CN109580485A - A kind of fixation device and application method for red blood cell super-resolution imaging - Google Patents
A kind of fixation device and application method for red blood cell super-resolution imaging Download PDFInfo
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- CN109580485A CN109580485A CN201910047576.6A CN201910047576A CN109580485A CN 109580485 A CN109580485 A CN 109580485A CN 201910047576 A CN201910047576 A CN 201910047576A CN 109580485 A CN109580485 A CN 109580485A
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- 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
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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
- G01N21/84—Systems specially adapted for particular applications
Abstract
A kind of fixation device and application method for red blood cell super-resolution imaging, belongs to the technical field that micro-processing technology intersects with bio-imaging.The device is fixed red blood cell and close to glass surface, realizes the red blood cell smooth to surface and carries out physics and fixes, so that the observation of total internal reflection super-resolution imaging is very convenient, has reached requirement adherent to sample during total internal reflection super-resolution imaging.The device does not need to carry out any chemical modification and processing, and easy to use, cost of manufacture is cheap.Each red blood cell fixes the small-sized of chamber, many repetitive units can be arranged on the fixation device that one piece of area is less than unitary coin, super-resolution shooting can be carried out to multiple red blood cells simultaneously, in addition several micro- channels can be arranged on a fixed device, super-resolution imaging can be carried out to the red blood cell in several source difference blood samples simultaneously namely on the same fixed device, substantially increase experiment flux.
Description
Technical field
The present invention relates to a kind of fixation devices and application method for red blood cell super-resolution imaging, belong to micro Process skill
The technical field that art is intersected with bio-imaging.
Background technique
Super-resolution imaging technology is the field developed rapidly in recent years, it is due to that can break through optical diffraction limit, in nanometer
Scale observes biological sample, becomes the powerful of cell analysis now.But current unimolecule positions super-resolution imaging, mostly
Based on total internal reflection techniques, the biological sample of technical requirements observation is observed close to glass sheet surface, for attached cell
It is easier to realize, but smooth for surfaces such as red blood cells and not adherent cell is observed, and is difficult to realize.
Fixation device proposed by the present invention for red blood cell super-resolution imaging is consolidated red blood cell using elastic material PDMS
It is scheduled in chamber, which may be implemented the super-resolution imaging based on total internal reflection, and have the characteristics that high throughput.
Summary of the invention
The present invention proposes a kind of fixation device for red blood cell super-resolution imaging, is made of a plurality of micro- channel, every
Micro- channel connects the fixed chamber of multiple red blood cells, and the fixed chamber of each red blood cell includes one section of entrance channel and red blood cell lock chamber
Room.The height of the fixed chamber of red blood cell is no more than one times of red blood cell dimension, and such red blood cell is inhaled into the fixed chamber of red blood cell
It is fixed afterwards by single layer.
Fixation device proposed by the present invention for red blood cell super-resolution imaging, device rapidoprint are PDMS(poly- two
Methylsiloxane) and slide, PDMS material porous breathable and flexible, in the fixed chamber of red blood cell by red blood cell pressure
Firmly, close to glass sheet substrate, super-resolution imaging is carried out.
This is used for the fixation device of red blood cell super-resolution imaging, and the fixed chamber height of red blood cell is 0.8 to 1.2um, does not surpass
One times for crossing red blood cell dimension；Size is between 30um*30um to 80um*80um, for being enriched with multiple red blood cells.It is each micro- logical
Road has multiple repetitive units, and high-throughput carry out red blood cell super-resolution imaging may be implemented.
A kind of fixation device for red blood cell super-resolution imaging, the device include the PDMS microcavity being arranged on slide
Room, a plurality of micro- channel structure is arranged in PDMS micro chamber, and micro- channel structure is connected using sample inlet with culture solution entrance
To entrance main channel, entrance main channel is arranged in bending, and entrance main channel passes through after being connected to the fixed chamber unit of multiple red blood cells
Export main channel connection outlet；The fixed chamber unit of the red blood cell is using the fixed chamber of red blood cell on fixed chamber main channel
Entrance connects the fixed chamber of red blood cell, fixed chamber main channel connection entrance main channel and outlet main channel；The red blood cell is solid
The thickness for determining chamber is no more than one times of red blood cell dimension, the fixed chamber of red blood cell with a thickness of 0.8-1.2um, red blood cell is fixed
The size of chamber is between 30um*30um to 80um*80um.Wherein, PDMS micro chamber is dimethyl silicone polymer micro chamber.
The different channels of each chip can also be passed through different blood samples, can simultaneously to the red blood cell in different blood samples into
Row super-resolution imaging comparative experiments.
