Dear Posters, this issue IMHO is really an old one and in former times also was intensively discussed by our predecessors in EM-techniques. It seems that the problem arises sometimes and not only the causes but also the sources how to overcome mostly are hidden as usual. But I am following now a very interesting and huge (and fast) reply thread on this and am happy about the many posters working with T(EM).

The problem: as was stated above, all previous explanations are right, more or less, but since we don’t know exactly whether Amy uses formvar-mounted grids or just picks up the ultrathin sections onto naked grids we might guess a little bit what the real culprit has been.

I agree with several posters about the “forces” which will be responsible for a good adherence to the copper grid (and normally these forces will hold the sections in their position after drying on the grid before staining them). Since meanwhile I am writing this there have been other postings too, I have to reply to the post of Elizabeth Martin concerning the use of a different wash bottle and another type of forceps, leading to floating off of sections. I have never seen this (32 years in doing staining ultrathin staining right that way she described) doing staining the sections always manually. So IMHO there are some points I will pull out of the discussion so far:

• Cleanliness of grids (and therefore doing such right, many possibilities and personal choices have been given. Usually we don’t know how long it took from fabrication to selling and usually you’ll purchase 3-5 packages of 100 grids each, so prolonged storage – on air – which will cause oxidation less to severe, but we will not see this macroscopically, sometimes there might be a slight oily film-deposit etc.),

• Choice of suited tweezers (antimagnetic), ‘shoes [mostly non insulating rubber sole] and personal protective cloth’ [synthetic lab coat material] - with the possibility of electrostatic loading when handling grids at the EM or at staining…. have you seen copper grids leaping aside when trying to fix the grids in between? I have, several times….)

• Placing steps and adhering sections to the grid (with or without formvar-foil mount) INCLUDING definite drying of the section unto the grid (at least wicking grids/sections with filter paper as described)

• Washing steps (dipping or “jet-stream-washing out of a squeeze-wash bottle, wicking again and afterwards more/less “dry” grids supposed to be stored until the next step into a grid-box, eventually drying again within a laminar air flow cabinet).

Since others have posted their methods for cleaning grids, I add here also perhaps interesting information:

Summary of MSA-Listserver-posts (2006, no mentioning of use of ultrasonic treatment): cleaning solutions used:

• conc. NaOH solution (some seconds) – -A.dest OR:

• 3% HCl in 95-96% EtOH(30-40 sec) – - wash in 95% EtOH --- A.dest OR:

• Household (NH3+) hydrous ammoniacal solution, 30 min-1 h, grids shiny/glossy --- A.dest. OR:

• flame of an alcohol burner (short) OR:

• 5-10% hydrochloric acid solution – gently shake – A. dest. Ethanol, Acetone, chloroform and/ or petroleumbenzine. OR:

• Just before use: dip grid into 1 N HCl, Acetone – A. dest. – use / mount sections immediately.

Short version: EM-Laboratory, Pathology, Salzburg (Austria):

cleaning of (old) Formvar-mounted grids:

• (1)Chloroform 100%, ultrasonic treatment ( 2 bathes, 3-5 min each)

• (2)Acetone (following, for fat-removal ( 1-2 bathes, 3-5 min each))

• (3)20 ml of 2N HCl + 96%EtOH ad 100 ml EV Ultrasonic bath 10-15 seconds until grids become shiny and glossy (not longer, since HCl tackles/pits copper)

• (4)Eventually neutralize by 1N NaOH, or NH3 (hydrous ammoniacal solution diluted (5-10 min, or concentrated1-3 min)

• (5)A. dest. (several times)

• (6)Afterwards shortly Acetone followed by a petroleumbenzine bath,

• (7)Dry from glass beaker over a sheet of lens paper

cleaning of un-mounted (naked) grids from package: starting from (3) above.

