoid space region of the mouse brain.
It can be seen that the photomagnetic bimodal indicator cell can achieve in vivo tracking under both MRI imaging and fluorescence imaging techniques, thereby reflecting the physiological condition of the organism from the cellular level. The photomagnetic bimodal indicating cell has the characteristics of both a fluorescent dye and a nuclear magnetic contrast agent, and two detection technologies can be combined. After being injected into the body of the animal, the biological compound can be used for determining the physiological state of the animal according to the distribution characteristics of the biological compound in the body.
In addition, the magneto-optical bimodal indicator cell positioned in the animal blood vessel can dynamically monitor the blood flow velocity of the blood vessel in real time under a laser confocal microscope and a nuclear magnetic imager, and can clearly observe the flowing process of the magneto-optical cytosphere in the blood vessel, so that the magneto-optical bimodal indicator cell can be used for imaging of vascular tissues. And because it exists in a cellular form, the problem of permeation through the blood vessel wall does not occur, thereby more accurately reflecting the physiological condition of the organism from the cellular level.
"exemplary" means "serving as an example, instance, or illustration" herein, and any embodiment described as "exemplary" herein should not be construed as a more preferred or advantageous solution.
In this context, "equal", "same", etc. are not strictly mathematical and/or geometric limitations, but also include errors that may be understood by a person skilled in the art and allowed for production or use, etc. Unless otherwise indicated, numerical ranges herein include not only the entire range within its two endpoints, but also several sub-ranges subsumed therein.
It should be understood that although the present description has been described in terms of various embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and those skilled in the art will recognize that the embodiments described herein may be combined as suitable to form other embodiments, as will be appreciated by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications such as combinations, divisions or repetitions of features, which do not depart from the technical spirit of the present invention, should be included in the scope of the present invention.
Claims (9)
1. A preparation method of a photomagnetic bimodal indicator cell is characterized by comprising the following steps:
suspending erythrocytes in cell isotonic solution, and adding a fluorescent dye and a nuclear magnetic contrast agent to obtain a staining mixture; and
and incubating the staining mixture, washing the cells in the staining mixture by using a cell isotonic solution, and finally harvesting the cells to obtain the photomagnetic bimodal indicator cell.
2. The method for preparing a magneto-optical bimodal indicator cell according to claim 1, wherein the fluorescent dye is selected from a combination of one or more of fluorite CH dipotassium salt, fluorescein isothiocyanate, rhodamine B and Alexa Fluor 594; the nuclear magnetic contrast agent is gadopentetate dimeglumine.
3. The method for preparing a magneto-optical bimodal indicator cell as claimed in claim 2, wherein the final concentration of fluorite CH dipotassium salt is not less than 0.001mM, the final concentration of fluorescein isothiocyanate is not less than 0.001mM, the final concentration of rhodamine B is not less than 0.005mM, and the final concentration of Alexa Fluor594 is not less than 0.005 mM; the final concentration of the gadopentetate dimeglumine is not less than 0.1 mM.
4. The method of claim 1, wherein the red blood cells are immobilized red blood cells, and the method comprises incubating live red blood cells in an isotonic solution of cells containing 4% paraformaldehyde at 20-25 ℃ for no less than 30 minutes.
5. The method for preparing magneto-optical bimodal indicator cell according to claim 4, wherein the method for obtaining the living red blood cell is: anticoagulating blood with anticoagulant, centrifuging to remove serum, washing cells in the blood with cell isotonic solution, and centrifuging to obtain the living red blood cells.
6. A magneto-optical bimodal indicator cell, characterized in that it is obtained by the method of any one of claims 1 to 5.
7. The method for detecting the magneto-optical bimodal indicator cell according to claim 6, comprising:
resuspending the photomagnetic bimodal indicator cells in cell isotonic solution, then placing the cell isotonic solution in a laser scanning confocal microscope for detection, and imaging through a fluorescence channel and an optical lens; wherein parameters of the fluorescent channel are determined from excitation and emission wavelengths of the fluorescent dye; and
placing the photomagnetic bimodal indicator cell in an EP tube, and diluting the indicator cell with cell isotonic solution until the cell number is 104To 108cell/mL, and then placing the EP tube in a magnetic resonance imaging system for imaging.
8. Use of the magneto-optical bimodal indicator cell according to claim 6 for in vivo tracking, comprising:
introducing the photomagnetic bimodal indicator cell into an animal;
placing the animal in a magnetic resonance imaging system for MRI imaging; and
placing said animal, tissue section of said animal, or cells of said animal in a confocal laser scanning microscope for detection, wherein parameters of said fluorescent channel are determined based on excitation and emission wavelengths of said fluorescent dye.
