<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Hi Reza,<div class=""><br class=""></div><div class=""><br class=""></div><div class="">Fred mentioned most of important tips we figured out. In addition to that, most of liposomes made of phospholipids tends to adsorb hydrophilic carbon film, grow discharged with air. As you can imagine, if you prepared a grid without thin carbon support, most of vesicles tends to adsorb thick carbon support film. So that you do need high concentration of liposomes to make them go into the holes. If you glow discharge under amyl amine, vesicles are not that much sticky to carbon film. This is one way to reduce the requirement of “high” concentration of liposomes. </div><div class=""><br class=""></div><div class="">You can test what is going on with SUV by sonication. This is most effective and easiest way to start with. Be sure to make transparent solution with socnication. 10mg/ml will be good lipid concentration to start with. If you have to make larger than 100 nm liposomes, you can make them with extruder. But most probably, you will see deformed, not circular vesicles because of the ice thickness.</div><div class=""><br class=""></div><div class="">As Fred mentioned, using thin carbon film is what we are doing for proteliposomes because the concentration is somehow limited after reconstitution. Don’t try to concentrate with ultracentrifuge. Most cases, pelleted liposomes are tends to sticky each other and the effective concentration will be reduced after that. If you concern about the noise from thin carbon film, you can try graphene oxide as well. Even though, it is not that easy to have good coverage of graphene oxide even with the best protocol so far we have.</div><div class=""><a href="https://figshare.com/articles/Graphene_Oxide_Grid_Preparation/3178669" class="">https://figshare.com/articles/Graphene_Oxide_Grid_Preparation/3178669</a></div><div class=""><br class=""></div><div class=""><br class=""></div><div class="">Hideki</div><div class=""><br class=""></div><div class=""><div class="">
<div class="">-----<br class="">Hideki Shigematsu Ph. D.<br class=""><br class="">Senior Scientist<br class="">RIKEN Center for Life Science Technologies</div><div class="">1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 Japan</div><div class="">Phone: +81-45-503-9204</div><div class=""><br class=""></div><div class=""><br class=""></div><br class="Apple-interchange-newline">
</div>
<br class=""><div><blockquote type="cite" class=""><div class="">2017/05/13 5:01、Sigworth, Frederick <<a href="mailto:fred.sigworth@yale.edu" class="">fred.sigworth@yale.edu</a>> のメール：</div><br class="Apple-interchange-newline"><div class="">
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" class="">
<div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">
Hi, Reza,
<div class=""><br class="">
</div>
<div class="">With Hideki Shigematsu we’ve done a lot of imaging of liposomes, and here are some more observations to add.</div>
<div class=""><br class="">
</div>
<div class="">1. The particular lipid makes a big difference. PC makes small liposomes (20-30 nm), PE makes big ones (1000 nm, obvious from the milky-looking suspension). Use lipids above their transition temperature. We like POPC, and even better, POPC+cholesterol.</div>
<div class="">2. The way you form them makes a difference. We dry the lipids, then hydrate them overnight (yields big, multilamellar structures) then sonicate (this gives SUVs) but then we solubilize in detergent and later remove detergent because we’re reconstituting
protein too.</div>
<div class="">3. Extrusion doesn’t work quite as expected. If we make big PE liposomes and extrude them, either 30nm or 50 nm filters give us 100 nm and larger liposomes. We prefer controlling size with lipid composition. Dynamic light scattering can give
a rough measure of size.</div>
<div class="">4. We use a thin carbon film spanning holes for liposomes to adsorb to. In the final prep applied to the grid the overall lipid concentration is a few mM.</div>
<div class=""><br class="">
</div>
<div class="">Fred Sigworth</div>
<div class=""><span style="orphans: 2; text-align: -webkit-auto; widows: 2;" class=""><br class="">
</span></div>
<div class=""><span style="orphans: 2; text-align: -webkit-auto; widows: 2;" class="">Professor</span></div>
<div class="">
<div class="">
<div style="font-family: Helvetica; font-style: normal; font-variant-ligatures: normal; font-variant-position: normal; font-variant-caps: normal; font-variant-numeric: normal; font-variant-alternates: normal; font-variant-east-asian: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">
Cell Molec Physiology</div>
<div style="font-family: Helvetica; font-style: normal; font-variant-ligatures: normal; font-variant-position: normal; font-variant-caps: normal; font-variant-numeric: normal; font-variant-alternates: normal; font-variant-east-asian: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">
<span style="text-align: -webkit-auto;" class="">Molec Biophys and Biochem</span></div>
<div style="font-family: Helvetica; font-style: normal; font-variant-ligatures: normal; font-variant-position: normal; font-variant-caps: normal; font-variant-numeric: normal; font-variant-alternates: normal; font-variant-east-asian: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">
Yale University<br class="">
</div>
</div>
<div class=""><br class="webkit-block-placeholder">
</div>
<div class=""><br class="">
</div>
<div class="">
<blockquote type="cite" class="">
<div class="">On May 12, 2017, at 3:39 PM, Kenneth Goldie <<a href="mailto:k.goldie@unibas.ch" class="">k.goldie@unibas.ch</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div class="">Dear Reza,<br class="">
<br class="">
a quick indication with having a good concentration of liposomes for making cryo grids with a reasonable dispersion is to have a solution that looks pretty milky. As white as milk is good! Adsorb undiluted. If not try to get the concentration up. Best results
work with liposomes of 100nm or less. Ideally they should be in water or low salt buffer. Avoid buffers with any additives as much as possible. No ethanol for example.<br class="">
My feeling is the liposomes sit nicely dispersed in the buffer drop when you add it to a glow discharged grid, and float there like balloons and do not settle as easily as say single particle protein type samples. The key is to adsorb for much longer times
than "usual". I usually use 5 minutes adsorbing on-grid in tweezers on a bench and not in the cryo-plunger chamber. If you still have problems or cannot increase the sample concentration you can adsorb for an hour or more or even overnight as long as you
keep the grid "wet". This is easily done by using a large petri dish that you can "humidify" by wetting several pieces of round filter paper in the bottom with water and add a lid. Place your grid with sample in there for the desired time (also sometimes helps
for low concentrations of protein samples, filaments or planar crystals). Then take it out and cryo-plunge. Being able to plunge from the backside (opposite the side you adsorbed on) can also up the concentration sometimes b<br class="">
y pulling the liposomes down onto the surface and sometimes producing a filter effect to get more in holes. They may tend to stick and pull off on the filter paper if you blot from the sample adsorbed side.
<br class="">
Holey films will result in more sample in holes as there is less background carbon (same goes for proteins!) for them to stick to. Even in low magnification, you should see the liposome spheres if you defocus a lot which helps you pinpoint them.<br class="">
<br class="">
Best of luck<br class="">
<br class="">
Ken<br class="">
<br class="">
-----------------------<br class="">
<br class="">
Ken Goldie, PhD<br class="">
Facility Manager, C-CINA, Biozentrum, University Basel<br class="">
Mattenstrasse 26 | D-BSSE | WRO-1058 | CH-4058 Basel | Switzerland<br class="">
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| Tel. +41-61-387 32 18| eMail. <a href="mailto:k.goldie@unibas.ch" class="">k.goldie@unibas.ch</a><br class="">
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1. Re: Liposomes in ice (Terje Dokland)<br class="">
<br class="">
<br class="">
----------------------------------------------------------------------<br class="">
<br class="">
Message: 1<br class="">
Date: Fri, 12 May 2017 12:00:20 -0500<br class="">
From: Terje Dokland <<a href="mailto:dokland@uab.edu" class="">dokland@uab.edu</a>><br class="">
To: Reza Khayat <<a href="mailto:rkhayat@ccny.cuny.edu" class="">rkhayat@ccny.cuny.edu</a>><br class="">
Cc: 3dem <<a href="mailto:3dem@ncmir.ucsd.edu" class="">3dem@ncmir.ucsd.edu</a>><br class="">
Subject: Re: [3dem] Liposomes in ice<br class="">
Message-ID: <<a href="mailto:842FF5C0-9F1E-43BF-9110-E7D234E089D8@uab.edu" class="">842FF5C0-9F1E-43BF-9110-E7D234E089D8@uab.edu</a>><br class="">
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<br class="">
Maybe you didn't get that many liposomes in the first place? We have had similar problems with extruded liposomes. It seemed that a lot of the lipid did not form liposomes at all, but remained in micellar form, contaminating the background.<br class="">
<br class="">
On May 12, 2017, at 9:30 AM, Reza Khayat wrote:<br class="">
<br class="">
<blockquote type="cite" class="">Hi,<br class="">
<br class="">
We are preparing extruded liposomes (200nm). Problem is we're getting very few liposomes in ice. The starting lipid concentration (DOPG:<a href="https://avantilipids.com/product/840475/" class="">https://avantilipids.com/product/840475/</a>) is 4mg/ml.<br class="">
<br class="">
Reza<br class="">
<br class="">
Reza Khayat, PhD<br class="">
Assistant Professor<br class="">
City College of New York<br class="">
Department of Chemistry<br class="">
New York, NY 10031<br class="">
From: Ludtke, Steven J <<a href="mailto:sludtke@bcm.edu" class="">sludtke@bcm.edu</a>><br class="">
Sent: Friday, May 12, 2017 10:27 AM<br class="">
To: Reza Khayat<br class="">
Subject: Re: [3dem] Liposomes in ice<br class="">
<br class="">
You'll have to define exactly what you mean by liposomes. Extruded SUVs are one of our favorite test/training specimens because they are so easy to prepare and freeze. When most people say liposome, they mean something much larger. You'll need to give some
details if you want useful advice. What are the lipid constituents, how large are the liposomes, how are they prepared, etc. Would also be useful to see a representative image or two.<br class="">
<br class="">
<blockquote type="cite" class="">On May 12, 2017, at 9:18 AM, Reza Khayat <<a href="mailto:rkhayat@ccny.cuny.edu" class="">rkhayat@ccny.cuny.edu</a>> wrote:<br class="">
<br class="">
Hi,<br class="">
<br class="">
We're trying to visualize liposomes in ice but are having some serious difficulty. Can anyone suggest some tricks that may be helpful? Thanks.<br class="">
<br class="">
Best wishes,<br class="">
Reza<br class="">
<br class="">
Reza Khayat, PhD<br class="">
Assistant Professor<br class="">
City College of New York<br class="">
Department of Chemistry<br class="">
New York, NY 10031<br class="">
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