Title of Invention	" A PROCESS FOR PREPARATION OF HOLEY/LACEY FILMS"
Abstract	A process for preparation of holey/lacey support film useful in electron microscopy said method comprising the steps cleaning cleaved mica sheet with alcohol, soaking the cleaved mica 2 square sheet in 0.2-0.3% polyvinyl resin for a period in the range of 1 to 5 minuts, incubating the soaked mica sheet at a temperature in the range of 45 to 70°C for a period of 10 to 40 minutes to obtain a film, floating the film on the surface of water and subsequently placing grids on the film, exposing the film under an infrared lamp having power in the range of 100 to 200 Watts for a period of 10 to 30 minutes to obtain the holey/lacey film. Optionally, reinforcing with a thin layer of carbon on the film and/or dissolving polyvinyl resin using organic solvent to obtain perforated bare carbon film.

Title of Invention

" A PROCESS FOR PREPARATION OF HOLEY/LACEY FILMS"

Abstract

A process for preparation of holey/lacey support film useful in electron microscopy said method comprising the steps cleaning cleaved mica sheet with alcohol, soaking the cleaved mica 2 square sheet in 0.2-0.3% polyvinyl resin for a period in the range of 1 to 5 minuts, incubating the soaked mica sheet at a temperature in the range of 45 to 70°C for a period of 10 to 40 minutes to obtain a film, floating the film on the surface of water and subsequently placing grids on the film, exposing the film under an infrared lamp having power in the range of 100 to 200 Watts for a period of 10 to 30 minutes to obtain the holey/lacey film. Optionally, reinforcing with a thin layer of carbon on the film and/or dissolving polyvinyl resin using organic solvent to obtain perforated bare carbon film.

