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Near-infrared dual-channel fluorescence living body microscopic imaging method

A technology of microscopic imaging and fluorescence imaging, applied in the field of biomedical imaging, can solve problems that have not been reported

Pending Publication Date: 2020-09-22
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the dual-channel fluorescence in vivo microscopy imaging technology combining the first and second near-infrared regions has not been reported at home and abroad.

Method used

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  • Near-infrared dual-channel fluorescence living body microscopic imaging method
  • Near-infrared dual-channel fluorescence living body microscopic imaging method
  • Near-infrared dual-channel fluorescence living body microscopic imaging method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] The schematic diagram of the near-infrared first zone / two-channel dual-channel fluorescence in vivo microscopic imaging device of the present invention is as follows figure 1 As shown, it consists of the following main components:

[0084] The near-infrared excitation light source is a semiconductor laser with a wavelength of 808nm and a maximum power of 5W;

[0085] Dichroic mirror 1 is an 830nm long-wave pass dichroic mirror;

[0086] The microscope lens is a 20x plan fluorescence objective lens;

[0087] The sleeve lens is a convex lens with focal length f=400mm;

[0088] Dichroic mirror 2 is a 950nm long-wave pass dichroic mirror;

[0089] Filter 1 is an 830-880nm bandpass filter;

[0090] Filter 2 is a 1000nm long-pass filter;

[0091] The near-infrared zone 1 detector is a water-cooled Si chip sCOMS camera;

[0092] The detector in the second near-infrared zone is a short-wave near-infrared camera with liquid nitrogen cooling InGaAs chip.

[0093] The physica...

Embodiment 2

[0095] The synthetic route of near-infrared fluorescent probe compound 7 (ICG-dextran 40) for labeling blood vessels is as follows:

[0096] Synthetic route 3:

[0097]

[0098] Reagents and conditions:

[0099] a. Acetic acid, acetic anhydride, glutadienal diphenylamine hydrochloride; reflux reaction for 1.5 hours;

[0100] b. acetic acid, pyridine; reflux reaction for 0.5 hours;

[0101] c. Dextran (40kD) anhydrous DMSO, CDI, 1,3-propanediamine; react at room temperature for 8 hours;

[0102] d. Anhydrous DMSO, EDC·HCl, NHS; react at room temperature overnight in the dark.

[0103] Synthesis of compound 2 (indole cyanine dye intermediate):

[0104] Compound 1 (0.2g, 0.56mmol) and glutadienal dianiline hydrochloride (0.18g, 0.63mmol) were dissolved in a mixed solution of acetic acid (5mL) and acetic anhydride (5mL), and refluxed for 90 minutes. Then it was cooled to room temperature, and the solvent was evaporated under reduced pressure below 40C. The reddish-brown o...

Embodiment 3

[0113] A fluorescent probe compound 8 in the second near-infrared region for labeling cells, its structure is as follows:

[0114]

[0115] Compound 8 (BPBBT) is a donor-acceptor NIR-II fluorescent molecule with AIE group. Weigh 1mg of the compound and 0.8mg DSPE-PEG 2000 Dissolved in 0.5mL tetrahydrofuran. The mixed solvent was added to 5 mL of ultrapure water and mixed. The mixed solution was sonicated with an ultrasonic cell pulverizer, the ultrasonic power was 100 W, and the ultrasonic time was 2 min. Afterwards, the mixed solution was poured into a round-bottomed flask, placed in a fume hood, and stirred at a high speed at 40° C. for 4 h evenly and at a high speed. Finally, a 30kD ultrafiltration tube was used to wash and concentrate to obtain a nanoprobe solution. The particle size, polymer dispersion index (PDI), drug loading and encapsulation efficiency (Table 1), and morphology ( Figure 4 ).

[0116] BPBBT exhibits fluorescence quenching in the benign polar ...

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Abstract

The invention belongs to the field of biomedical images, and relates to a near-infrared first-region and second-region dual-channel fluorescence living body microscopic imaging technology, which comprises the following steps: carrying out real-time microscopic observation on living body tissues and cells of animals by adopting an imaging device and a fluorescence probe combination, wherein the imaging device comprises a near-infrared light source, a microscope lens, a dichroscope, an optical filter, a sleeve lens, a near-infrared first-region fluorescence detector and a near-infrared second-region fluorescence detector; the fluorescent probe is a probe combination for emitting near-infrared first-region fluorescence and near-infrared second-region fluorescence, and the probe for emitting near-infrared second-region fluorescence is a fluorescent nano-probe constructed by utilizing near-infrared second-region fluorescence molecules containing aggregation-induced emission groups; the probe for emitting near-infrared first-region fluorescence is an indocyanine derivative. The imaging device and the probe combination provided by the invention can realize real-time microscopic observation of living tissues and cells of animals.

Description

technical field [0001] The invention belongs to the field of biomedical imaging, and specifically relates to a near-infrared dual-channel fluorescent living microscopic imaging technology, which includes a device for performing near-infrared first-zone / two-channel dual-channel fluorescent microscopic imaging on living animals, emitting near-infrared Fluorescent probes that emit fluorescence in the second region and can label cells, and fluorescent probes that emit fluorescence in the first region of near-infrared and that can label blood vessels or cells. Background technique [0002] The prior art discloses that two-photon microscopic imaging technology is the main research method for observing cell morphology, movement and function in living tissues, and it can reveal the interaction between cells and cells, blood vessels, and matrix components on time and space scales; however, Due to the emission wavelength of traditional fluorescent dyes between 400-800nm, as well as th...

Claims

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Application Information

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IPC IPC(8): G01N21/64C09K11/06
CPCG01N21/6458C09K11/06G01N2021/6463C09K2211/145C09K2211/1466C09K2211/1051C09K2211/1092C09K2211/1007
Inventor 魏国光高帅陆伟
Owner FUDAN UNIV
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