CN103151231B - Attached cell ESEM carrier and preparation method - Google Patents

Abstract

The present invention discloses a kind of attached cell ESEM carrier, there is scanning electron microscope example holder (1), scanning electron microscope example holder (1) is fixed with by conductive adhesive layer (2) base plate (3) of TC superficial cell culture dish, the base plate (3) of TC superficial cell culture dish is covered with cellular layer (4), cellular layer (4) has conducting film (5), bus (6) is connected across conducting film (5) and conductive adhesive layer (2).Its preparation method directly uses TC superficial cell culture dish cultured cell, substitute ethanol with the tert-butyl alcohol and make dehydrating agent, finally prepare attached cell ESEM carrier with the base plate of the TC superficial cell culture dish being covered with cellular layer, there is the advantages such as simple, quick and easy.

Description

Attached cell ESEM carrier and preparation method

Technical field

The present invention relates to a kind of attached cell ESEM carrier and preparation method, especially a kind of simple to operate, cost is low, avoid cellular morphology to change, ensure the attached cell ESEM carrier of experiment effect and preparation method.

Background technology

At present, at medical domain, the surface topography observing attached cell with ESEM (electron microscope) is very general.Due to ESEM be utilize fine focusing electron beam to eject when sample surfaces scans various physical signallings to be modulated into picture, therefore attached cell must be prepared into can for the carrier of scanning electron microscopic observation, i.e. attached cell ESEM carrier.Existing attached cell ESEM carrier is take glass slide as culture plate mostly, its preparation method be substantially by glass slide through ultraviolet disinfection, poly-D-lysine bag by after put into Tissue Culture Dish, carry out cell chulture.When after cell chulture to desired density, in culture dish, add buffer solution clean cell, and then with pH7.2 ~ 7.4, concentration is the glutaraldehyde fixed cell of 2.5%, after again cleaning cell, with the ethanol of variable concentrations gradient to cell dehydration, drying and vacuumize process.The glass slide being covered with cell is taken out, and by conductive adhesive layer, glass slide is fixed in scanning electron microscope example holder, a bus is drawn from conductive adhesive layer side, the other end of bus is placed on cell face, with ion sputtering film coating instrument plating conductive layer, the other end of bus and conductive layer are fixedly connected.There are the following problems:

1. the cell attachment due to glass slide is poor, for improving cell attachment, must carry out bag and being processed, waste time and energy before cultivation to wave carrier piece;

2. glass slide is through repeatedly dewatering and fragility increase after dry process, and easy fracture, causes the change of cellular morphology, especially can cause the cells Dendritic fracture with longer dendron; Cell surface also can cover the thick tunicle of one deck because lipid separates out simultaneously, and the profile of attached cell is indistinctly manifested, causes attached cell shrinkage, subside time serious, all phenomenons all can affect experimental result;

3. all need the glass slide being covered with cellular layer to take out from Tissue Culture Dish, because the adhesive force of glass slide and culture dish bottom surface is large, therefore it is comparatively difficult to take out wave carrier piece, easily on cell face, produces cut at every turn;

4. be difficult to distinguish cell face and glass slide bottom surface, occur the phenomenon ventricumbent for cell glass slide be fixed on sample carrier sometimes.

TC superficial cell culture dish (Corning tissue-culturetreatedculturedishes) is existing cell culture vessel, with transparent, there is the pure polystyrene manufacture of certain toughness moral form, inner surface covalent bond hydrogel layer, there is hydrophily and neutral charge, there is the persistence of surface wettability and the stability of cell adsorption, as special cell chulture utensil, there is good compatibility to cell.But, up to now not about the relevant report directly preparing attached cell ESEM carrier with TC superficial cell culture dish.

Summary of the invention

The present invention is the above-mentioned technical problem in order to solve existing for prior art, provide a kind of especially a kind of simple to operate, cost is low, avoid cellular morphology to change, ensure the attached cell ESEM carrier of experiment effect and preparation method.

Technical solution of the present invention is: a kind of attached cell ESEM carrier, there is scanning electron microscope example holder, scanning electron microscope example holder is fixed with by conductive adhesive layer the base plate of TC superficial cell culture dish, the base plate of TC superficial cell culture dish is covered with cellular layer, cellular layer there is conducting film, bus cross-over connection conducting film and conductive adhesive layer.

A preparation method for described attached cell ESEM carrier, is characterized in that carrying out as follows successively:

A. cell is cultured to desired density in TC superficial cell culture dish;

B. use pH7.2 ~ 7.4, concentration be 2.5% glutaraldehyde fixed cell;

C. use the PBS buffer solution for cleaning cell 3 times of 0.1mol/L, then soak cell 12 ~ 15 hours with the PBS buffer solution of 0.1mol/L;

D. 1 × PBS buffer solution for cleaning cell 1 time is used;

E. in cell, add the tert-butyl alcohol from low to high according to concentration gradient, often add a tert-butyl alcohol, softly blow and beat 3 ~ 5 times, leave standstill 10 minutes, 1 × PBS buffer solution for cleaning cell 1 time, until add the tert-butyl alcohol for the last time; When directly making tert-butyl alcohol crystallization under liquid nitrogen environment after adding the tert-butyl alcohol for the last time; Described concentration gradient is 10%, 25%, 50%, 60%, 70%, 80%, 90%, 100%;

F. TC superficial cell culture dish is inserted in vacuum machine and vacuumize;

G. TC superficial cell culture dish sidewall is cut off, and the base plate of the culture dish being covered with cellular layer is cut to the size needed for electron microscopic observation and shape and by conductive adhesive layer, the base plate of culture dish is fixed in scanning electron microscope example holder, a bus is drawn from conductive adhesive layer side;

H. the other end of bus is placed on cellular layer, with ion sputtering film coating instrument plating conductive layer, the other end of bus and conductive layer is fixedly connected.