As to the question: “Clean” or “Not to clean” see e.g. Special grid cleaning procedures: cf.

http://www.2spi.com/catalog/grids/grid-cleaning.html

and: To clean or not to clean TEM grids before use cf.

http://www.2spi.com/catalog/grids/cleaning-of-TEM-grids-before-use.html

(no financial interest, no affiliations, there might also be information on this matter from other dealing companies and EM-suppliers).

Have a nice weekend!

Most importantly, the grids must be very clean before they are used to pick up sections. I routinely clean a new batch of grids with two changes of acetone followed by two changes of ethanol. Each change is done about 2 min in a beaker placed in a sonicator. Make sure the reagents are fresh and uncontaminated. After cleaning pour off the solvent and invert the beaker over filter paper and place in an oven at about 50 centigrade and allow to dry until the grids release from the top of the beaker and are completely dry on the filter paper. If necessary, additional adhesion of the section can be gained by lightly flaming the grid prior to collecting sections. To do this, wet the grid with clean distilled water and pass through a Bunsen burner flame until the water is gone. Then dip the grid in pure ethanol and rinse well in distilled water. Lastly, it is best to pick the sections up on the dull side of the grid as this can help adhesion. And finally if you are still having problems, once you have collected sections on grids, place in a 50 centigrade oven for 1 hour prior to staining. Using some or all of the above methods should definitely help you with section adhesion.

You ever tried a simple glow discharge of the grids, to make the foil more hydrophilic. This would be a more or less comparable effect as heating the foil. But the method is cleaner and better controlled.

What is the thickness of the sections? If the sections are too thick, they will came off as well.

Epon sections are self-adherent on copper grids as long as they are thin enough. Ultrathin sections of 20 - 100 nm are picked up from the water surface and dried. Even semithin sections up to about 0.5µm are self-adherent. But, if sections become thicker they might fly away in the microscope. In this case you can still use socalled sandwich grids, which are double copper grids with a lash for closure. Some electron microscopes allow electron transmission of semithin sections. in this case we use sandwich grids.

Carol's answer above is very useful I'm certain, but, for me, I always assume (naively maybe) that the grids are clean when I receive them from the supplier. Anyway, two things I would advise: (1) when I immunogold label sections, and they have to go through lots of immunoreagent changes etc., and are thus maybe liable to be lost, then I always use 'sticky' grids (the latter are made very simply and in seconds: simply place about 5" of sellotape in about 5ml of chloroform in a small glass tube, shake for a few seconds and then remove the sellotape - dip your grids in this solution briefly, then dry them down on filter paper, and you have your 'sticky grids'); (2) when it comes to rinsing after staining, I simply fill the wells of a 24-well microtitre plate to convexity with distilled water, and rinse the grids by floating, sections side down, on the top of the water (4 X c.5min for uranyl acetate; 5 X c.5min for Pb citrate). But remember to place the grids section side up when you remove them from the final rinse and dry them on filter paper, otherwise you will lose them for sure! Good luck :-)

When sections do come off the grids a reason might be that the grids were not rinsed well enough with alcohol 70% before using them, or the sections themselves are not smooth enough because of a bad knife.

Making sure the grid is clean is an important consideration; holding the grid in the forceps, we use a 5 second agitation in acetone followed by 5 seconds in ethanol and finally water.

Picking the sections up on the shiny side of grids is better than the dull side as there is more surface contact for adhesion.

The diagnostic staff in our unit also place the dried grids in a 50C oven for an hour,as suggested by Carol.

Finally, you may wish to check that your lead citrate is no too alkaline as this can sometimes cause problems.

With these procedures, we almost never have any problems with section loss.

Well, we don't have this sort of problems because, I think, we always cover the grid with a formvar film, then proceed for a carbon coating to harden this film; it is very adherent to sections and they remain firmly attached.