Full Text	Field of the Invention: The invention relates to a process for preparation of holey/lacey support film useful in electron microscopy. Specimen support is of utmost importance in electron microscopy. The resolution depends on the thinness of support film. Ideally there should not be any specimen support for achieving highest resolution. One way of achieving this goal is to use holey or lacey support film. Lacey film is support film with large number of holes in it while holey film is support film with fewer holes in it. In cryo-electron microscopy of suspended particles use of lacey support film is the method of choice. In polymer science also lacey polymer films have important applications. Background and prior art: Holey or lacey films are generally prepared using one of the following methods with or without any modifications. Bayer M.E. and Anderson, T.F. 1963, Experentia, 19, 1-3 Bayer and Anderson prepared holey films by dipping a glass slide into 0.3 to 0.4% formvar-coated slide before the formvar evaporates [Bayer M.E. and Anderson, T.F. 1963, Experentia, 19, 1-3. Experentia, 19, 1-3. The preparation of holey films for electron microscopy]. Breathing causes holes to be formed in the formvar film. The formvar film was then scored with a razor blade, floated onto a water surface and the grids were coated standard techniques. Harris W.T. 1962. Nature, 196,499-500. Holey films for electron microscopy Harris suspended glycerol in formvar solution and used the suspension for casting film on glass slide [Harris W.J. 1962. Nature, 196, 499-500. Holey films for electron microscopy]. Glycerol causes holes to be formed in the formvar. This film was then floated over water surface and mounted by standard techniques. Fukami, A. and Adachi, K. 1965. J. Electron Microsc, 14,112-118 Fukami and Adachi made a glass slide hydrophobic first and then allowed to form water droplets on the slides by condensation [Fukami, A. and Adachi, K. 2965. /. Electron Microsc, 14, 112-118]. The glass slide was then coated with a layer of plastic film, which was then stripped off and used to coat grids. Additional reinforcement can be done with the evaporation of a thin layer of carbon. All the methods described above have some drawbacks. For example, in the method of Bayer and Anderson breathing onto the film-covered slide is not a very reproducible method. Because the amount of water droplets that can be deposited by breathing varies from person to person. And the whole method is not very reproducible. The method of Harris is not very reproducible either because mixing of glycerol with water to give hole of useful size is a tricky art. In the method of Fukami and Adachi making the glass slide hydrophobic consistently is not a very reproducible method. Also forming holes of useful size by condensation depends on a number of parameters. To overcome the difficulty of the above stated method, the Applicant has developed a method of preparation of holey/ lacey film, which does not use any glass slide, there is no need to form holes by breathing or by condensation or by mixing something with plastic solution and is very reproducible. Object of the invention: The objective of the present invention is to develop a method of preparing holey/lacey films. Still in another object of the present invention is to develop a method of preparing holey/lacey films, which reproducible. Statement of the present invention: Accordingly, the present invention deals with a method for the preparation of holey/lacey support film for electron microscopy said method comprising the steps: a) cleaning cleaved mica sheet with alcohol, b) soaking the cleaved mica sheet in 0.2-0.3% polyvinyl resin for a period in the range of 1 to 5 minutes, c) incubating the soaked mica sheet at a temperature in the range of 45 to 70°C for a period in the range of 10 to 40 minutes to obtain a film, d) floating the film on the surface of water and subsequently placing grids on the film, e) exposing the film under an infrared lamp for a period in the range of 10 to 30 minutes to obtain the holey/lacey film. f) optionally, reinforcing with a thin layer of carbon on the film and/or dissolving polyvinyl resin using organic solvent to obtain perforated bare carbon film. Brief description of drawings Figure I: Electron micrograph of the holey/lacey film prepared by the present method. The photomicrograph shows the distribution of the holes in the support film. Detailed description of the invention: Accordingly, the present invention deals with a method for the preparation of holey/lacey support film for electron microscopy said method comprising the steps: a) cleaning cleaved mica sheet with alcohol, b) soaking the cleaved mica sheet in 0.2-0.3% polyvinyl resin for a period in the range of 1 to 5 minutes, c) incubating the soaked mica sheet at a temperature in the range of 45 to 70°C for a period in the range of 10 to 40 minutes to obtain a film, d) floating the film on the surface of water and subsequently placing grids on the film, e) exposing the film under an infrared lamp for a period in the range of 10 to 30 minutes to obtain the holey /lacey film. f) optionally, reinforcing with a thin layer of carbon on the film and/or dissolving polyvinyl resin using organic solvent to obtain perforated bare carbon film. In another embodiment of the present invention the area of cleaved mica is 2 square inches. Yet in another embodiment of the present invention polyvinyl resin is selected from a group comprising polyvinyl butyral and polyvinyl formal. Still in another embodiment of the present invention wherein the power of infrared lamp is in the range of 100 to 200 Watts. Further in another embodiment of the present invention the distance