Unlock instant, AI-driven research and patent intelligence for your innovation.

Fluorescence imaging method for observing biomacromolecules on surface of living cell membrane and near living cell membrane

A technology of biological macromolecules and cell membranes, applied in the fields of fluorescence/phosphorescence, analytical materials, material excitation analysis, etc., can solve the problems such as the inability to effectively obtain the normal distance and the inability to observe living cells, achieve high-throughput rapid screening, improve The effect of resolution

Active Publication Date: 2021-06-22
INST OF PHYSICS - CHINESE ACAD OF SCI
View PDF5 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fluorescence resonance energy transfer method requires that the position of the receptor is completely known, and it can only be used to simply observe the point-to-point distance between two molecules, which is difficult to achieve on the flowing living cell membrane. Fluidity, the molecules on the membrane will continue to diffuse and move along the tangential direction of the membrane surface, while the molecular movement that is really related to the membrane function is along the normal direction (that is, the radial direction) of the membrane. In this case, the conventional FRET The method cannot effectively obtain the change of the normal distance of the molecule from the membrane surface
[0005] Electron microscopes in the prior art have relatively high resolution, but usually they can only observe fixed cell samples at low temperature or in a vacuum environment, so they cannot directly observe living cells

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescence imaging method for observing biomacromolecules on surface of living cell membrane and near living cell membrane
  • Fluorescence imaging method for observing biomacromolecules on surface of living cell membrane and near living cell membrane
  • Fluorescence imaging method for observing biomacromolecules on surface of living cell membrane and near living cell membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] In this embodiment, the movement information of the cell membrane surface and nearby biomacromolecules under the action of the target drug is measured from the fluorescence lifetime information. Use Liss-Rhod-PE (a green fluorescent-labeled phospholipid macromolecule) to label the human lung cancer epithelial cell A549 cell membrane, and add 5 mM (substance amount, millimole) blue dextran (BlueDextran-5) dissolved on the outside of the cell membrane (quencher) colorless DMEM medium, the purpose is to calculate the radial movement of the clusters or individuals of the green fluorescently labeled phospholipid macromolecules near the surface of the living cell membrane. In this example, a fluorescence lifetime microscope is used to observe the cell membrane surface of the phospholipid bilayer structure, and it can be observed that there are two different fluorescence lifetime distributions of the fluorescence of the green fluorescently labeled phospholipid macromolecules on...

Embodiment 2

[0085] In this embodiment, the movement information of a fluorescently labeled single human antimicrobial peptide macromolecule near the cell membrane phospholipid bilayer is observed from the fluorescence intensity information. In this example, human-derived antibacterial peptide LL-37 labeled with 5nM tetramethylrhodamine (a green fluorescent molecule) was used to infect human lung cancer epithelial cell A549 cell membrane, and 5mM blue dextran (Blue Dextran-5, etc.) quencher in colorless DMEM medium, the purpose is to calculate the radial movement of a single macromolecule of the human antimicrobial peptide LL-37 near the surface of the living cell membrane. When observing, the amount of fluorescent molecular labeling is 1 μm 2 No more than 1 fluorescent molecule shall prevail in the region (i.e. 10 -12 piece / m 2 ), the determination process is realized by total internal reflection microscopy (TIRF), such as observing a 1μm 2 If there is more than one fluorescent molecul...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a fluorescence imaging method for observing biomacromolecules on the surface of a living cell membrane and near the surface of the living cell membrane. Based on a fluorescence resonance energy transfer principle, the radial displacement of the macromolecules in the cell membrane is measured by using a total internal reflection or confocal illumination mode. The method comprises a matching rule of specific fluorescence donors and receptors, a fluorescence labeling scheme of living cells and a calculation process of measured data. According to the method, the motion state and conformation change of the cell membrane and biomolecules near the cell membrane can be observed at a sub-nanometer scale with relatively high time and spatial resolution, meanwhile, the cell membrane is not damaged, the observation of living cells can be maintained, and the screening of the membrane receptor related drugs can be rapidly completed at high flux.

Description

technical field [0001] The invention relates to the technical field of observing biomacromolecules with a fluorescence imaging method. Specifically, the invention relates to a fluorescence imaging method for in situ observation of macromolecular conformation and position changes in the cell membrane and on the cell membrane surface of living cells. Background technique [0002] The cell membrane is a selectively permeable barrier that separates the cell from the external environment. It controls the flow of information and the entry and exit of substances. Membrane proteins are the targets of many drugs. Advancing the study of membrane-associated processes relies on new technologies capable of measuring the position and conformational changes of biomolecules in membranes. Most membrane-associated processes occur within the cell membrane (membrane intercalation) or within ten nanometers of the membrane. [0003] In the prior art, fluorescent methods are usually used to obser...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N21/64
CPCG01N21/6458
Inventor 侯文清陆颖马东飞贺小龙李明
Owner INST OF PHYSICS - CHINESE ACAD OF SCI