I have never experienced such a problem, however Formvar grids help. The proceure for staining is important, as Christopher wrote the pH of the lead citrate solution has to be controlled. I've never used a cleaning procedure for the grids. The thickness of the sections is of primary importance for the final results in TEM analysis, if this is not kept under control then all the rest is useless

In addition to clear explanations and suggestions to keep the epoxy resin thin (50-200nm thick) sections on copper grids, it is important to know the epoxy resin are hydrophobic and even after adhered as thin plastic sheet on the copper grid surface, can be released under hydrophilic repulsive action of water during handling. All previous explanations are right and should be taken in mind and two additional tricky points can improve the results. All EM grids (copper, nickel, gold, platinum, etc) have the matt/shiny sides and I recommend always use the matt rough surface side rather than the shiny smooth side to better stick the sections. Take special attention during washing steps by dribbled water and be sure the water drops is running parallel to the grid surface – never drop directly over the surface. The hydrophilic tension of water against hydrophobic resin can remove it if the drops hit the section from “back side”, or disrupt the sections if the resin was not well embedded in the samples and make small holes that easily disrupt the ultrathin section. Taking in account the sum of all well addressed compliance steps and procedure you will hopefully solve this issue.

The answer to the question in the title is hydrophobic forces. Bare metals and the resin sections are hydrophobic, so you are dealing with weak short range attractive forces. Once a nice flat section has made contact with a bare metal grid - or better one coated with a continuous sheet of hydrophobic formvar - the adhesion should be secure.

Amy - the main problems with your protocol seem to be your not ensuring the grids are clean (critical and pointed out by several others) and perhaps "dribbling" water on them for rinsing. That could help dislodge the sections especially if adhesion is already weak due to dirty grids. (Of course it's better the section fall off on your bench than in the EM). Good practice is to gently plop the grid (sections facing down) on a droplet of the stain solution and carefully transfer serially to the next drops of stain or rinse water.

Sections are hydrophobic. I have 3 little beakers line up. Before picking up sections, I pick up the grid with forceps and dip in 0.1 N HCl, then distiller water, finally 95-100% Ethanol. Then place grid on filter paper in a glass Petri dish. I have done this for over 25 years. Simple, and it works.

We used to clean the all prior to use with acetone, then drip old Formvar solution over them (separated on a piece of filter paper in a small glass Petri dish) and let them dry. I usually left them in the Petri dish and picked them up as I used them. They stayed clean and didn't stick to each other.

i generally clean grids by sonicating in acetone before using them. Another helpful material is the 'Quick Coat G' pen from EMS. It's a light adhesive that you can apply to the grids just before picking up sections. I find it useful if the sections are going to be run through a series of treatments before they get to the microscope. It's probably the same as the cellotape method mentioned above but a little more convenient. I find it especially useful when working with slot grids.

I would also support the suggestion of Dietmar, using glow discharge before taking up the sections. This not only makes the grids (coated or uncoated) more hydrophilic, it also cleans and sterilizes the surfaces. And those glow discharge units do not have to be bought in each case, sometimes the workshops facility at your institute should be able to build something comparable, included in a small vacuum chamber.

Andreas, do you want the grids more hydrophillic? The biological expoxy-based sections are hydrophobic. Maybe you are doing other types of microscopy.

I think Amy has her answer and we may be going off into a tangent and even confusing matters a bit. Forces holding the sections to grid are hydrophobic. If Amy follows standard protocols and the helpful tips re how to ensure the grids are clean, she'll be fine True that all this is taking place in water, and surface tension can create problems as well as solve some (spreading of the sections at the air-water interface is what irons out the wrinkles). But Amy didn't say she was having problems enticing the sections onto the grids, just keeping them there. : )

This is something I never had a problem with until last week. I had not sectioned for a few years but knew that I should always wash the grids well between and after staining. I was using a different wash bottle than before. I actually washed most of my sections off the grid! First time ever! I use curved forceps and wash the water over the forceps rather than hitting the sections directly. However, the water was too forceful and washed more than stain off the grids. Therefore, be careful in washing the grids with staining. It appears that I am washing the forcep tips rather than the grid, but the water washes over the sections. The water stays in between the tips of the forceps, but a use filter paper, cut in pie shaped sections, and gently put the filter paper sections between the forceps tips to wick the water away, then gently push the grids onto the filter paper. Hopefully this answers Amy's original question.