between the film and the infrared lamp is in the range of 5 to 20 cms. In another embodiment of the present invention wherein the grids are of 200 to 400 mesh size. A polyvinyl resin solution of strength 0.2-0.3 % is prepared either in chloroform or in ethylene dichloride preferably in a brown bottle. Pioloform was obtained from Agar Scientific Co., U.K. Pioloform and formvar/butvar are plastics. Freshly cleaved mica sheet is cut into rectangles. The preferred size is 1 x2 inch (approximately) although other sizes can also be used. The cleaved surface is cleaned with rectified spirit and is allowed to dry at room temperature (23-26 °C and relative humidity 40-70%). The mica sheet is then dipped vertically downwards in the pioloform (or formvar/butvar) solution for 2-3 minutes, withdrawn from the solution and is dried at room temperature. The mica sheet with the pioloform (or formvar/butvar) film on it is now placed in an oven at 45 to 70 °C for 10-40 minutes. On incubation the holes are formed but the holes are still covered with a very fine film. The size of the holes and their number depends on the temperature and time of incubation. After incubation the edges of the pioloform film on the mica are scored with a sharp razor blade. The pioloform film is then allowed to float on the surface of the water taken in a beaker. The scoring helps release of the film onto the water. Grids of 200-400 meshes (other mesh sizes can also be used) are put on the floating film. This film with the grids on it is then picked up on a filter paper and is placed on a glass slide. This glass slide with the pioloform-coated grids is then allowed to dry under an infrared lamp (100200 W). The distance between the infrared light source and filter paper (with the film and grids) should be 5 - 20 cm. The drying needs to be carried out for 10-30 minutes. This drying is required to rupture the thin film still covering the holes. Care should be taken during the drying process as prolong drying causes the film to rupture. The grids can be used now if grid with bare plastic film with holes in it is needed. Otherwise the grids are placed in a high vacuum evaporator and are covered with 10-20 nm thick carbon layer. The polyvinyl resin film may be removed by dissolving it in chloroform or in any other organic solvent if grid with bare carbon film with holes in it is needed; otherwise the polyvinyl resin can be left as it is. The grids are now ready for use. The introduction of mica has several advantages over the use of glass slide. Mica does not need any treatment for rendering it hydrophobic. Also there is no need for water droplets to be formed on the slide either by condensation or by breathing. Further the polyvinyl resin solution does not need to be mixed with glycerol or anything else for the formation of hole. The method is thus much simpler than the previously used methods for preparing holey/ lacey films. Also the method requires much less skill in the formation process of the holes. The method is highly reproducible. Example The fact that the present method works well is evident from the electron microscopic studies on the holes prepared by the present technique. Figure 1 shows the holes and their distribution in the film prepared by the present technique. The mica sheet was dipped in 0.2% pioloform film and incubated at 50 °C for 10 min. The pioloform film was then floated onto the water surface, grids were lined up onto the film and was picked up on a filter paper. The filter paper was placed on a glass slide and dried under an infrared lamp of 150W at a distance of 8 cms for 25 mins. The filter paper with the grids on it was placed in a high vacuum evaporator and a thin layer (10 nm approximately) of carbon was deposited on it. The grids were examined in a FBI transmission electron microscope; model Tecnai 12 BioTwin and photomicrographs were taken. Advantages: The present technique is a very simple one, needs little skill, requires few chemicals and is reproducible. The introduction of mica has several advantages over glass slide for casting plastic film. The advantages of the technique are summarized below. a) Mica does not need any treatment for rendering it hydrophobic. b) Also there is no need for water droplets to be formed on the mica sheet either by condensation or by breathing. c) The polyvinyl resin solution does not need to be mixed with glycerol or anything else for the formation of hole. The method is thus much simpler than the previously used methods for preparing holey/ lacey films. d) The method is very reproducible. WE CLAIM; 1. A process for preparation of holey/lacey support film useful in electron microscopy said method comprising the steps: a) cleaning cleaved mica sheet with alcohol, b) soaking the cleaved mica 2 square sheet in 0.2-0.3% polyvinyl resin for a period of 1 to 5 minuts, c) incubating the soaked mica sheet at a temperature of 45 to 70°C for a period of 10 to 40 minutes to obtain a film, d) floating the film on the surface of water and subsequently placing grids on the film, e) exposing the film under an infrared lamp having power in the range of 100 to 200 Watts for a period of 10 to 30 minutes to obtain the holey/lacey film, f) Optionally, reinforcing with a thin layer of carbon on the film and/or dissolving polyvinyl resin using organic solvent to obtain perforated bar carbon film. 2. A method as claimed in claim 1, wherein polyvinyl resin is selected from a group comprising polyvinyl butyral and polyvinyl formal. 3. A method as claimed in claim 1, wherein the step (e) the distance between the film and the infrared lamp is in the range of 5 to 20 cms. 4. A method as claimed in claim 1, wherein in step (d), the grids are of 200 to 400 mesh size.

