Hi Gerrit, how are you?

I would also like to have a killer protocol for negative staining of liposomes. Here are some considerations from my guessing and our experience:

* There may not be such a thing as "pure" liposomes. The charge of lipids will contribute to the binding, so if you can afford adding some charged lipids, that should help. If you do not need to use a synthetic lipid mixture, the Folch fraction is a good starting point.

* If you do not need the albumin for the experiment, you may want to leave it out, as it will bind to lipids and change the morphology of your liposomes, like you observed.

* We used Formvar/Carbon Coated grids and did not have a problem with poor binding.

* We used 20 sec 2% phosphotungstic acid (PTA) for negative staining and had ok results (with still room for improvement).

Hope this is helpful, all the best, Sven

Hi Sven,

thanks for the advice, especially on Formvar and PTA. So far have used 2 % uranly acetate on carbon coated grids.

With "pure liposomes" I was refering to synthetic liposomes (e.g. DOPC/DOPS) without any proteins. Due to the PS they were charged. We also tried Folch extract and other synthetic compositions (+/- SM, PIP2, PE) but with similar results, i.e. almost no liposomes at all on the grid if we left out the albumin. In the same experiment we used dynamin as a positive contro and saw nice decorated and tubulated liposomes in the absence of albumin.

Best

Gerrit

Hi Gerrit, 

I found avoiding excessive treatment of liposome sample is essential. Spot 20 ul of liposomes in phosphate-free buffer onto a grid, glow-discharged for 30 seconds. Allow 15-30 seconds for adsorption, then blot sample away and transfer it into a drop of 1%UA for 30 seconds. Blot contrasting agent and air dry the grid. 

I would suggest trying higher concentrations of liposomes, up to 0.5 mg/ml.

You can render grid's surface hydrophilic using poly-L Lysine.

I also agree with Sven's notion about liposome's charge. I consistently see higher on-greed recovery for charged liposomes. 

Hope this helps.

Best wishes, 

Oleksiy

Unfortunately the TEM analysis of liposomes suffer very often from the presence of artifacts, The current litterature is plenty of pictures showing anything but no liposome at all.

I strongly suggest to find a collaboration with a research group performing cryo-TEM or SEM analysis after freeze fracture.

I agree with Claudio Nastruzzi, collaborate with a cryo-TEM group and image your liposomes unstained in the frozen hydrated state. With conventional TEM you will always be biased if you are interested in size or form because they will deform due to adsorption and your negative stain (uranyl salts, phosphotungstic acid, methylamine-vanadate  etc. ) will add to the size. If you just want to identify your liposomes e.g. in a formulation you may try ruthenium-stains which are quite specific for membrane lipids such as phosphatidyl-cholines 

I agree with both Werner and Claudio. I have looked at liposomes for a couple of groups and now always use cryo-EM. Although the grid preparation is more difficult its relatively straight forward and since you are not after high resolution (more measuring diameter) you can collect at more modest magnification to increase your field of view.  Grid preparation and data collection can be done in a day. We find a number of artefacts in negative staining for liposomes. 

Can someone please provide articles or links to articles on the protocol of negative staining of liposomes and actually getting 'satisfying' results. Thank you in advance.

Dear all,

I would kindly revisit this thread. In particular, I found this paper that has been published http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2999936/?tool=pmcentrez

showing the staining of liposomes that are commercially purchased. If you dig deep enough, they also made a JOVE video  see here

http://www.jove.com/video/51087/optimized-negative-staining-high-throughput-protocol-for-examining

The author describes its method using uranyl formate - but from what I understand, it's the uranyl ion that binds to the liposomes/proteins to cause electron to scatter in TEM - so does the formate or acetate really make such a difference in morphology?

To Gerrit; yes, I also observed those weird deformed stuff in self-extruded liposomes (as attached). I have no experience in DLS.

I also tried negative staining with osmium tetraoxide (vapour) at 200-fold dilution - I could not see any liposomes on my grids.

Still desperately trying again with osmium tetraoxide - but I think I almost feel like I should give up on neg-staining and invest in cryoEM somewhere instead.

Best,

http://www.jove.com/video/51087/optimized-negative-staining-high-throughput-protocol-for-examining

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2999936/?tool=pmcentrez

Here is an image of a commercial liposome preparation in negative staining using 2% UAc which shows that successful negative staining can be achieved but will of course depend on several things such as the type of liposomes. The preparation was done on a glow discharged commercial formvar stabilized 400 mesh carbon grid. In my experience care should be taken finding the optimal "on-grid" concentration and also how the stain is blotted off i.e. too thick stain embedding leads to deformation and too thin results in dehydration effects and flattening.

Lars Haag, this is a very nice image.

Have you tried your protocol on other non-commercially isolated liposomes?

If possible, could you please send me the detailed protocol that you used for staining these liposomes in the image?

All the best

Lars Haag, could you please send me too the detailed protocol regarding the staining of these liposomes? Also, could you mention the size of these liposomes?

Thank you,

Anca

Lars Haag Thank You for the nice micrographs. This indeed looks great, almost all vesicles seem to be intact. Is there any way you could provide us with the (staining) protocol? Thank You a lot in advance.

I found an image of liposomes recorded with cryo-EM (credit: Department of Pharmaceutical Technology and Biopharmacy, University of Freiburg), which looks very similar, but apart from the fact that all vesicles are intact as well, they kept their round / spherical shape. In the NS image most vesicles experienced some deformation and are not perfectly round anymore, obviously.

Lars Haag , could you please also send me too the detailed protocol regarding the staining of these liposomes?

Thanks

Sybil