The technique I use is to pass the copper grids over a flame on an alcohol lamp. Although this weakens the grid, it makes it easier to pick up the sections in the water trough. I've tried many of the other methods mentioned already, and they work, but take more time.

We always clean or grids our grids in an ultrasonic cleaner using the following solution: 7% acetic acid + 1% tween 80 + 92% dH2O clean for 1 min, followed by 3 rinses in dH2O (1 min ea) followed by 3 rinses in acetone (1 min ea). I use a loop to put my sections on grids then I allow the sections 1-2 h to dry down before I post-stain. When post-staining, I gently dip my grids into several beakers of dddH2O. By using the above techniques, I haven't lost sections in years.

@ Elizabeth: I think I explained it the wrong way. I mean, when you try to fish the sections, it helps to have hydrophilic grids, because you can put them under water much easier.

As was already mentioned, grids should be clean and hydrophilic. Then, when drying, water surface tension force can push section against a grid hard enough to engage van der Waals forces – the main cause of section adhesion to a grid.

So far I never had detached sections. May be my “protocol” will be useful for somebody.

I never use coated grids unless it is absolutely necessary (for mineralized tissue, of when edges of grids should be observed). Coated grids I always treat with glow discharge to make them hydrophilic (some of spatter coaters have glow discharge mode; when buying spatter coater I chose one with glow discharge). Copper grids: dip in 2M HCL, dip in acetone, dip in di water, catch section, and let it dry completely before staining. After staining I was grids in two beakers (200 ml each, plenty of water). I wave forceps with a grid for 15 sec in each of beakers. Works fine so far. Dripping water along a grid looks a bit complicated and disruptive for me.

Dear Posters, this issue IMHO is really an old one and in former times also was intensively discussed by our predecessors in EM-techniques. It seems that the problem arises sometimes and not only the causes but also the sources how to overcome mostly are hidden as usual. But I am following now a very interesting and huge (and fast) reply thread on this and am happy about the many posters working with T(EM).

The problem: as was stated above, all previous explanations are right, more or less, but since we don’t know exactly whether Amy uses formvar-mounted grids or just picks up the ultrathin sections onto naked grids we might guess a little bit what the real culprit has been.

I agree with several posters about the “forces” which will be responsible for a good adherence to the copper grid (and normally these forces will hold the sections in their position after drying on the grid before staining them). Since meanwhile I am writing this there have been other postings too, I have to reply to the post of Elizabeth Martin concerning the use of a different wash bottle and another type of forceps, leading to floating off of sections. I have never seen this (32 years in doing staining ultrathin staining right that way she described) doing staining the sections always manually. So IMHO there are some points I will pull out of the discussion so far:

• Cleanliness of grids (and therefore doing such right, many possibilities and personal choices have been given. Usually we don’t know how long it took from fabrication to selling and usually you’ll purchase 3-5 packages of 100 grids each, so prolonged storage – on air – which will cause oxidation less to severe, but we will not see this macroscopically, sometimes there might be a slight oily film-deposit etc.),

• Choice of suited tweezers (antimagnetic), ‘shoes [mostly non insulating rubber sole] and personal protective cloth’ [synthetic lab coat material] - with the possibility of electrostatic loading when handling grids at the EM or at staining…. have you seen copper grids leaping aside when trying to fix the grids in between? I have, several times….)