Full Text

Field of the Invention:
The invention relates to a process for preparation of holey/lacey support film useful in electron microscopy. Specimen support is of utmost importance in electron microscopy. The resolution depends on the thinness of support film. Ideally there should not be any specimen support for achieving highest resolution. One way of achieving this goal is to use holey or lacey support film. Lacey film is support film with large number of holes in it while holey film is support film with fewer holes in it. In cryo-electron microscopy of suspended particles use of lacey support film is the method of choice. In polymer science also lacey polymer films have important applications.
Background and prior art:
Holey or lacey films are generally prepared using one of the following methods with or without any modifications.
Bayer M.E. and Anderson, T.F. 1963, Experentia, 19, 1-3
Bayer and Anderson prepared holey films by dipping a glass slide into 0.3 to 0.4% formvar-coated slide before the formvar evaporates [Bayer M.E. and Anderson, T.F. 1963, Experentia, 19, 1-3. Experentia, 19, 1-3. The preparation of holey films for electron microscopy]. Breathing causes holes to be formed in the formvar film. The formvar film was then scored with a razor blade, floated onto a water surface and the grids were coated standard techniques.
Harris W.T. 1962. Nature, 196,499-500. Holey films for electron microscopy
Harris suspended glycerol in formvar solution and used the suspension for casting film on glass slide [Harris W.J. 1962. Nature, 196, 499-500. Holey films for electron microscopy]. Glycerol causes holes to be formed in the formvar. This film was then floated over water surface and mounted by standard techniques.
Fukami, A. and Adachi, K. 1965. J. Electron Microsc, 14,112-118
Fukami and Adachi made a glass slide hydrophobic first and then allowed to form water droplets on the slides by condensation [Fukami, A. and Adachi, K. 2965. /. Electron Microsc, 14, 112-118]. The glass slide was then coated with a layer of plastic film, which was then stripped off and used to coat grids. Additional reinforcement can be done with the evaporation of a thin layer of carbon.
All the methods described above have some drawbacks. For example, in the method of Bayer and Anderson breathing onto the film-covered slide is not a very reproducible method. Because the amount of water droplets that can be deposited by breathing varies from person to person. And the whole method is not very reproducible. The method of Harris is not very reproducible either because mixing of glycerol with water to give hole of useful size is a tricky art. In the method of Fukami and Adachi making the glass slide hydrophobic consistently is not a very reproducible method. Also forming holes of useful size by condensation depends on a number of parameters.
To overcome the difficulty of the above stated method, the Applicant has developed a method of preparation of holey/ lacey film, which does not use any glass slide, there is no need to form holes by breathing or by condensation or by mixing something with plastic solution and is very reproducible.
Object of the invention:
The objective of the present invention is to develop a method of preparing holey/lacey films.
Still in another object of the present invention is to develop a method of preparing holey/lacey films, which reproducible.
Statement of the present invention:
Accordingly, the present invention deals with a method for the preparation of holey/lacey support film for electron microscopy said method comprising the steps:
a) cleaning cleaved mica sheet with alcohol,
b) soaking the cleaved mica sheet in 0.2-0.3% polyvinyl resin for a period
in the range of 1 to 5 minutes,
c) incubating the soaked mica sheet at a temperature in the range of 45 to
70°C for a period in the range of 10 to 40 minutes to obtain a film,
d) floating the film on the surface of water and subsequently placing grids
on the film,
e) exposing the film under an infrared lamp for a period in the range of 10
to 30 minutes to obtain the holey/lacey film.
f) optionally, reinforcing with a thin layer of carbon on the film and/or
dissolving polyvinyl resin using organic solvent to obtain perforated
bare carbon film.
Brief description of drawings
Figure I: Electron micrograph of the holey/lacey film prepared by the present method. The photomicrograph shows the distribution of the holes in the support film.
Detailed description of the invention:
Accordingly, the present invention deals with a method for the preparation of holey/lacey support film for electron microscopy said method comprising the steps:
a) cleaning cleaved mica sheet with alcohol,
b) soaking the cleaved mica sheet in 0.2-0.3% polyvinyl resin for a period
in the range of 1 to 5 minutes,
c) incubating the soaked mica sheet at a temperature in the range of 45 to
70°C for a period in the range of 10 to 40 minutes to obtain a film,
d) floating the film on the surface of water and subsequently placing grids
on the film,
e) exposing the film under an infrared lamp for a period in the range of 10
to 30 minutes to obtain the holey /lacey film.
f) optionally, reinforcing with a thin layer of carbon on the film and/or
dissolving polyvinyl resin using organic solvent to obtain perforated
bare carbon film.
In another embodiment of the present invention the area of cleaved mica is 2 square inches.
Yet in another embodiment of the present invention polyvinyl resin is selected from a group comprising polyvinyl butyral and polyvinyl formal.
Still in another embodiment of the present invention wherein the power of infrared lamp is in the range of 100 to 200 Watts.
Further in another embodiment of the present invention the distance between the film and the infrared lamp is in the range of 5 to 20 cms.
In another embodiment of the present invention wherein the grids are of 200 to 400 mesh size.
A polyvinyl resin solution of strength 0.2-0.3 % is prepared either in chloroform or in ethylene dichloride preferably in a brown bottle. Pioloform was obtained from Agar Scientific Co., U.K. Pioloform and formvar/butvar are plastics. Freshly cleaved mica sheet is cut into rectangles. The preferred size is 1 x2 inch (approximately) although other sizes can also be used. The cleaved surface is cleaned with rectified spirit and is allowed to dry at room temperature (23-26 °C and relative humidity 40-70%). The mica sheet is then dipped vertically downwards in the pioloform (or formvar/butvar) solution for 2-3 minutes, withdrawn from the solution and is dried at room temperature. The mica sheet with the pioloform (or formvar/butvar) film on it is now placed in an oven at 45 to 70 °C for 10-40 minutes. On incubation the holes are formed but the holes are still covered with a very fine film. The size of the holes and their number depends on the temperature and time of incubation.
After incubation the edges of the pioloform film on the mica are scored with a sharp razor blade. The pioloform film is then allowed to float on the surface of the water taken in a beaker. The scoring helps release of the film onto the water. Grids of 200-400 meshes (other mesh sizes can also be used) are put on the floating film. This film with the grids on it is then picked up on a filter paper and is placed on a glass slide. This glass slide with the pioloform-coated grids is then allowed to dry under an infrared lamp (100200 W). The distance between the infrared light source and filter paper (with the film and grids) should be 5 - 20 cm. The drying needs to be carried out for 10-30 minutes. This drying is required to rupture the thin film still covering the holes. Care should be taken during the drying process as prolong drying causes the film to rupture. The grids can be used now if grid with bare
plastic film with holes in it is needed. Otherwise the grids are placed in a high vacuum evaporator and are covered with 10-20 nm thick carbon layer. The polyvinyl resin film may be removed by dissolving it in chloroform or in any other organic solvent if grid with bare carbon film with holes in it is needed; otherwise the polyvinyl resin can be left as it is. The grids are now ready for use.
The introduction of mica has several advantages over the use of glass slide. Mica does not need any treatment for rendering it hydrophobic. Also there is no need for water droplets to be formed on the slide either by condensation or by breathing. Further the polyvinyl resin solution does not need to be mixed with glycerol or anything else for the formation of hole. The method is thus much simpler than the previously used methods for preparing holey/ lacey films. Also the method requires much less skill in the formation process of the holes. The method is highly reproducible.
Example
The fact that the present method works well is evident from the electron microscopic studies on the holes prepared by the present technique. Figure 1 shows the holes and their distribution in the film prepared by the present technique. The mica sheet was dipped in 0.2% pioloform film and incubated at 50 °C for 10 min. The pioloform film was then floated onto the water surface, grids were lined up onto the film and was picked up on a filter paper. The filter paper was placed on a glass slide and dried under an infrared lamp of 150W at a distance of 8 cms for 25 mins. The filter paper with the grids on it was placed in a high vacuum evaporator and a thin layer (10 nm approximately) of carbon was deposited on it. The grids were examined in a FBI transmission electron microscope; model Tecnai 12 BioTwin and photomicrographs were taken.

