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Saturated resonance energy transfer super-resolution probe and preparation method and application thereof

A resonance energy transfer and super-resolution technology, applied in the field of super-resolution imaging, can solve the problem that the lateral spatial resolution cannot meet the requirements of observing the fine structure of cells, and can solve the problems of multi-color imaging and live cell imaging. Thermal and chemical stability, the effect of improving resolution

Inactive Publication Date: 2017-01-04
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the difficulty that the lateral spatial resolution of the traditional optical microscope in the prior art cannot meet the requirements for the observation of fine structures in cells, and to provide a method for super-resolution of saturated resonance energy transfer (SFM) ) probes, which produce extremely high FRET efficiency, can be applied to common laser confocal microscopes, break through the optical diffraction limit, and achieve SFM nanoscale resolution. SFM ultra-high resolution multicolor imaging of live cells

Method used

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  • Saturated resonance energy transfer super-resolution probe and preparation method and application thereof
  • Saturated resonance energy transfer super-resolution probe and preparation method and application thereof
  • Saturated resonance energy transfer super-resolution probe and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Alexa 555-V HH -GFP probe preparation and application

[0048] 1. Preparation of fluorescent molecularly labeled heavy chain single domain antibody V HH

[0049] 1. Take 50 μL of heavy chain single domain antibody V HH into dialysis tubing (purchased from Pierce, molecular weightcutoff = 3500 Da) and slip the tubing onto the buoy. Place the buoy in 20mL containing dialysate (0.2M NaHCO 3 , PH=8.2) in a plastic beaker, take it out after rotating at low speed for 2h, and get V HH solution.

[0050] 2. Dissolving the fluorescent dye. Dissolve 1 mg of Alexa 555 into 100 μL of DMSO to obtain a solution of Alexa 555, which is stored at -80°C. At this time, the concentration of Alexa 555 was 8 mM (the molecular mass of Alexa 555 was 1250).

[0051] 3. Take 5 μL of the Alexa555 solution obtained in step 2 and add 50 μL of the V HH In solution (i.e. the molar mass of Alexa 555 relative to V HH 10-fold excess), incubated at 25°C for 2 hours to obtain Incubatio...

Embodiment 2

[0089] Example 2 Alexa 546-V HH -GFP probe preparation and application

[0090] 1. Preparation of fluorescent molecularly labeled heavy chain single domain antibody V HH

[0091] 1. Take 50 μL of heavy chain single domain antibody V HH into dialysis tubing (purchased from Pierce, molecular weightcutoff = 3500 Da) and slip the tubing onto the buoy. Place the buoy in 20mL containing dialysate (0.2M NaHCO 3 , pH=8.2) in a plastic beaker, take it out after rotating at low speed for 2h, and get V HH solution.

[0092] 2. Dissolving the fluorescent dye. Dissolve 1 mg of Alexa 546 into 100 μL of DMSO to obtain a solution of Alexa 546, which is stored at -80°C. At this time, the concentration of Alexa 546 was 8.6 mM (the molecular mass of Alexa 546 was 1159).

[0093] 3. Take 5 μL of the Alexa546 solution obtained in step 2, and add 50 μL of the V obtained in step 1 HH In solution (i.e. the molar mass of Alexa 546 relative to V HH 10-fold excess), incubated at 25°C for 2 hou...

Embodiment 3

[0129] Example 3 Atto550-V HH -GFP probe preparation and application

[0130] 1. Preparation of fluorescent molecularly labeled heavy chain single domain antibody V HH

[0131] 1. Take 50 μL of heavy chain single domain antibody V HH into dialysis tubing (purchased from Pierce, molecular weightcutoff = 3500 Da) and slip the tubing onto the buoy. Place the buoy in 20mL containing dialysate (0.2M NaHCO 3 , PH=8.2) in a plastic beaker, take it out after rotating at low speed for 2h, and get V HH solution.

[0132] 2. Dissolving the fluorescent dye. Dissolve 1 mg of Atto550 in 100 μL of DMSO to obtain Atto550 solution and store it at -80°C. At this time, the concentration of Atto550 was 12.6 mM (the molecular mass of Atto550 was 791).

[0133] 3. Take 5 μL of the Atto550 solution obtained in step 2, and add 50 μL of the V obtained in step 1 HH In solution (ie the molar mass of Atto550 relative to V HH 10-fold excess), and incubated at 25°C for 2 hours to obtain Incubatio...

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Abstract

The invention discloses a saturated resonance energy transfer super-resolution probe and a preparation method and application thereof. The probe comprises following components: fluorescent protein, heavy chain single domain antibodies VHH which are connected with the fluorescent protein and resist the fluorescent protein, and organic fluorescent molecules which are connected with the heavy chain single domain antibodies VHH. The organic fluorescent molecules and the fluorescent protein mutually serve as a saturated resonance energy transfer donor and a saturated resonance energy transfer receptor. The probe can achieve the super-resolution imaging of the fluorescent protein in cells conveniently, and also can carry out the saturated resonance energy transfer super-resolution imaging of multicolor and live cells. The probe greatly simplifies the marking steps, and solves a problem of the imaging of multicolor and live cells.

Description

technical field [0001] This field relates to the field of super-resolution imaging, in particular to a saturation resonance energy transfer super-resolution probe and its preparation method and application. Background technique [0002] Optical microscopy imaging technology has attracted much attention because it can directly observe cells without damage and in real time, and then reveal the inner mechanism of complex life phenomena. It is an important technical means widely used in the field of biology. Its development runs through the development of cell biology and is one of the main driving forces in the field of life sciences. It can be considered that the resolution of optical cell imaging microscopy systems largely limits and determines how small the microscopic scale of biology can be studied. Cells and subcellular structures are mostly at the nanometer scale. For example, the diameter of the important organelle ribosome is only 25 nanometers, and the diameter of pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01Q60/00
Inventor 侯尚国邓素辉程亚黄庆樊春海
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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