9. The use of the magneto-optical bimodal indicator cell in vivo tracking as claimed in claim 8, wherein the magneto-optical bimodal indicator cell is introduced into the occipital cisterna of an animal, and the tissue section is the brain tissue of the animal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811548358.2A CN111334467A (en) | 2018-12-18 | 2018-12-18 | Photomagnetic bimodal indicator cell, preparation method thereof, detection method thereof and application thereof in-vivo tracing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811548358.2A CN111334467A (en) | 2018-12-18 | 2018-12-18 | Photomagnetic bimodal indicator cell, preparation method thereof, detection method thereof and application thereof in-vivo tracing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111334467A true CN111334467A (en) | 2020-06-26 |
Family
ID=71181332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811548358.2A Pending CN111334467A (en) | 2018-12-18 | 2018-12-18 | Photomagnetic bimodal indicator cell, preparation method thereof, detection method thereof and application thereof in-vivo tracing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111334467A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4762701A (en) * | 1986-10-31 | 1988-08-09 | Smithkline Beckman Corporation | In vivo cellular tracking |
| US20090274630A1 (en) * | 2008-05-01 | 2009-11-05 | National Health Research Institutes | Red blood cell-derived vesicles as a nanoparticle drug delivery system |
| US20100061937A1 (en) * | 2006-07-03 | 2010-03-11 | Universita Degli Studi Di Urbino "Carlo Bo" | Delivery of contrasting agents for magnetic resonance imaging |
| CN106390143A (en) * | 2015-07-17 | 2017-02-15 | 中国科学院大连化学物理研究所 | Tumor-targeted nuclear magnetic resonance/fluorescence dual-mode imaging contrast agent, preparation and applications thereof |
-
2018
- 2018-12-18 CN CN201811548358.2A patent/CN111334467A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4762701A (en) * | 1986-10-31 | 1988-08-09 | Smithkline Beckman Corporation | In vivo cellular tracking |
| US20100061937A1 (en) * | 2006-07-03 | 2010-03-11 | Universita Degli Studi Di Urbino "Carlo Bo" | Delivery of contrasting agents for magnetic resonance imaging |
| US20090274630A1 (en) * | 2008-05-01 | 2009-11-05 | National Health Research Institutes | Red blood cell-derived vesicles as a nanoparticle drug delivery system |
| CN106390143A (en) * | 2015-07-17 | 2017-02-15 | 中国科学院大连化学物理研究所 | Tumor-targeted nuclear magnetic resonance/fluorescence dual-mode imaging contrast agent, preparation and applications thereof |
Non-Patent Citations (1)
| Title |
|---|
| GIUSEPPE FERRAUTO等: "Generation of multiparametric MRI maps by using Gd-labelled- RBCs reveals phenotypes and stages of murine prostate cancer", 《SCIENTIFIC REPORTS》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102148747B1 (en) | Methods and compositions for preparing biological specimens for microscopic analysis | |
| KR100298682B1 (en) | For in vivo cell tracing of cyanine dye-labeled cells or for measuring lifetime of in vivo cells | |
| Pilatus et al. | Intracellular volume and apparent diffusion constants of perfused cancer cell cultures, as measured by NMR | |
| US20180045622A1 (en) | Method and System for Imaging and Analysis of a Biological Specimen | |
| CN105229466B (en) | Parallel plate flow chambers and its application method with port | |
| US20070258886A1 (en) | Methods for assessing cell labeling | |
| CN111450264A (en) | Bimodal nanoprobe targeting glioblastoma multiforme and preparation method thereof | |
| Venter et al. | A manganese porphyrin-based T1 contrast agent for cellular MR imaging of human embryonic stem cells | |
| Li et al. | Intraoperative detection of micrometastases in whole excised lymph nodes using fluorescent paired-agent imaging principles: identification of a suitable staining and rinsing protocol | |
| CN102350002B (en) | Glioma-targeting molecule magnetic resonance contrast agent as well as preparation method and application thereof | |
| Kim et al. | Magneto-acoustic protein nanostructures for non-invasive imaging of tissue mechanics in vivo | |
| Addington et al. | Siloxane nanoprobes for labeling and dual modality functional imaging of neural stem cells | |
| CN111334467A (en) | Photomagnetic bimodal indicator cell, preparation method thereof, detection method thereof and application thereof in-vivo tracing | |
| Joshi et al. | Single cell analysis of phagocytosis, phagosome maturation, phagolysosomal leakage, and cell death following exposure of macrophages to silica particles | |
| CN115389544B (en) | Method and application for measuring water molecule outflow rate across cell membrane, and method and system for measuring glioma magnetic resonance imaging marker | |
| Petersen et al. | Bioreactor and probe system for magnetic resonance microimaging and spectroscopy of chondrocytes and neocartilage | |
| CN104597016B (en) | It is inverted laser confocal microscope animals and plants vivo observation device and method | |
| CN109954147B (en) | Application of nanogold probe in preparation of early diagnosis reagent for kidney injury | |
| Kim et al. | Modularized dynamic cell culture platform for efficient production of extracellular vesicles and sequential analysis | |
| Firatligil-Yildirir et al. | Homing Choices of Breast Cancer Cells Revealed by Tissue Specific Invasion and Extravasation Lab-on-a-chip Platforms | |
| CN204359693U (en) | Be inverted laser confocal microscope animals and plants vivo observation device | |
| Chowdhury et al. | Molecular imaging with aquaporin-based reporter genes: quantitative considerations from Monte Carlo diffusion simulations | |
| CN107320737A (en) | Alzheimer disease senile plaque expelling nano target MR diagnosis contrast medium and preparation method | |
| CN109276723B (en) | Spherical pomegranate-structured nanoparticles and preparation method and application thereof | |
| JP2023183408A (en) | Quantitative evaluation method of human brain axon density based on magnetic resonance diffusion tensor imaging |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |