EMS GRIDS & TEM SUPPORT FILMS CATALOG EDITION V TEM SUPPORT FILMS


C-flat™ Family of Holey Carbon, Gold Grids for S/TEM and Cryo-TEM (continued) Holey Carbon Grids for Cryo-TEM


Cryo Preparation Using C-flat™ Overview


C-flat™ is a holey carbon support film, manufactured using a patent pending semiconductor- based technology without plastics, resists or other soft materials. As a result, the carbon films are flat, uniform and free of residues or plastics. C-flat™ is designed to be an "out of the box" solution, and should require minimal sample preparation. Extensive plasma cleaning is not needed, and could potentially thin the carbon, making it too fragile for blotting or freezing.


Plasma Preparation


If you are using C-flat™ for the first time, it is recommended that no plasma preparation be used initially. As with any carbon film, plasma preparation is sometimes necessary to make the surface more hydrophilic. If your initial results dictate making the films more hydrophilic, below are some guidelines for preparation using several common systems.


Fischione Model 1020 • 25% Oxygen/75% Argon


• Use 5 grid holder and dampening shield • Plasma clean grids for 10-30 seconds


Note: It is recommended that the dampening shield be used when cleaning C-flat™ using the Fischione Model 1020 plasma cleaner. The shield will dampen the effect of the plasma, reducing the erosion rate of the carbon while allowing the film to become more hydrophilic.


Gatan Solarus™ • 25% Oxygen/75% Argon


• Place grids on a support (e.g. glass slide) • Set slide in the bottom of the chamber • Set RF power to 25 watts


• Plasma clean grids for 10-20 seconds Glow Discharge


These systems vary widely depending on the manufacturer. Typically, keep the glow from the plasma dim and the clean time approximately 10-30 seconds.


Plunge Freezing Recommended settings for plunge freezing with the Vitrobot™


• Temperature: 4°C • Humidity: 100% (can vary between 90–100%) • Blot Time: 3-5 seconds • Volume on Grid: 3µL (can vary) • Drain Time: 0 seconds • Offset: 0 for regular samples, –1 for viscous


When using the Vitrobot™, it is recommended that the filter paper be changed regularly (generally after freezing 4-5 grids or 10 minutes, whichever comes first.) The filter paper can become saturated in the high humidity environment of the chamber.


Working with Viscous Samples


Generally, lowering the volume of solution on the grid can help to eliminate the need for multiple blots, which can damage the carbon film. As little as 1µL of solution can cover a 3mm grid area if the pipette tip is used to spread the drop, but reducing the volume to 1.5 or 2.0µL will help as well. Once the sample is on the grid, it should be blotted within a few minutes before further evaporation occurs. If a Vitrobot™ is used, changing the offset from 0mm to -1 or -2mm can also help.


Frozen-Hydrated Bacteriophage Capsid (data acquired on CF-1.2/1.3-4C)


Hydrophilicity/Hydrophobicity


Increasing the hydrophilicity of the carbon film will help a droplet spread evenly over the carbon, rather than pool on the surface. The most common method for achieving this is by plasma or glow discharge; recommended settings for various equipment are given. Keep in mind that C-flat™ is manufactured without any plastics or soft materials in the process, therefore plasma or glow discharge steps are only needed to make the surface more hydrophilic, not to clean. For this reason, a lower power and time is generally used.


Adding Carbon to C-flat™


Many C-flat™ parts are now offered in both the standard as well as a thicker carbon film, designed to give each lab the option to choose not only the most appropriate hole geometry and size, but also the ideal carbon thickness for their application. In addition, carbon can be added to C-flat™ either to thicken the existing hole pattern, or as a thin continuous overlay across the hole pattern. Overlays are often used when particles have a strong affinity towards the carbon material.


Keeping the Carbon Intact


C-flat™ is designed to be an “out of the box” solution. Extensive sample preparation steps are generally not required, and often carbon that is torn or broken is a sign of plasma cleaning that is too long and/or at too high a power setting. Please refer to the suggestions on plasma cleaner settings, as well as on working with viscous samples.


YOU MAY NEED... zzz Plunging Tweezers for the CP3 (CryoplungeTM 3) These custom tweezers specifically made to fit the Gatan CryoplungeTM Description CP3690 Plunging Tweezers zzzVitrobot™ Filter Paper


This special Filter Paper has an outer diameter of 55 mm, with an inner diameter of 20 mm. Made from Grade 595 paper.


Cat No. 71166-65 Description Vitrobot™ Filter Paper Qty. 100/pk 45


Qty. each


,


an instrument used in the preparation of frozen hydrated specimens for cryoEM. Cat No.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92