The implementation method of the fixation device of the red blood cell super-resolution imaging, comprising the following steps:
(1) pass through the method for soft lithographic and punching preparation PDMS microcavity cell structure；
(2) PDMS microcavity cell structure is by the way that after plasma treatment, picture surface is swept away, and is put on clean slide；
(3) it will be passed through in chip after blood sample concussion uniformly, since PDMS is after plasma processing to the wellability of liquid and right
The characteristic that gas absorbs, blood are absorbed into the fixed chamber of red blood cell automatically；
(4) since the height of the fixed chamber of red blood cell is no more than one times of red blood cell dimension, red blood cell is in cell lock chamber
It is fixed in room by single layer；
(5) micro-fluidic chip for being loaded with red blood cell, which is placed on microscope, carries out super-resolution imaging.
Application for the fixation device of red blood cell super-resolution imaging, in total internal reflection super-resolution imaging.
The utility model has the advantages that the fixation device is made of a plurality of micro- channel, every micro- channel connects the fixed chamber of multiple red blood cells,
The fixed chamber of each red blood cell includes one section of entrance channel and red blood cell fixed chamber.After plasma processing using PDMS material
Wellability to liquid and the characteristic to gas absorption, after blood sample is added in main channel, red blood cell can be automatically drawn into and be fixed on
Red blood cell fixes chamber.The time that blood fills up cell chamber is about 2-3 minutes, substitutes the culture solution of main channel later, also not
The cell of cell chamber can be taken out of.
Since red blood cell is fixed and close to glass surface, so that the observation of total internal reflection super-resolution imaging is very convenient；
It solves the problems, such as to be difficult to since erythrocyte surface is smooth to remain stationary during super-resolution imaging, is highly suitable for red blood cell
The research work of relevant super-resolution imaging.The fixation device may be implemented the red blood cell progress physics smooth to surface and fix,
It is able to solve adherent to sample requirement during total internal reflection super-resolution imaging.The device does not need to carry out any chemical modification
And processing, easy to use, cost of manufacture is cheap.It can be to the red blood cell in adult blood sample, pneumonia using the fixation device
The form progress super-resolution shooting and analysis of the red blood cell of child's blood sample, the adult blood sample red blood cell being long placed in, can also be to red thin
The micropore that erythrocyte surface is formed during cell lysis is swollen carries out super-resolution imaging shooting.The size of the fixed chamber of each red blood cell
Very little can arrange many repetitive units on the chip that one piece of area is less than unitary coin, can be simultaneously to multiple red blood cells
Super-resolution shooting is carried out, in addition can arrange several micro- channels on a fixed device, that is, in the same fixed device
On can simultaneously in several source difference blood samples red blood cell carry out super-resolution imaging, substantially increase experiment flux.
Detailed description of the invention
Fig. 1 is a kind of structure chart of fixation device for red blood cell super-resolution imaging.
Fig. 2 is micro- channel junction composition of fixed device.
Fig. 3 is the main view of the fixed chamber unit of red blood cell.
Fig. 4 is the side view of the fixed chamber unit of red blood cell.
Fig. 5 is to carry out super-resolution imaging figure to adult human red blood cells using fixed device.
Fig. 6 is the super-resolution imaging figure of pneumonia child's red blood cell.
Fig. 7 is the super-resolution imaging figure of adult erythrocyte surface swelling position.
Fig. 8 is the super-resolution imaging figure for being long placed in adult erythrocyte.
In figure: 1, slide, 2, PDMS micro chamber, 3, micro- channel structure, 4, sample inlet, 5, culture solution entrance, 6, entrance
Main channel, 7, the fixed chamber unit of red blood cell, the fixed chamber of 7a, red blood cell, 7b, fixed chamber main channel, 7c, red blood cell are fixed
Chamber ingress, 8, outlet main channel, 9, outlet.
Specific embodiment
Embodiment 1
Fig. 1-Fig. 4 is the structure chart of the fixation device for red blood cell super-resolution imaging.There are four micro- channels in the fixation device
3, indicate there is the fixed chamber repetitive unit of 90 red blood cells on every channel with red and blue respectively.Each red blood cell is fixed
Chamber with a thickness of 1um, chamber size 50um*50um.Experimentation is as follows, passes through the method and punching preparation PDMS of soft lithographic
Microcavity cell structure, PDMS microcavity cell structure are swept away by picture surface after plasma treatment and are put on clean slide, blood sample is shaken
It is passed through in sample inlet 4 after swinging uniformly, since PDMS micro chamber 2 is after plasma processing to the wellability of liquid and to gas
The characteristic of absorption, blood sample enters entrance main channel 6 from sample inlet 4 and is connected to fixed chamber main channel 7b, then passes through fixation
Chamber ingress 7c is connected to the fixed chamber 7a of red blood cell.Since the thickness of the fixed chamber of red blood cell is no more than the one of red blood cell dimension
Times, therefore red blood cell is fixed in the fixed chamber 7a of cell by single layer.Being loaded with the fixation device of red blood cell, to be placed in microscope enterprising
Row super-resolution imaging.
Embodiment 2
Fig. 5 is to carry out super-resolution imaging figure to adult human red blood cells using fixed device proposed by the present invention.Wherein, figure a is general
5 red blood cells of logical wide field imaging, figure b are 5 red blood cells of super-resolution imaging, and figure c characterizes super-resolution imaging positioning accuracy
20nm, figure d show that the chip can keep red blood cell motionless in 80s, carry out super-resolution imaging, two kinds of dyestuffs dii and c180
It can realize.Scale bar 2um.
Embodiment 3
The fixation device can be used for the super-resolution imaging of pneumonia child's red blood cell
(1) pass through the method for soft lithographic and punching preparation PDMS microcavity cell structure；
(2) PDMS microcavity cell structure is by the way that after plasma treatment, picture surface is swept away, and is put on clean slide；
(3) it will be passed through in chip after the blood concussion uniformly of pneumonia child, since PDMS is after plasma processing to liquid
Wellability and the characteristic absorbed to gas, blood are absorbed into the fixed chamber of red blood cell automatically；
(4) since the height of the fixed chamber of red blood cell is no more than one times of red blood cell dimension, red blood cell is inhaled into red blood cell
It is fixed after fixed chamber by single layer；
(5) micro-fluidic chip for being loaded with red blood cell, which is placed on super-resolution microscope, carries out imaging.Here wherein three are shown
The red blood cell super resolution image of pneumonia child, as shown in Figure 6.
Embodiment 4
Super-resolution imaging using fixed device proposed by the present invention for adult erythrocyte surface swelling position.It will be adult red thin
After born of the same parents fix in the chips, water is passed through in chip main channel, water can diffuse into red blood cell lock chamber by red blood cell entrance
Room, red blood cell will receive the influence of osmotic pressure, be swollen, using super-resolution imaging, cell when can shoot red blood cell swelling
The aperture of film surface, as shown in Figure 7.
Embodiment 5
The fixation device can also be used in the super-resolution imaging for being long placed in adult erythrocyte
(1) pass through the method for soft lithographic and punching preparation PDMS microcavity cell structure；
(2) PDMS microcavity cell structure is by the way that after plasma treatment, picture surface is swept away, and is put on clean slide；
(3) it will be passed through in chip after the adult blood sample concussion uniformly of refrigeration 3 months (being long placed in), since PDMS is at plasma
To the wellability of liquid and the characteristic absorbed to gas after reason, blood is absorbed into the fixed chamber of red blood cell automatically；
(4) since the height of the fixed chamber of red blood cell is no more than one times of red blood cell dimension, red blood cell is inhaled into red blood cell
It is fixed after fixed chamber by single layer；
(5) micro-fluidic chip for being loaded with red blood cell, which is placed on super-resolution microscope, carries out imaging.Here wherein three are shown
The adult erythrocyte's super resolution image being long placed in, as shown in Figure 8.
The above experiment content has obtained national young Projects, and (item number: 21804016) and central colleges and universities are basic
The support of scientific research expense (item number: DUT17LK43).
Claims (3)
1. a kind of fixation device for red blood cell super-resolution imaging, it is characterised in that: the device includes being arranged on slide (1)
PDMS micro chamber (2), a plurality of micro- channel structure (3) of setting, micro- channel structure are entered using sample in PDMS micro chamber (2)
Mouth (4) and culture solution entrance (5) are connected to entrance main channel (6), and entrance main channel (6) arrange in bending, entrance main channel
(6) multiple red blood cells fixed chamber unit (7) are connected to by outlet main channel (8) connection outlet (9)；The red blood cell lock chamber
Chamber unit (7) is using the fixed chamber of red blood cell fixed chamber ingress (7c) connection red blood cell on fixed chamber main channel (7b)
(7a), fixed chamber main channel (7b) connection entrance main channel (6) and outlet main channel (8)；The fixed chamber of the red blood cell
The thickness of (7a) is no more than one times of red blood cell dimension, red blood cell fixed chamber (7a) with a thickness of 0.8-1.2um, red blood cell is solid
The size of chamber (7a) is determined between 30um*30um to 80um*80um.
2. a kind of application method of fixation device for red blood cell super-resolution imaging according to claim 1, including with
Lower step:
(1) it will be passed through in sample inlet (4) after blood sample concussion uniformly, since PDMS micro chamber (2) is right after plasma processing
The wellability of liquid and the characteristic absorbed to gas, blood sample enter entrance main channel (6) from sample inlet (4) and are connected to fixation
Chamber main channel (7b), then red blood cell is connected to by fixed chamber ingress (7c) and fixes chamber (7a)；
(2) since the thickness of the fixed chamber of red blood cell is no more than one times of red blood cell dimension, red blood cell is in cell lock chamber
It is fixed in room (7a) by single layer；
(3) the fixation device for being loaded with red blood cell, which is placed on microscope, carries out super-resolution imaging.
3. a kind of application of fixation device for red blood cell super-resolution imaging according to claim 1, it is characterised in that:
The device is applied in total internal reflection super-resolution imaging.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113945549A (en) * | 2021-10-18 | 2022-01-18 | 国科温州研究院(温州生物材料与工程研究所) | Comprehensive analysis method applied to red blood cell super-resolution imaging and single molecule tracking |
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