Ideas for good, unusual labels for fixed cells?

[Steffen Dietzel]

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Dear listers,

we are about to set up a multi-color experiment as proof of principle in
formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.

We are planning on DNA stain, phalloidin as actin stain, some GFP label
(amplified with nanobodies), and two primary antibodies from rabbit and
mouse, e.g. for mitochondria , ER, nuclear pores or the like with the
fluorochrome on the secondary ab. The actual labeled structures do not
matter so much, as long as they give good signals of specific structures
(i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from
goat (or donkey)

We would like to stain one or two additional structures, but we are not
sure how to label them. Antibodies from rat have a tendency to
cross-react with mouse antibodies, that would not be ideal. (to be
precise: the secondary antibody cross reacts to the other species, of
course.)

Does anybody have some ideas for additional good labels? An antibody
from chicken (or other species) that nicely delineates the Golgi, ER,
Mitochondria, focal adhesion points, nuclear pores, splicing factors,
etc, would be great.

Any good antibody with a cellular target where the secondary will not
cross react with mouse or rabbit ab would be of interest. Or some good
non-antibody labeling of cellular structures, like phalloidin, just for
something other than actin or DNA, that gives decent signals in fixed
cells.

Of course on could go for labeling of various primaries from the same
species. But antibody labeling is not exactly our core competence and it
would add one more layer where things can go wrong. Plus, we might not
get the fluorochromes that we plan to use.

Hoping on your experience

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Sylvie Le Guyader]

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Hi Steffen



Does your scientific question (i.e. what do you want to measure in the images) requires you to label DNA and actin? If you do not specifically need them for your analysis, you can skip labelling them and use these channels for something else.

Mitochondria can be labelled with dyes without using antibodies.

Why do you need to counter stain GFP?



Multiplexing is another option. See this paper: https://pubmed.ncbi.nlm.nih.gov/30072512/



Med vänlig hälsning / Best regards



Sylvie



@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Blickagången 16,

Room 7362 (lab)/7840 (office)

14157 Huddinge, Sweden

mobile: [phone]

LCI website

Follow our microscopy blog!



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel
Sent: 04 December 2020 14:53
To: [hidden email]
Subject: Ideas for good, unusual labels for fixed cells?



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Dear listers,



we are about to set up a multi-color experiment as proof of principle in formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.



We are planning on DNA stain, phalloidin as actin stain, some GFP label (amplified with nanobodies), and two primary antibodies from rabbit and mouse, e.g. for mitochondria , ER, nuclear pores or the like with the fluorochrome on the secondary ab. The actual labeled structures do not matter so much, as long as they give good signals of specific structures (i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from goat (or donkey)



We would like to stain one or two additional structures, but we are not sure how to label them. Antibodies from rat have a tendency to cross-react with mouse antibodies, that would not be ideal. (to be

precise: the secondary antibody cross reacts to the other species, of

course.)



Does anybody have some ideas for additional good labels? An antibody from chicken (or other species) that nicely delineates the Golgi, ER, Mitochondria, focal adhesion points, nuclear pores, splicing factors, etc, would be great.



Any good antibody with a cellular target where the secondary will not cross react with mouse or rabbit ab would be of interest. Or some good non-antibody labeling of cellular structures, like phalloidin, just for something other than actin or DNA, that gives decent signals in fixed cells.



Of course on could go for labeling of various primaries from the same species. But antibody labeling is not exactly our core competence and it would add one more layer where things can go wrong. Plus, we might not get the fluorochromes that we plan to use.



Hoping on your experience



Steffen



--

------------------------------------------------------------

Steffen Dietzel, PD Dr. rer. nat

Ludwig-Maximilians-Universität München

Biomedical Center (BMC)

Head of the Core Facility Bioimaging



Großhaderner Straße 9

D-82152 Planegg-Martinsried

Germany



https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioimaging.bmc.med.uni-muenchen.de%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=iyY%2FMZT%2FlYRNz8JaFLGfad0cJ%2FDS3prHXLvhYOuOh3w%3D&amp;reserved=0



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[ICIT]

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Dear Steffen,

if there's only need to get some structures to the specimen how about using some live dyes, like MitoTracker Red, LysoTracker, live-tubulin, there are quite a few nice new ones around in Thermo shop.

Alternatively direct labeling of primaries?

Cheers,

Mika

[Rhonda Powell]

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Dr Dietzel,

I’ve been working on 6 and 7 color slides and while they’re certainly not perfect (yet), I’d definitely be willing to talk more about what I have in the works!

Rhonda Reigers Powell
[hidden email]
Research Lab Manager
Clemson Light Imaging Facility
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Steffen Dietzel <[hidden email]>
Sent: Friday, December 4, 2020 9:07 AM
To: [hidden email]
Subject: Ideas for good, unusual labels for fixed cells?

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Dear listers,

we are about to set up a multi-color experiment as proof of principle in
formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.

We are planning on DNA stain, phalloidin as actin stain, some GFP label
(amplified with nanobodies), and two primary antibodies from rabbit and
mouse, e.g. for mitochondria , ER, nuclear pores or the like with the
fluorochrome on the secondary ab. The actual labeled structures do not
matter so much, as long as they give good signals of specific structures
(i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from
goat (or donkey)

We would like to stain one or two additional structures, but we are not
sure how to label them. Antibodies from rat have a tendency to
cross-react with mouse antibodies, that would not be ideal. (to be
precise: the secondary antibody cross reacts to the other species, of
course.)

Does anybody have some ideas for additional good labels? An antibody
from chicken (or other species) that nicely delineates the Golgi, ER,
Mitochondria, focal adhesion points, nuclear pores, splicing factors,
etc, would be great.

Any good antibody with a cellular target where the secondary will not
cross react with mouse or rabbit ab would be of interest. Or some good
non-antibody labeling of cellular structures, like phalloidin, just for
something other than actin or DNA, that gives decent signals in fixed
cells.

Of course on could go for labeling of various primaries from the same
species. But antibody labeling is not exactly our core competence and it
would add one more layer where things can go wrong. Plus, we might not
get the fluorochromes that we plan to use.

Hoping on your experience

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

https://urldefense.proofpoint.com/v2/url?u=http-3A__www.bioimaging.bmc.med.uni-2Dmuenchen.de&d=DwIDaQ&c=Ngd-ta5yRYsqeUsEDgxhcqsYYY1Xs5ogLxWPA_2Wlc4&r=RdGdqH-W1QDLa4-EXdnHz2R7U86lC-mLh-KZ0_bf2Qc&m=lE48QQlW3OxcyRhqAk7yfNGWA3c7daXS07men34IDRU&s=nmZR4747sJ-3OksMNjcgY8VnRJUKgozzAm5UO6l3ZlE&e=

[Zdenek Svindrych-2]

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Hi Steffen,

Can you transfect for more stuff, e.g. BFP, mPum, etc..? Or Halo-tag,
Snap-tag?
Or you can switch to chicken secondaries (chicken anti-rat, chicken
anti-rabbit, chicken anti-goat)...
As mentioned, there are goot ER/lyso/mito/whatever tracker dyes,
SiR-tubulin (and others). There are also membrane-specific dyes.

Directly labeling primaries may be risky / expensive (if something goes
wrong and you lose your protein), but some are commercially available.
People have also tried pre-incubating the primary ab with labeled protein A
(protein G), see
https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cbic.201800743

Best, zdenek

On Fri, Dec 4, 2020 at 9:07 AM Steffen Dietzel <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear listers,
>
> we are about to set up a multi-color experiment as proof of principle in
> formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.
>
> We are planning on DNA stain, phalloidin as actin stain, some GFP label
> (amplified with nanobodies), and two primary antibodies from rabbit and
> mouse, e.g. for mitochondria , ER, nuclear pores or the like with the
> fluorochrome on the secondary ab. The actual labeled structures do not
> matter so much, as long as they give good signals of specific structures
> (i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from
> goat (or donkey)
>
> We would like to stain one or two additional structures, but we are not
> sure how to label them. Antibodies from rat have a tendency to
> cross-react with mouse antibodies, that would not be ideal. (to be
> precise: the secondary antibody cross reacts to the other species, of
> course.)
>
> Does anybody have some ideas for additional good labels? An antibody
> from chicken (or other species) that nicely delineates the Golgi, ER,
> Mitochondria, focal adhesion points, nuclear pores, splicing factors,
> etc, would be great.
>
> Any good antibody with a cellular target where the secondary will not
> cross react with mouse or rabbit ab would be of interest. Or some good
> non-antibody labeling of cellular structures, like phalloidin, just for
> something other than actin or DNA, that gives decent signals in fixed
> cells.
>
> Of course on could go for labeling of various primaries from the same
> species. But antibody labeling is not exactly our core competence and it
> would add one more layer where things can go wrong. Plus, we might not
> get the fluorochromes that we plan to use.
>
> Hoping on your experience
>
> Steffen
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Steffen Dietzel]

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Hi all,

thanks for your fast answers, this list is amazing.

Sylvie, we are not having a biological-function-type of scientific
question with this, the goal is to show that we can use a certain
(hopefully large) number of fluorochromes without too much cross-talk.
In this scheme, the DNA stain and actin label are/carry two of these
fluorochromes. So yes, we need them. Or label something else instead.
The problem for once is not the number of fluorescent channels for
excitation/detection, but how to attach so many different labels without
cross-labeling (cross-labeling as in anti-rat also detects mouse-ab). We
have shown for the fluorochromes we plan to use that they work in single
or two color stains. Now we are trying to combine them all.

The GFP does not deal well with some of the procedures we apply to that
preparation. The nanobodies allow us to tag it with an organic
fluorochrome without 'wasting' mouse or rabbit as primary antibody species.
Zdenek, I expect we would have the same problem with additional/other
FPs, thus that is not very promising

Right now we seem to be able to combine the following five labels

1 - DNA
2-Actin
3-GFP-nanobody (say, lamin-GFP)
4-primary mouse ab plus secondary from goat (e.g. for nuclear pores)
5-primary rabbit ab plus secondary from goat (eg. for focal adhesions)

what could be number 6 (eg. for mitochondria)? and possibly 7 (ER,
Glogi, ...)? The first thing that came to mind was another species for
the primaries where the secondary very unlikely cross-targets mouse and
rabbit antibodies, therefore the mention of chicken in the previous
mail. But we are not aware of a chicken antibody against a cellular
target, plus catalogs don't really tell you how good they perform.
Therefore, tapping into the knowledge of this list seems wise.

Sylvie, Mika, Zdenek: Mitochondria labeling without ab - Tracker dyes,
SiR-Tubulin, etc: My limited experience with tracker dyes is that they
do not perform very well on fixed cells. (Except for actin stains, but
we already have actin). Are some of those dyes fixable or can be applied
in fixed cells? Any recommendations? It might work, if the fluorochrome
properties fit with the rest of our scheme.

The multiplexing paper Sylvie linked uses sequential labeling with
elution/removal of the antibodies inbetween. But our point is to use
many fluorochromes at the same time.

Zdenek, direct ab labeling could possibly work. But as you write, it is
high risk, especially without experience. So I'd rather avoid it.
Commercially available ones, I didn't find any with good cellular
targets. But maybe I was looking at the wrong places?

Best

Steffen

Am 04.12.2020 um 15:23 schrieb Sylvie Le Guyader:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Steffen
>
>
>
> Does your scientific question (i.e. what do you want to measure in the images) requires you to label DNA and actin? If you do not specifically need them for your analysis, you can skip labelling them and use these channels for something else.
>
> Mitochondria can be labelled with dyes without using antibodies.
>
> Why do you need to counter stain GFP?
>
>
>
> Multiplexing is another option. See this paper: https://pubmed.ncbi.nlm.nih.gov/30072512/
>
>
>
> Med vänlig hälsning / Best regards
>
>
>
> Sylvie
>
>
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader, PhD
>
> Live Cell Imaging Facility Manager
>
> Karolinska Institutet- Bionut Dpt
>
> Blickagången 16,
>
> Room 7362 (lab)/7840 (office)
>
> 14157 Huddinge, Sweden
>
> mobile: [phone]
>
> LCI website
>
> Follow our microscopy blog!
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel
> Sent: 04 December 2020 14:53
> To: [hidden email]
> Subject: Ideas for good, unusual labels for fixed cells?
>
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
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> Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=wQTXNyPORAs64aCBls305ZPRU9QsDnZtKp%2BalYsUQyA%3D&amp;reserved=0 and include the link in your posting.
>
> *****
>
>
>
> Dear listers,
>
>
>
> we are about to set up a multi-color experiment as proof of principle in formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.
>
>
>
> We are planning on DNA stain, phalloidin as actin stain, some GFP label (amplified with nanobodies), and two primary antibodies from rabbit and mouse, e.g. for mitochondria , ER, nuclear pores or the like with the fluorochrome on the secondary ab. The actual labeled structures do not matter so much, as long as they give good signals of specific structures (i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from goat (or donkey)
>
>
>
> We would like to stain one or two additional structures, but we are not sure how to label them. Antibodies from rat have a tendency to cross-react with mouse antibodies, that would not be ideal. (to be
>
> precise: the secondary antibody cross reacts to the other species, of
>
> course.)
>
>
>
> Does anybody have some ideas for additional good labels? An antibody from chicken (or other species) that nicely delineates the Golgi, ER, Mitochondria, focal adhesion points, nuclear pores, splicing factors, etc, would be great.
>
>
>
> Any good antibody with a cellular target where the secondary will not cross react with mouse or rabbit ab would be of interest. Or some good non-antibody labeling of cellular structures, like phalloidin, just for something other than actin or DNA, that gives decent signals in fixed cells.
>
>
>
> Of course on could go for labeling of various primaries from the same species. But antibody labeling is not exactly our core competence and it would add one more layer where things can go wrong. Plus, we might not get the fluorochromes that we plan to use.
>
>
>
> Hoping on your experience
>
>
>
> Steffen
>
>
>
> --
>
> ------------------------------------------------------------
>
> Steffen Dietzel, PD Dr. rer. nat
>
> Ludwig-Maximilians-Universität München
>
> Biomedical Center (BMC)
>
> Head of the Core Facility Bioimaging
>
>
>
> Großhaderner Straße 9
>
> D-82152 Planegg-Martinsried
>
> Germany
>
>
>
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioimaging.bmc.med.uni-muenchen.de%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=iyY%2FMZT%2FlYRNz8JaFLGfad0cJ%2FDS3prHXLvhYOuOh3w%3D&amp;reserved=0
>
>
>
> När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.
>
>
> Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[ICIT]

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Steffen,
indeed, the live dyes come in various forms. We've had good experiences with fixing LysoTracker Red, for example.

After having expensive tests with placebo antibodies from a well-known US company our source for primary antibodies was Abcam. Many antibodies can be bought directly conjugated and usually worked well (out of a few hundred antibodies we had problems with maybe a handful). I don't know if they still have the test program of purchasing only 1/10th of the amount.

Anyway, mostly we conjugated primaries ourselves using standard chemistry and with a Sigma spin column labeling kit.

Looking forward for your solution, so please keep us updated...and make one slide for me!

Cheers,

Mika

[Michael Abanto]

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Hi,

if memory serve me well… i believe there are facs companies validating their facs abs for multiplex imaging, like this one (i haven’t used them):
https://www.miltenyibiotec.com/CH-en/products/macs-antibodies/antibodies-for-microscopy-and-imaging.html.

I would be surprised if other companies (abcam?) haven’t done the same or checked their facs abs under the scope.

Similarly think there are purchasable and published recipes for DNA barcoding. Barcoding would overcome the primary-secondary problem. Since so much is purchasable or published you wouldnt have to worry (as much) about the efficacy, validation etc. I think you’d be safe with popular/obvious targets like tubulin.

https://www.nature.com/articles/s41467-019-12372-6#Sec2
and the published/purchaseable codex panels from Nolan lab/akoya.


hope that helps.

Mike



On Dec 4, 2020, at 4:43 PM, Steffen Dietzel <[hidden email]<mailto:[hidden email]>> wrote:

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Hi all,

thanks for your fast answers, this list is amazing.

Sylvie, we are not having a biological-function-type of scientific question with this, the goal is to show that we can use a certain (hopefully large) number of fluorochromes without too much cross-talk. In this scheme, the DNA stain and actin label are/carry two of these fluorochromes. So yes, we need them. Or label something else instead. The problem for once is not the number of fluorescent channels for excitation/detection, but how to attach so many different labels without cross-labeling (cross-labeling as in anti-rat also detects mouse-ab). We have shown for the fluorochromes we plan to use that they work in single or two color stains. Now we are trying to combine them all.

The GFP does not deal well with some of the procedures we apply to that preparation. The nanobodies allow us to tag it with an organic fluorochrome without 'wasting' mouse or rabbit as primary antibody species.
Zdenek, I expect we would have the same problem with additional/other FPs, thus that is not very promising

Right now we seem to be able to combine the following five labels

1 - DNA
2-Actin
3-GFP-nanobody (say, lamin-GFP)
4-primary mouse ab plus secondary from goat (e.g. for nuclear pores)
5-primary rabbit ab plus secondary from goat (eg. for focal adhesions)

what could be number 6 (eg. for mitochondria)? and possibly 7 (ER, Glogi, ...)? The first thing that came to mind was another species for the primaries where the secondary very unlikely cross-targets mouse and rabbit antibodies, therefore the mention of chicken in the previous mail. But we are not aware of a chicken antibody against a cellular target, plus catalogs don't really tell you how good they perform. Therefore, tapping into the knowledge of this list seems wise.

Sylvie, Mika, Zdenek: Mitochondria labeling without ab - Tracker dyes, SiR-Tubulin, etc: My limited experience with tracker dyes is that they do not perform very well on fixed cells. (Except for actin stains, but we already have actin). Are some of those dyes fixable or can be applied in fixed cells? Any recommendations? It might work, if the fluorochrome properties fit with the rest of our scheme.

The multiplexing paper Sylvie linked uses sequential labeling with elution/removal of the antibodies inbetween. But our point is to use many fluorochromes at the same time.

Zdenek, direct ab labeling could possibly work. But as you write, it is high risk, especially without experience. So I'd rather avoid it. Commercially available ones, I didn't find any with good cellular targets. But maybe I was looking at the wrong places?

Best

Steffen

Am 04.12.2020 um 15:23 schrieb Sylvie Le Guyader:
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Hi Steffen



Does your scientific question (i.e. what do you want to measure in the images) requires you to label DNA and actin? If you do not specifically need them for your analysis, you can skip labelling them and use these channels for something else.

Mitochondria can be labelled with dyes without using antibodies.

Why do you need to counter stain GFP?



Multiplexing is another option. See this paper: https://pubmed.ncbi.nlm.nih.gov/30072512/



Med vänlig hälsning / Best regards



Sylvie



@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Blickagången 16,

Room 7362 (lab)/7840 (office)

14157 Huddinge, Sweden

mobile: [phone]

LCI website

Follow our microscopy blog!



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel
Sent: 04 December 2020 14:53
To: [hidden email]
Subject: Ideas for good, unusual labels for fixed cells?



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Dear listers,



we are about to set up a multi-color experiment as proof of principle in formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.



We are planning on DNA stain, phalloidin as actin stain, some GFP label (amplified with nanobodies), and two primary antibodies from rabbit and mouse, e.g. for mitochondria , ER, nuclear pores or the like with the fluorochrome on the secondary ab. The actual labeled structures do not matter so much, as long as they give good signals of specific structures (i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from goat (or donkey)



We would like to stain one or two additional structures, but we are not sure how to label them. Antibodies from rat have a tendency to cross-react with mouse antibodies, that would not be ideal. (to be

precise: the secondary antibody cross reacts to the other species, of

course.)



Does anybody have some ideas for additional good labels? An antibody from chicken (or other species) that nicely delineates the Golgi, ER, Mitochondria, focal adhesion points, nuclear pores, splicing factors, etc, would be great.



Any good antibody with a cellular target where the secondary will not cross react with mouse or rabbit ab would be of interest. Or some good non-antibody labeling of cellular structures, like phalloidin, just for something other than actin or DNA, that gives decent signals in fixed cells.



Of course on could go for labeling of various primaries from the same species. But antibody labeling is not exactly our core competence and it would add one more layer where things can go wrong. Plus, we might not get the fluorochromes that we plan to use.



Hoping on your experience



Steffen



--

------------------------------------------------------------

Steffen Dietzel, PD Dr. rer. nat

Ludwig-Maximilians-Universität München

Biomedical Center (BMC)

Head of the Core Facility Bioimaging



Großhaderner Straße 9

D-82152 Planegg-Martinsried

Germany



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--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Konstantín Levitskiy]

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Hi Steffen.
Could you work with a time-resolved fluorescence on your equipment?
Bets regards,
Konstantin

________________________________
De: Confocal Microscopy List <[hidden email]> en nombre de Mika Ruonala <[hidden email]>
Enviado: viernes, 4 de diciembre de 2020 17:16
Para: [hidden email] <[hidden email]>
Asunto: Re: Ideas for good, unusual labels for fixed cells?

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Steffen,
indeed, the live dyes come in various forms. We've had good experiences with fixing LysoTracker Red, for example.

After having expensive tests with placebo antibodies from a well-known US company our source for primary antibodies was Abcam. Many antibodies can be bought directly conjugated and usually worked well (out of a few hundred antibodies we had problems with maybe a handful). I don't know if they still have the test program of purchasing only 1/10th of the amount.

Anyway, mostly we conjugated primaries ourselves using standard chemistry and with a Sigma spin column labeling kit.

Looking forward for your solution, so please keep us updated...and make one slide for me!

Cheers,

Mika

[Zdenek Svindrych-2]

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Steffen,
what I was trying to say (among other things) is that if you can't find
good chicken primaries, move to chichen secondaries and find a good goat
primary.
That will give you one more target.
Or try the protein-A trick, that would let you eliminate the offenting
secondary antibody.
Btw, many of the "trackers" are fixable, but that does not mean they work
in fixed cells...
zdenek

On Fri, Dec 4, 2020 at 10:43 AM Steffen Dietzel <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> thanks for your fast answers, this list is amazing.
>
> Sylvie, we are not having a biological-function-type of scientific
> question with this, the goal is to show that we can use a certain
> (hopefully large) number of fluorochromes without too much cross-talk.
> In this scheme, the DNA stain and actin label are/carry two of these
> fluorochromes. So yes, we need them. Or label something else instead.
> The problem for once is not the number of fluorescent channels for
> excitation/detection, but how to attach so many different labels without
> cross-labeling (cross-labeling as in anti-rat also detects mouse-ab). We
> have shown for the fluorochromes we plan to use that they work in single
> or two color stains. Now we are trying to combine them all.
>
> The GFP does not deal well with some of the procedures we apply to that
> preparation. The nanobodies allow us to tag it with an organic
> fluorochrome without 'wasting' mouse or rabbit as primary antibody species.
> Zdenek, I expect we would have the same problem with additional/other
> FPs, thus that is not very promising
>
> Right now we seem to be able to combine the following five labels
>
> 1 - DNA
> 2-Actin
> 3-GFP-nanobody (say, lamin-GFP)
> 4-primary mouse ab plus secondary from goat (e.g. for nuclear pores)
> 5-primary rabbit ab plus secondary from goat (eg. for focal adhesions)
>
> what could be number 6 (eg. for mitochondria)? and possibly 7 (ER,
> Glogi, ...)? The first thing that came to mind was another species for
> the primaries where the secondary very unlikely cross-targets mouse and
> rabbit antibodies, therefore the mention of chicken in the previous
> mail. But we are not aware of a chicken antibody against a cellular
> target, plus catalogs don't really tell you how good they perform.
> Therefore, tapping into the knowledge of this list seems wise.
>
> Sylvie, Mika, Zdenek: Mitochondria labeling without ab - Tracker dyes,
> SiR-Tubulin, etc: My limited experience with tracker dyes is that they
> do not perform very well on fixed cells. (Except for actin stains, but
> we already have actin). Are some of those dyes fixable or can be applied
> in fixed cells? Any recommendations? It might work, if the fluorochrome
> properties fit with the rest of our scheme.
>
> The multiplexing paper Sylvie linked uses sequential labeling with
> elution/removal of the antibodies inbetween. But our point is to use
> many fluorochromes at the same time.
>
> Zdenek, direct ab labeling could possibly work. But as you write, it is
> high risk, especially without experience. So I'd rather avoid it.
> Commercially available ones, I didn't find any with good cellular
> targets. But maybe I was looking at the wrong places?
>
> Best
>
> Steffen
>
> Am 04.12.2020 um 15:23 schrieb Sylvie Le Guyader:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Steffen
> >
> >
> >
> > Does your scientific question (i.e. what do you want to measure in the
> images) requires you to label DNA and actin? If you do not specifically
> need them for your analysis, you can skip labelling them and use these
> channels for something else.
> >
> > Mitochondria can be labelled with dyes without using antibodies.
> >
> > Why do you need to counter stain GFP?
> >
> >
> >
> > Multiplexing is another option. See this paper:
> https://pubmed.ncbi.nlm.nih.gov/30072512/
> >
> >
> >
> > Med vänlig hälsning / Best regards
> >
> >
> >
> > Sylvie
> >
> >
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> >
> > Sylvie Le Guyader, PhD
> >
> > Live Cell Imaging Facility Manager
> >
> > Karolinska Institutet- Bionut Dpt
> >
> > Blickagången 16,
> >
> > Room 7362 (lab)/7840 (office)
> >
> > 14157 Huddinge, Sweden
> >
> > mobile: [phone]
> >
> > LCI website
> >
> > Follow our microscopy blog!
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Steffen Dietzel
> > Sent: 04 December 2020 14:53
> > To: [hidden email]
> > Subject: Ideas for good, unusual labels for fixed cells?
> >
> >
> >
> > *****
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=8nn%2FDn93e7V4nRKirMzOjjaCvupL2eGOfDsJt5BFTGA%3D&amp;reserved=0
> >
> > Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=wQTXNyPORAs64aCBls305ZPRU9QsDnZtKp%2BalYsUQyA%3D&amp;reserved=0
> and include the link in your posting.
> >
> > *****
> >
> >
> >
> > Dear listers,
> >
> >
> >
> > we are about to set up a multi-color experiment as proof of principle in
> formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.
> >
> >
> >
> > We are planning on DNA stain, phalloidin as actin stain, some GFP label
> (amplified with nanobodies), and two primary antibodies from rabbit and
> mouse, e.g. for mitochondria , ER, nuclear pores or the like with the
> fluorochrome on the secondary ab. The actual labeled structures do not
> matter so much, as long as they give good signals of specific structures
> (i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from
> goat (or donkey)
> >
> >
> >
> > We would like to stain one or two additional structures, but we are not
> sure how to label them. Antibodies from rat have a tendency to cross-react
> with mouse antibodies, that would not be ideal. (to be
> >
> > precise: the secondary antibody cross reacts to the other species, of
> >
> > course.)
> >
> >
> >
> > Does anybody have some ideas for additional good labels? An antibody
> from chicken (or other species) that nicely delineates the Golgi, ER,
> Mitochondria, focal adhesion points, nuclear pores, splicing factors, etc,
> would be great.
> >
> >
> >
> > Any good antibody with a cellular target where the secondary will not
> cross react with mouse or rabbit ab would be of interest. Or some good
> non-antibody labeling of cellular structures, like phalloidin, just for
> something other than actin or DNA, that gives decent signals in fixed cells.
> >
> >
> >
> > Of course on could go for labeling of various primaries from the same
> species. But antibody labeling is not exactly our core competence and it
> would add one more layer where things can go wrong. Plus, we might not get
> the fluorochromes that we plan to use.
> >
> >
> >
> > Hoping on your experience
> >
> >
> >
> > Steffen
> >
> >
> >
> > --
> >
> > ------------------------------------------------------------
> >
> > Steffen Dietzel, PD Dr. rer. nat
> >
> > Ludwig-Maximilians-Universität München
> >
> > Biomedical Center (BMC)
> >
> > Head of the Core Facility Bioimaging
> >
> >
> >
> > Großhaderner Straße 9
> >
> > D-82152 Planegg-Martinsried
> >
> > Germany
> >
> >
> >
> >
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioimaging.bmc.med.uni-muenchen.de%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=iyY%2FMZT%2FlYRNz8JaFLGfad0cJ%2FDS3prHXLvhYOuOh3w%3D&amp;reserved=0
> >
> >
> >
> > När du skickar e-post till Karolinska Institutet (KI) innebär detta att
> KI kommer att behandla dina personuppgifter. Här finns information om hur
> KI behandlar personuppgifter<
> https://ki.se/medarbetare/integritetsskyddspolicy>.
> >
> >
> > Sending email to Karolinska Institutet (KI) will result in KI processing
> your personal data. You can read more about KI’s processing of personal
> data here<https://ki.se/en/staff/data-protection-policy>.
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Arvydas Matiukas]

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________________________________
Dear confocal experts,

A year ago I started more general discussion on confocal lifetime that I found very useful. Now I would like to know how Cores estimate useful/competitive lifetime and means to maximize it. It though does not help to know that there exist 30-year old confocals because they won't help researchers to get new grants.

Specifically, how I can corroborate my estimate of the remaining lifetime of 3 years/10k hrs of the currently 6.5 y.o./13k hrs confocal. My estimate is based on the increased part breakage rate, image quality degradation (that is somewhat restored after PM but still not to the level of the brand new machine), and observation of full lifecycle of other confocal that died after 17 year/17k hrs. The useful/competitive lifetime may be even shorter especially if manufacturer support ends: e.g. nobody wanted to use the 14 y.o. machine performing below specs after the brand new confocal became available.
I received an interesting suggestion from NIH experts to obtain the best service contract and run confocal 24/7=8760 hrs/year. If I followed this recommendation I expect my confocal to be "dead" for useful/competitive imaging in about 2 years at 8.5 years/28k hrs (when the manufacturer ends the support as well). Unfortunately this time may be too short to receive a replacement SIG award (>3years at 32% success rate), and the suggestion would leave many labs without local confocal.

Please advise how your Core estimates the timing of the old confocal replacement. Do you know any standards, recommendations, lifetime statistics that I could refer to. Do you know of specific usage & maintenance regime that maximizes the useful lifetime hrs?
Any thoughts and/or advice is greatly appreciated. You can reply via listserv or privately to my email.

Thanks,
Arvydas
******************************



Arvydas Matiukas, Ph.D.

Director of  Neuroscience  Microscopy Core

Manager of NRB Shared Research Equipment

SUNY Upstate Medical University
Neuroscience & Physiology Dept

Room 3607 IHP

505 Irving Ave

Syracuse, NY 13210


Email: [hidden email]

[Craig Brideau]

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In my own experience managing the consumables is fairly important. Many
confocals come with banks of diode lasers which the controller fires up the
moment the system is powered on. While this lets the lasers warm up and
stabilize, the lasers will be "burning" the entire time the system is on
even if the current user does not require a specific wavelength. It is
better to be able to activate the lasers individually only as they are
needed. Running a diode laser or LED full power only to cover it with a
shutter is wasteful unless you are experiencing stability issues during
warm-up.
The detectors also wear depending on use. "Classic" photomultiplier tubes
tend to last 5-10 years as long as they are not over exposed frequently. I
find newer GaAsP PMTs are more sensitive and tend to last about 2-3 years.
They are also about $5k per tube to replace so this can be a significant
expense for a many-channel system.
Barring accidents or abuse/misuse most of a typical confocal is good for
about 8-10 years. The computer may be an issue depending on the operating
system, and you can always have the misfortune of a failed drive or power
supply, although this happens less than it used to. Also consider how you
will be managing your data, for instance if you are using portable hard
drives or USB sticks, will an effective anti-virus be available for the
computer's OS in eight years time? If you have it on the network for data
transfer will you still be able to get security updates for the operating
system? If the computer fails will you be able to get replacement parts?

My experiences: On a confocal over 10 years had 405, 561, 638nm diode
lasers and 488 argon laser fail. 561 after 4 years, the rest in the
remaining six. These were early generation diode lasers so the lifetime is
about half of what you should expect from a contemporary diode laser. I
replaced all of these lasers except the 561 with modern diode lasers (argon
replaced with single-line 488 diode laser) and all these replacements have
been operating for about 4 years with no issues.

Another confocal: 457/476/488/514 argon laser quit after 6 years. Had it
retubed because we used the 457 and 514 laser lines as well as the 488, so
didn't make sense to replace with single-wavelength diode laser. Retubed
unit still going 4 years later at factory-rated output power providing all
four lines.

2P/confocal system: Ti:Saph laser died. ~$100k+ to replace so mothballed 2P
functionality and used only as a confocal. Separate 2P system purchased
later to provide separate 2P functionality.

Widefield camera system: Exfo white light source died after 5 years,
replaced with discrete four-color LED system, upgraded to 6-LED system

Hope this helps.
Craig

On Fri, Dec 4, 2020 at 11:50 AM Arvydas Matiukas <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>
>
> ________________________________
> Dear confocal experts,
>
> A year ago I started more general discussion on confocal lifetime that I
> found very useful. Now I would like to know how Cores estimate
> useful/competitive lifetime and means to maximize it. It though does not
> help to know that there exist 30-year old confocals because they won't help
> researchers to get new grants.
>
> Specifically, how I can corroborate my estimate of the remaining lifetime
> of 3 years/10k hrs of the currently 6.5 y.o./13k hrs confocal. My estimate
> is based on the increased part breakage rate, image quality degradation
> (that is somewhat restored after PM but still not to the level of the brand
> new machine), and observation of full lifecycle of other confocal that died
> after 17 year/17k hrs. The useful/competitive lifetime may be even shorter
> especially if manufacturer support ends: e.g. nobody wanted to use the 14
> y.o. machine performing below specs after the brand new confocal became
> available.
> I received an interesting suggestion from NIH experts to obtain the best
> service contract and run confocal 24/7=8760 hrs/year. If I followed this
> recommendation I expect my confocal to be "dead" for useful/competitive
> imaging in about 2 years at 8.5 years/28k hrs (when the manufacturer ends
> the support as well). Unfortunately this time may be too short to receive a
> replacement SIG award (>3years at 32% success rate), and the suggestion
> would leave many labs without local confocal.
>
> Please advise how your Core estimates the timing of the old confocal
> replacement. Do you know any standards, recommendations, lifetime
> statistics that I could refer to. Do you know of specific usage &
> maintenance regime that maximizes the useful lifetime hrs?
> Any thoughts and/or advice is greatly appreciated. You can reply via
> listserv or privately to my email.
>
> Thanks,
> Arvydas
> ******************************
>
>
>
> Arvydas Matiukas, Ph.D.
>
> Director of  Neuroscience  Microscopy Core
>
> Manager of NRB Shared Research Equipment
>
> SUNY Upstate Medical University
> Neuroscience & Physiology Dept
>
> Room 3607 IHP
>
> 505 Irving Ave
>
> Syracuse, NY 13210
>
>
> Email: [hidden email]
>

[Benjamin Smith]

*****
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*****

A few other failure modes I've encountered:

Confocal systems:

1) Failure of the AOM (such as the transducer detaching from the crystal).
As expected, this will take out all of your visible laser lines, and so
will brick a system unless replaced by the vendor.

2) Laser fiber coupler going out of alignment (often when people think they
have a laser failure, this is what has really happened).  This is again
mostly a problem if the vendor used a proprietary coupler and chooses to no
longer support your system.

Two photon systems:

1) Failure of the recirculating chiller pump.  I have seen this many times,
and is due to the thinning of a teflon bushing inside the pump, causing the
impeller to scrape on the housing.  Cheap to fix, but hard to get to and
requires a thorough system flush to get the resulting plastic bits out of
the cooling loop.

2) Motherboard failure.  This is the same for all PCBs where thermal
cycling of the board stresses the vias and, if you are unlucky, a critical
via fails open and bricks the board.  The general expected half-life for a
well manufactured board is around 10 years, but this of course depends on
the number of thermal cycles across that time (the 10 year rating is
usually for motherboards assuming one thermal cycle per day):
https://www.dfrsolutions.com/hubfs/Resources/services/Reliable-Plated-Through-Via-Design-Fabrication.pdf

3) LBO and diode temp recalibration.  This isn't a failure mode per se, but
it is a required maintenance procedure for Ti:Sapphire lasers as the laser
ages or else the laser will become unstable and won't be able to tune to
certain wavelengths.  Unfortunately, modern laser systems block you from
being able to do this maintenance yourself, so you will need to pay
a vendor to perform this routine maintenance task for you assuming your
system is still supported.

Some ways to maximize the useful lifetime are:
1) Keep the room temperature and humidity within the optimal range
specified in the datasheet for your scope,
2) Keep the temperature as stable as possible, especially reducing the rate
of temperature cycling (such as an AC unit repeatedly toggling on and off).
3) Regularly clean all dust off both the surface and the inside your system
(inducing inside the laser bay, computers, etc.), also making sure to clean
any air filters,
4) Make sure to keep all cooling fluids topped up (one gotcha I ran across
was that the scan head on one system also used a recirculating liquid
cooler that needed to be regularly topped off that was hidden in the laser
bay),
5) As Craig suggested, reduce the on-time of diode lasers (these can be
toggled on and off safely),
6) Make sure light detectors are never saturated (detector sensitivity
gradually diminishes the more they are abused),
7) Carefully train users, and make sure they master simple tasks before
progressing to more complicated ones that run a higher risk of damaging the
microscope.
8) Survey the building vibrations in the area you are going to install the
microscope, and make sure you install the scope in a node if possible.
Also, make sure the prevalent frequencies are within the range filtered by
your air-table.  Note that this solution is not perfect, as building
re-construction (such as adding or relocating an air handler) can
dramatically change the locations of nodes and antinodes.  Because of this,
the best location for a microscope will almost always be in the basement.
Your physics department should have the needed sensor and spectrum analyzer
if you ask nicely.
9) Run the system off of UPSs to protect it from power surges, brown outs,
and outages; all of which can be very damaging to electronics.

As I was hinting at earlier, perhaps the biggest issue to consider when
estimating serviceable lifetimes for modern systems is planned
obsolescence.  By not disclosing service manuals, locking down
diagnostic/alignment/calibration tools and software, using proprietary
parts and serial protocols, and no longer supporting older models,
otherwise repairable systems can become bricked (or at least VERY hard to
bring back) once the vendor decides to no longer support the model.  I've
noticed this issue becoming more and more prevalent over the last 10
years, even to the point of having anonymized markings on chips
on circuit boards.  Therefore, with careful maintenance and well trained
users, there is a reasonable chance a modern confocal will no longer be
usable due to it no longer being supported by the vendor before actually
suffering an irreparable failure.  Fortunately, there are now a couple of
partially open-source vendors that have come into the two-photon microscope
market, and if anyone knows of a similar vendor for single photon systems,
I would love to know.

Better yet, you could also build your own system, which turns out to be
surprisingly easy for a two-photon microscope (especially thanks to
ScanImage), but a noticeably more complicated for a single photon system.

Cheers,
   Ben Smith



On Fri, Dec 4, 2020 at 11:31 AM Craig Brideau <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> In my own experience managing the consumables is fairly important. Many
> confocals come with banks of diode lasers which the controller fires up the
> moment the system is powered on. While this lets the lasers warm up and
> stabilize, the lasers will be "burning" the entire time the system is on
> even if the current user does not require a specific wavelength. It is
> better to be able to activate the lasers individually only as they are
> needed. Running a diode laser or LED full power only to cover it with a
> shutter is wasteful unless you are experiencing stability issues during
> warm-up.
> The detectors also wear depending on use. "Classic" photomultiplier tubes
> tend to last 5-10 years as long as they are not over exposed frequently. I
> find newer GaAsP PMTs are more sensitive and tend to last about 2-3 years.
> They are also about $5k per tube to replace so this can be a significant
> expense for a many-channel system.
> Barring accidents or abuse/misuse most of a typical confocal is good for
> about 8-10 years. The computer may be an issue depending on the operating
> system, and you can always have the misfortune of a failed drive or power
> supply, although this happens less than it used to. Also consider how you
> will be managing your data, for instance if you are using portable hard
> drives or USB sticks, will an effective anti-virus be available for the
> computer's OS in eight years time? If you have it on the network for data
> transfer will you still be able to get security updates for the operating
> system? If the computer fails will you be able to get replacement parts?
>
> My experiences: On a confocal over 10 years had 405, 561, 638nm diode
> lasers and 488 argon laser fail. 561 after 4 years, the rest in the
> remaining six. These were early generation diode lasers so the lifetime is
> about half of what you should expect from a contemporary diode laser. I
> replaced all of these lasers except the 561 with modern diode lasers (argon
> replaced with single-line 488 diode laser) and all these replacements have
> been operating for about 4 years with no issues.
>
> Another confocal: 457/476/488/514 argon laser quit after 6 years. Had it
> retubed because we used the 457 and 514 laser lines as well as the 488, so
> didn't make sense to replace with single-wavelength diode laser. Retubed
> unit still going 4 years later at factory-rated output power providing all
> four lines.
>
> 2P/confocal system: Ti:Saph laser died. ~$100k+ to replace so mothballed 2P
> functionality and used only as a confocal. Separate 2P system purchased
> later to provide separate 2P functionality.
>
> Widefield camera system: Exfo white light source died after 5 years,
> replaced with discrete four-color LED system, upgraded to 6-LED system
>
> Hope this helps.
> Craig
>
> On Fri, Dec 4, 2020 at 11:50 AM Arvydas Matiukas <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> >
> >
> > ________________________________
> > Dear confocal experts,
> >
> > A year ago I started more general discussion on confocal lifetime that I
> > found very useful. Now I would like to know how Cores estimate
> > useful/competitive lifetime and means to maximize it. It though does not
> > help to know that there exist 30-year old confocals because they won't
> help
> > researchers to get new grants.
> >
> > Specifically, how I can corroborate my estimate of the remaining lifetime
> > of 3 years/10k hrs of the currently 6.5 y.o./13k hrs confocal. My
> estimate
> > is based on the increased part breakage rate, image quality degradation
> > (that is somewhat restored after PM but still not to the level of the
> brand
> > new machine), and observation of full lifecycle of other confocal that
> died
> > after 17 year/17k hrs. The useful/competitive lifetime may be even
> shorter
> > especially if manufacturer support ends: e.g. nobody wanted to use the 14
> > y.o. machine performing below specs after the brand new confocal became
> > available.
> > I received an interesting suggestion from NIH experts to obtain the best
> > service contract and run confocal 24/7=8760 hrs/year. If I followed this
> > recommendation I expect my confocal to be "dead" for useful/competitive
> > imaging in about 2 years at 8.5 years/28k hrs (when the manufacturer ends
> > the support as well). Unfortunately this time may be too short to
> receive a
> > replacement SIG award (>3years at 32% success rate), and the suggestion
> > would leave many labs without local confocal.
> >
> > Please advise how your Core estimates the timing of the old confocal
> > replacement. Do you know any standards, recommendations, lifetime
> > statistics that I could refer to. Do you know of specific usage &
> > maintenance regime that maximizes the useful lifetime hrs?
> > Any thoughts and/or advice is greatly appreciated. You can reply via
> > listserv or privately to my email.
> >
> > Thanks,
> > Arvydas
> > ******************************
> >
> >
> >
> > Arvydas Matiukas, Ph.D.
> >
> > Director of  Neuroscience  Microscopy Core
> >
> > Manager of NRB Shared Research Equipment
> >
> > SUNY Upstate Medical University
> > Neuroscience & Physiology Dept
> >
> > Room 3607 IHP
> >
> > 505 Irving Ave
> >
> > Syracuse, NY 13210
> >
> >
> > Email: [hidden email]
> >
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Konstantín Levitskiy]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

I'm agree with you. Many of "fixable" trackers are not fixable at all. I put once data in this list about Mito Tracker Deep Red, with coexpressing an GFP mitochondria protein. In vivo they matched exactly, but fixing in the same preparation MitoTracker went to any other structure (could be mitochondria-like), but not in mitochondria.
Best regards,
Konstantin

________________________________
De: Confocal Microscopy List <[hidden email]> en nombre de Zdenek Svindrych <[hidden email]>
Enviado: viernes, 4 de diciembre de 2020 17:50
Para: [hidden email] <[hidden email]>
Asunto: Re: Ideas for good, unusual labels for fixed cells?

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Steffen,
what I was trying to say (among other things) is that if you can't find
good chicken primaries, move to chichen secondaries and find a good goat
primary.
That will give you one more target.
Or try the protein-A trick, that would let you eliminate the offenting
secondary antibody.
Btw, many of the "trackers" are fixable, but that does not mean they work
in fixed cells...
zdenek

On Fri, Dec 4, 2020 at 10:43 AM Steffen Dietzel <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> thanks for your fast answers, this list is amazing.
>
> Sylvie, we are not having a biological-function-type of scientific
> question with this, the goal is to show that we can use a certain
> (hopefully large) number of fluorochromes without too much cross-talk.
> In this scheme, the DNA stain and actin label are/carry two of these
> fluorochromes. So yes, we need them. Or label something else instead.
> The problem for once is not the number of fluorescent channels for
> excitation/detection, but how to attach so many different labels without
> cross-labeling (cross-labeling as in anti-rat also detects mouse-ab). We
> have shown for the fluorochromes we plan to use that they work in single
> or two color stains. Now we are trying to combine them all.
>
> The GFP does not deal well with some of the procedures we apply to that
> preparation. The nanobodies allow us to tag it with an organic
> fluorochrome without 'wasting' mouse or rabbit as primary antibody species.
> Zdenek, I expect we would have the same problem with additional/other
> FPs, thus that is not very promising
>
> Right now we seem to be able to combine the following five labels
>
> 1 - DNA
> 2-Actin
> 3-GFP-nanobody (say, lamin-GFP)
> 4-primary mouse ab plus secondary from goat (e.g. for nuclear pores)
> 5-primary rabbit ab plus secondary from goat (eg. for focal adhesions)
>
> what could be number 6 (eg. for mitochondria)? and possibly 7 (ER,
> Glogi, ...)? The first thing that came to mind was another species for
> the primaries where the secondary very unlikely cross-targets mouse and
> rabbit antibodies, therefore the mention of chicken in the previous
> mail. But we are not aware of a chicken antibody against a cellular
> target, plus catalogs don't really tell you how good they perform.
> Therefore, tapping into the knowledge of this list seems wise.
>
> Sylvie, Mika, Zdenek: Mitochondria labeling without ab - Tracker dyes,
> SiR-Tubulin, etc: My limited experience with tracker dyes is that they
> do not perform very well on fixed cells. (Except for actin stains, but
> we already have actin). Are some of those dyes fixable or can be applied
> in fixed cells? Any recommendations? It might work, if the fluorochrome
> properties fit with the rest of our scheme.
>
> The multiplexing paper Sylvie linked uses sequential labeling with
> elution/removal of the antibodies inbetween. But our point is to use
> many fluorochromes at the same time.
>
> Zdenek, direct ab labeling could possibly work. But as you write, it is
> high risk, especially without experience. So I'd rather avoid it.
> Commercially available ones, I didn't find any with good cellular
> targets. But maybe I was looking at the wrong places?
>
> Best
>
> Steffen
>
> Am 04.12.2020 um 15:23 schrieb Sylvie Le Guyader:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Steffen
> >
> >
> >
> > Does your scientific question (i.e. what do you want to measure in the
> images) requires you to label DNA and actin? If you do not specifically
> need them for your analysis, you can skip labelling them and use these
> channels for something else.
> >
> > Mitochondria can be labelled with dyes without using antibodies.
> >
> > Why do you need to counter stain GFP?
> >
> >
> >
> > Multiplexing is another option. See this paper:
> https://pubmed.ncbi.nlm.nih.gov/30072512/
> >
> >
> >
> > Med vänlig hälsning / Best regards
> >
> >
> >
> > Sylvie
> >
> >
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> >
> > Sylvie Le Guyader, PhD
> >
> > Live Cell Imaging Facility Manager
> >
> > Karolinska Institutet- Bionut Dpt
> >
> > Blickagången 16,
> >
> > Room 7362 (lab)/7840 (office)
> >
> > 14157 Huddinge, Sweden
> >
> > mobile: [phone]
> >
> > LCI website
> >
> > Follow our microscopy blog!
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Steffen Dietzel
> > Sent: 04 December 2020 14:53
> > To: [hidden email]
> > Subject: Ideas for good, unusual labels for fixed cells?
> >
> >
> >
> > *****
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=8nn%2FDn93e7V4nRKirMzOjjaCvupL2eGOfDsJt5BFTGA%3D&amp;reserved=0
> >
> > Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=wQTXNyPORAs64aCBls305ZPRU9QsDnZtKp%2BalYsUQyA%3D&amp;reserved=0
> and include the link in your posting.
> >
> > *****
> >
> >
> >
> > Dear listers,
> >
> >
> >
> > we are about to set up a multi-color experiment as proof of principle in
> formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.
> >
> >
> >
> > We are planning on DNA stain, phalloidin as actin stain, some GFP label
> (amplified with nanobodies), and two primary antibodies from rabbit and
> mouse, e.g. for mitochondria , ER, nuclear pores or the like with the
> fluorochrome on the secondary ab. The actual labeled structures do not
> matter so much, as long as they give good signals of specific structures
> (i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from
> goat (or donkey)
> >
> >
> >
> > We would like to stain one or two additional structures, but we are not
> sure how to label them. Antibodies from rat have a tendency to cross-react
> with mouse antibodies, that would not be ideal. (to be
> >
> > precise: the secondary antibody cross reacts to the other species, of
> >
> > course.)
> >
> >
> >
> > Does anybody have some ideas for additional good labels? An antibody
> from chicken (or other species) that nicely delineates the Golgi, ER,
> Mitochondria, focal adhesion points, nuclear pores, splicing factors, etc,
> would be great.
> >
> >
> >
> > Any good antibody with a cellular target where the secondary will not
> cross react with mouse or rabbit ab would be of interest. Or some good
> non-antibody labeling of cellular structures, like phalloidin, just for
> something other than actin or DNA, that gives decent signals in fixed cells.
> >
> >
> >
> > Of course on could go for labeling of various primaries from the same
> species. But antibody labeling is not exactly our core competence and it
> would add one more layer where things can go wrong. Plus, we might not get
> the fluorochromes that we plan to use.
> >
> >
> >
> > Hoping on your experience
> >
> >
> >
> > Steffen
> >
> >
> >
> > --
> >
> > ------------------------------------------------------------
> >
> > Steffen Dietzel, PD Dr. rer. nat
> >
> > Ludwig-Maximilians-Universität München
> >
> > Biomedical Center (BMC)
> >
> > Head of the Core Facility Bioimaging
> >
> >
> >
> > Großhaderner Straße 9
> >
> > D-82152 Planegg-Martinsried
> >
> > Germany
> >
> >
> >
> >
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioimaging.bmc.med.uni-muenchen.de%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf6c6a0a408804d5d254d08d8985df4ee%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637426876623894076%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=iyY%2FMZT%2FlYRNz8JaFLGfad0cJ%2FDS3prHXLvhYOuOh3w%3D&amp;reserved=0
> >
> >
> >
> > När du skickar e-post till Karolinska Institutet (KI) innebär detta att
> KI kommer att behandla dina personuppgifter. Här finns information om hur
> KI behandlar personuppgifter<
> https://ki.se/medarbetare/integritetsskyddspolicy>.
> >
> >
> > Sending email to Karolinska Institutet (KI) will result in KI processing
> your personal data. You can read more about KI’s processing of personal
> data here<https://ki.se/en/staff/data-protection-policy>.
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Sylvie Le Guyader]

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Hi Arvydas
Over the past 12 years (8 heavy microscopes), we have had normal although not very common equipment failure in lasers, scanning mirrors, rotating parts like filter or objective turrets, stage...  But I considers these to be normal wear and tear.
Our strategy to get the best out of a microscope is:
- We educate our users as well as possible and giving them clear routines about objective care, laser routines, saturation of camera and detectors, reporting anything strange or any accident...
- To eliminate the risk of bumping in the objective, we cap z on all our microscopes and have a preferred multiwell plate source (zellKontakt, no personal interest) for their low skirt.
- All our microscopes have a cage incubator. The incubator is kept at 37 deg all the time except when an experiment specifically needs a different temperature. This keeps the microscope body at a constant temperature which I think prolongs the microscope body lifetime.
- Indeed we turn lasers on individually whenever we can. We have a 2h policy for argon lasers (leave them on unless no one else is going to use them in the coming 2h).
- 1 service visit per year is a must. The technicians usually tell us a few years in advance when something starts aging.
- Software and firmware updates every 3 years max and changing the computer max every 5 years. This means that all firmware are updated. No big jumps in updates is really important.
- I find that the 10000h mark is more for me a sign that it is time to inject good money in a system than trash it. By investing I mean doing a serious upgrade when major parts are changed to a newer version and the computer is changed. This gives the microscope a second life at around 30-20% of the cost of a new system.
Med vänlig hälsning / Best regards
Sylvie
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Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
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From: Confocal Microscopy List <[hidden email]> on behalf of Arvydas Matiukas <[hidden email]>
Sent: Friday, December 4, 2020 7:48:47 PM
To: [hidden email] <[hidden email]>
Subject: Estimating & Maximizing the useful confocal lifetime

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Dear confocal experts,

A year ago I started more general discussion on confocal lifetime that I found very useful. Now I would like to know how Cores estimate useful/competitive lifetime and means to maximize it. It though does not help to know that there exist 30-year old confocals because they won't help researchers to get new grants.

Specifically, how I can corroborate my estimate of the remaining lifetime of 3 years/10k hrs of the currently 6.5 y.o./13k hrs confocal. My estimate is based on the increased part breakage rate, image quality degradation (that is somewhat restored after PM but still not to the level of the brand new machine), and observation of full lifecycle of other confocal that died after 17 year/17k hrs. The useful/competitive lifetime may be even shorter especially if manufacturer support ends: e.g. nobody wanted to use the 14 y.o. machine performing below specs after the brand new confocal became available.
I received an interesting suggestion from NIH experts to obtain the best service contract and run confocal 24/7=8760 hrs/year. If I followed this recommendation I expect my confocal to be "dead" for useful/competitive imaging in about 2 years at 8.5 years/28k hrs (when the manufacturer ends the support as well). Unfortunately this time may be too short to receive a replacement SIG award (>3years at 32% success rate), and the suggestion would leave many labs without local confocal.

Please advise how your Core estimates the timing of the old confocal replacement. Do you know any standards, recommendations, lifetime statistics that I could refer to. Do you know of specific usage & maintenance regime that maximizes the useful lifetime hrs?
Any thoughts and/or advice is greatly appreciated. You can reply via listserv or privately to my email.

Thanks,
Arvydas
******************************



Arvydas Matiukas, Ph.D.

Director of  Neuroscience  Microscopy Core

Manager of NRB Shared Research Equipment

SUNY Upstate Medical University
Neuroscience & Physiology Dept

Room 3607 IHP

505 Irving Ave

Syracuse, NY 13210


Email: [hidden email]



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Hi,

I also observed this behaviour of MitoTracker Deep Red in primary alveolar cells. The labeled structure after fixation looked more like the actin cytoskeleton.  

However, we have had good experience with MitoTracker Orange CMTMRos after PFA fixation. See for example: 10.1038/s41598-017-18468-7  

Best regards,  
Andreas