The present invention directly prepares attached cell ESEM carrier with TC superficial cell culture dish cultured cell with the base plate of the TC superficial cell culture dish being covered with cellular layer, has the advantages such as simple, quick and easy, specific as follows:

1., because TC superficial cell culture dish has good compatibility to cell, be therefore processed without the need to carrying out bag, simple to operate, time saving and energy saving;

2.TC superficial cell culture dish has certain toughness, though through repeatedly dehydration and dry process also not easy fracture, avoid causing cellular morphology change because of fracture, guarantee experiment effect;

3. substitute ethanol with the tert-butyl alcohol and make dehydrating agent, not only there is better dehydrating effect, and the tert-butyl alcohol can form crystallization under liquid nitrogen environment, to keep the proterties of cell to greatest extent, avoid cellular morphology to change vacuumizing under state;

4. overcome and the glass slide being covered with cellular layer is taken out existing technological deficiency from Tissue Culture Dish;

5. due to be finally will cut off TC superficial cell culture dish sidewall and be covered with cell base plate be fixed on sample carrier, cell face is just very clear, there will not be the phenomenon be fixed on sample carrier that to be faced down by cell.

Accompanying drawing explanation

Fig. 1 is the structural representation of the embodiment of the present invention.

Embodiment

Carry out as follows successively:

A. cell is cultured to desired density in 35mmTC superficial cell culture dish;

B. use pH7.2 ~ 7.4, concentration be 2.5% glutaraldehyde fixed cell;

C. use the PBS buffer solution for cleaning cell 3 times of 0.1mol/L, then soak cell 12 ~ 15 hours with the PBS buffer solution of 0.1mol/L;

D. 1 × PBS buffer solution for cleaning cell 1 time is used;

E. in cell, add the tert-butyl alcohol from low to high according to concentration gradient, often add a tert-butyl alcohol, softly blow and beat 3 ~ 5 times, leave standstill 10 minutes, 1 × PBS buffer solution for cleaning cell 1 time, until add the tert-butyl alcohol for the last time; Just without the need to blowing and beating, leaving standstill after adding the tert-butyl alcohol for the last time, but directly under liquid nitrogen environment, make tert-butyl alcohol crystallization; The concentration gradient of the described tert-butyl alcohol is 10%, 25%, 50%, 60%, 70%, 80%, 90%, 100%;

F. TC superficial cell culture dish is inserted in vacuum machine and vacuumize, extract the tert-butyl alcohol etc. of crystallization out;

G. TC superficial cell culture dish sidewall is cut off, and the base plate of the culture dish being covered with cellular layer is cut to the size needed for electron microscopic observation and shape and by conductive adhesive layer, culture dish base plate (cellular layer upward) is fixed in scanning electron microscope example holder, a bus is drawn from conductive adhesive layer side;

H. the other end of bus is placed on cellular layer, with ion sputtering film coating instrument plating conductive layer, the other end of bus and conductive layer is fixedly connected.

The attached cell ESEM carrier produced as shown in Figure 1, there is scanning electron microscope example holder 1, scanning electron microscope example holder 1 is fixed with by conductive adhesive layer 2 base plate 3 of TC superficial cell culture dish, the base plate 3 of TC superficial cell culture dish is covered with cellular layer 4, cellular layer 4 has conducting film 5, bus 6 is connected across conducting film 5 and conductive adhesive layer 2.

Claims (1)

1. the preparation method of an attached cell ESEM carrier, described attached cell ESEM carrier has scanning electron microscope example holder (1), scanning electron microscope example holder (1) is fixed with by conductive adhesive layer (2) base plate (3) of TC superficial cell culture dish, the base plate (3) of TC superficial cell culture dish is covered with cellular layer (4), cellular layer (4) there is conducting film (5), bus (6) cross-over connection conducting film (5) and conductive adhesive layer (2), is characterized in that carrying out as follows successively:

A. cell is cultured to desired density in TC superficial cell culture dish;

B. use pH7.2 ~ 7.4, concentration be 2.5% glutaraldehyde fixed cell;

C. use the PBS buffer solution for cleaning cell 3 times of 0.1mol/L, then soak cell 12 ~ 15 hours with the PBS buffer solution of 0.1mol/L;

D. 1 × PBS buffer solution for cleaning cell 1 time is used;

E. in cell, add the tert-butyl alcohol from low to high according to concentration gradient, often add a tert-butyl alcohol, softly blow

Make a call to 3 ~ 5 times, leave standstill 10 minutes, 1 × PBS buffer solution for cleaning cell 1 time, until add the tert-butyl alcohol for the last time; When directly making tert-butyl alcohol crystallization under liquid nitrogen environment after adding the tert-butyl alcohol for the last time; Described concentration gradient is 10%, 25%, 50%, 60%, 70%, 80%, 90%, 100%;

F. TC superficial cell culture dish is inserted in vacuum machine and vacuumize;

G. TC superficial cell culture dish sidewall is cut off, and the base plate of the culture dish being covered with cellular layer is cut to the size needed for electron microscopic observation and shape and by conductive adhesive layer, the base plate of culture dish is fixed in scanning electron microscope example holder, a bus is drawn from conductive adhesive layer side;

H. the other end of bus is placed on cellular layer, with ion sputtering film coating instrument plating conductive layer, the other end of bus and conductive layer is fixedly connected.