• Placing steps and adhering sections to the grid (with or without formvar-foil mount) INCLUDING definite drying of the section unto the grid (at least wicking grids/sections with filter paper as described)

• Washing steps (dipping or “jet-stream-washing out of a squeeze-wash bottle, wicking again and afterwards more/less “dry” grids supposed to be stored until the next step into a grid-box, eventually drying again within a laminar air flow cabinet).

Since others have posted their methods for cleaning grids, I add here also perhaps interesting information:

Summary of MSA-Listserver-posts (2006, no mentioning of use of ultrasonic treatment): cleaning solutions used:

• conc. NaOH solution (some seconds) – -A.dest OR:

• 3% HCl in 95-96% EtOH(30-40 sec) – - wash in 95% EtOH --- A.dest OR:

• Household (NH3+) hydrous ammoniacal solution, 30 min-1 h, grids shiny/glossy --- A.dest. OR:

• flame of an alcohol burner (short) OR:

• 5-10% hydrochloric acid solution – gently shake – A. dest. Ethanol, Acetone, chloroform and/ or petroleumbenzine. OR:

• Just before use: dip grid into 1 N HCl, Acetone – A. dest. – use / mount sections immediately.

Short version: EM-Laboratory, Pathology, Salzburg (Austria):

cleaning of (old) Formvar-mounted grids:

• (1)Chloroform 100%, ultrasonic treatment ( 2 bathes, 3-5 min each)

• (2)Acetone (following, for fat-removal ( 1-2 bathes, 3-5 min each))

• (3)20 ml of 2N HCl + 96%EtOH ad 100 ml EV Ultrasonic bath 10-15 seconds until grids become shiny and glossy (not longer, since HCl tackles/pits copper)

• (4)Eventually neutralize by 1N NaOH, or NH3 (hydrous ammoniacal solution diluted (5-10 min, or concentrated1-3 min)

• (5)A. dest. (several times)

• (6)Afterwards shortly Acetone followed by a petroleumbenzine bath,

• (7)Dry from glass beaker over a sheet of lens paper

cleaning of un-mounted (naked) grids from package: starting from (3) above.

As to the question: “Clean” or “Not to clean” see e.g. Special grid cleaning procedures: cf.

http://www.2spi.com/catalog/grids/grid-cleaning.html

and: To clean or not to clean TEM grids before use cf.

http://www.2spi.com/catalog/grids/cleaning-of-TEM-grids-before-use.html

(no financial interest, no affiliations, there might also be information on this matter from other dealing companies and EM-suppliers).

Have a nice weekend!

Sections must always be positioned vertically and never horizontally when ever we proceed to the contrast of copper grids. Thus, the sections do not separate. Even the movement to immerse the copper grids must be vertical.

Are you sure you do not loose the section while you washing with strong dropping water?

Thank you for all the comments and advice. We work with uncoated copper grids. The problem was that my PI used 300 mesh grids which had been sitting for at least a year. I had washed 200 mesh grids and those worked fine. I have learned much from this discussion.

If you follow Elizabeth M Martin and Renee Lopez-Smith procedures you will no lost section any more.

First you have to prepare the grides to be coated with formvar film and dry by putting inverted on filterpaper in a petridish. then before woorking you can fitch the section on the coated grides and it will never droe especialy you have to leave it to dry befor staining

S i assure you it will never droped again by staining must be in a petridish having wax embeded on its bottom and the stain must be put on the surface as a drope so the gride will be floated on the surface of the stain and washeng will be by putting the grid on the back and the washing solution will be sucked by the filter paper

Several factors are involved in the detachment of ultra-thin sections from grid surface! The sections remain more firm if mounted on the dull side of the grid. Thicker sections, more than 100nm, tend to get detached while washing. Mesh size of grid also matters, sections remain more stable if mesh size is 300 or more. The size of sections also interfere with the stability of sections, larger sections are supported by many grid bars, therefore remain more stable. Finally, washing should be gentle, rather than using a jet of water better rinse gently several times (30 sec) in a beaker of water by holding grids at their rim with tweezers.