Advantages:
The present technique is a very simple one, needs little skill, requires few chemicals and is reproducible. The introduction of mica has several advantages over glass slide for casting plastic film. The advantages of the technique are summarized below.
a) Mica does not need any treatment for rendering it hydrophobic.
b) Also there is no need for water droplets to be formed on the mica sheet
either by condensation or by breathing.
c) The polyvinyl resin solution does not need to be mixed with glycerol or
anything else for the formation of hole. The method is thus much simpler
than the previously used methods for preparing holey/ lacey films.
d) The method is very reproducible.

WE CLAIM;
1. A process for preparation of holey/lacey support film useful in electron microscopy said method comprising the steps:
a) cleaning cleaved mica sheet with alcohol,
b) soaking the cleaved mica 2 square sheet in 0.2-0.3% polyvinyl resin for a period of 1 to 5 minuts,
c) incubating the soaked mica sheet at a temperature of 45 to 70°C for a period of 10 to 40 minutes to obtain a film,
d) floating the film on the surface of water and subsequently placing grids on the film,
e) exposing the film under an infrared lamp having power in the range of 100 to 200 Watts for a period of 10 to 30 minutes to obtain the holey/lacey film,
f) Optionally, reinforcing with a thin layer of carbon on the film and/or dissolving polyvinyl resin using organic solvent to obtain perforated bar carbon film.

2. A method as claimed in claim 1, wherein polyvinyl resin is selected from a group comprising polyvinyl butyral and polyvinyl formal.
3. A method as claimed in claim 1, wherein the step (e) the distance between the film and the infrared lamp is in the range of 5 to 20 cms.
4. A method as claimed in claim 1, wherein in step (d), the grids are of 200 to 400 mesh size.

Documents:

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Patent Number

279193

Indian Patent Application Number

2844/DEL/2005

PG Journal Number

03/2017

Publication Date

20-Jan-2017

Grant Date

13-Jan-2017

Date of Filing

25-Oct-2005

Name of Patentee

INDIAN COUNCIL OF MEDICAL RESEARCH

Applicant Address

V. RAMALINAGASWAMI BHAWAN, ANSARI NAGAR POST BOX 4911, NEW DELHI-110 029,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	AMAR N GHOSH	DIVISION OF ELECTRON MICROSCOPY, NATIONAL INSTITUTE OF CHOLERA AND ENTERIC DISEASES (ICMR), P-33, C.I.T. ROAD, SCHEME XM, BELEGHATA, KOLKATA-700 040,INDIA.
2	ANINDITO SEN	DIVISION OF ELECTRON MICROSCOPY, NATIONAL INSTITUTE OF CHOLERA AND ENTERIC DISEASES (ICMR), P-33, C.I.T. ROAD, SCHEME XM, BELEGHATA, KOLKATA-700 040,INDIA.

PCT International Classification Number

H01J 37/20

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA