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Measuring cell volume by negative staining

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Sylvie Le Guyader

Measuring cell volume by negative staining

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Dear list

I remember reading a paper where the authors wanted to precisely measure the volume of cells . They added a fluorophore to the medium, acquired a z stack and segmented the negative areas in the image to find the cells.
I cannot find this paper. Would anyone have a reference?

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
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Room 7362 (lab)/7840 (office)
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Nicolai.Urban@mpfi.org

Re: Measuring cell volume by negative staining

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Hi Sylvie,

I am not sure this is the paper you are looking for, but in 2018 Valentin Nägerl's group published a paper on "super-resolution shadow imaging" (SUSHI), in which they measured the extracellular space in brain tissue. Very nice paper, with lots of different applications, but maybe not what you had in mind.
The reference is here:
https://www.ncbi.nlm.nih.gov/pubmed/29474910
DOI: 10.1016/j.cell.2018.02.007

Best,
Nicolai

>>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
Dr. Nicolai T. Urban
Imaging Center @ Max Planck Florida Institute
Jupiter, 33458 FL, USA

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: Thursday, 10 October 2019 10:31
To: [hidden email]
Subject: Measuring cell volume by negative staining

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Dear list

I remember reading a paper where the authors wanted to precisely measure the volume of cells . They added a fluorophore to the medium, acquired a z stack and segmented the negative areas in the image to find the cells.
I cannot find this paper. Would anyone have a reference?

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: +46 (0) 73 733 5008
LCI website<https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fki.se%2Fen%2Fbionut%2Fwelcome-to-the-lci-facility&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cec8e60ca6c0c4182dde908d74d8fcf21%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C637063152397133838&sdata=gyVy9P4OsYlvffWf35mSoEPxRdy9UEbAZW0WwbWzhMc%3D&reserved=0>
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Mark Cannell-2

Re: Measuring cell volume by negative staining

In reply to this post by Sylvie Le Guyader

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Hi

We used that method in 1999 to measure t-tubes and cell surface invaginations DOI: 10.1161/01.res.84.3.266 and even extended it more recently. I think that for cell volume measurement it was subsequently called "volume exclusion microscopy"

HTH Mark

On 10/10/19, 3:40 PM, "Confocal Microscopy List on behalf of Sylvie Le Guyader" <[hidden email] on behalf of [hidden email]> wrote:

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Dear list

I remember reading a paper where the authors wanted to precisely measure the volume of cells . They added a fluorophore to the medium, acquired a z stack and segmented the negative areas in the image to find the cells.
I cannot find this paper. Would anyone have a reference?

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: +46 (0) 73 733 5008
LCI website<https://ki.se/en/bionut/welcome-to-the-lci-facility>
Follow our microscopy blog!<http://microscopykarolinska.se/>

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Patrick Happel

Re: Measuring cell volume by negative staining

In reply to this post by Sylvie Le Guyader

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Dear Sylvie!

Am Donnerstag, den 10.10.2019, 14:31 +0000 schrieb Sylvie Le Guyader:
>
> I remember reading a paper where the authors wanted to precisely
> measure the volume of cells . They added a fluorophore to the medium,
> acquired a z stack and segmented the negative areas in the image to
> find the cells.
> I cannot find this paper. Would anyone have a reference?

Maybe this one?

https://doi.org/10.1117/1.2138011

HTH
Patrick
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Sylvie Le Guyader

Re: Measuring cell volume by negative staining

In reply to this post by Nicolai.Urban@mpfi.org

mmodel

Re: Measuring cell volume by negative staining

In reply to this post by Sylvie Le Guyader

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Hi Sylvie,

you will find references to this and other methods in

Model M.A. Methods for cell volume measurement. Cytometry A 2018, 93(3) 281-296

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Sylvie Le Guyader <[hidden email]>
Sent: Thursday, October 10, 2019 10:31 AM
To: [hidden email] <[hidden email]>
Subject: Measuring cell volume by negative staining

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Dear list

I remember reading a paper where the authors wanted to precisely measure the volume of cells . They added a fluorophore to the medium, acquired a z stack and segmented the negative areas in the image to find the cells.
I cannot find this paper. Would anyone have a reference?

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: +46 (0) 73 733 5008
LCI website<https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fki.se%2Fen%2Fbionut%2Fwelcome-to-the-lci-facility&data=02%7C01%7Cmmodel%40KENT.EDU%7C4a39cc2f203b4f7e617b08d74d8fd07a%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637063152394291978&sdata=SYIHr28eUJIY0h7qydFyANKJ19EeUK9F315gvDb2Ltk%3D&reserved=0>
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mmodel

Re: Measuring cell volume by negative staining

In reply to this post by Sylvie Le Guyader

JOEL B. SHEFFIELD

Re: Measuring cell volume by negative staining

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To Sylvie's comment about permeability of the fixed cells. Have you
thought of using fluorescent dextran of different molecular weights as an
extracellular stain?

Joel

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
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On Thu, Oct 10, 2019 at 11:55 AM MODEL, MICHAEL <[hidden email]> wrote:

Jeremy Adler-4

Re: Measuring cell volume by negative staining

In reply to this post by Sylvie Le Guyader

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You could try fluorescent microspheres - I would expect 0.5um to be excluded from fixed cells.
Your fixed cells are likely to shrink in the Z axis - understating their volume.

Jeremy Adler
BioVis
Uppsala U

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: den 10 oktober 2019 17:15
To: [hidden email]
Subject: Re: Measuring cell volume by negative staining

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But I forgot to mention that our cells are fixed and permeabilized which makes it more complicated. We need to find a dye that is large enough to stay outside. :-/

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: +46 (0) 73 733 5008
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of [hidden email]
Sent: 10 October 2019 16:53
To: [hidden email]
Subject: Re: Measuring cell volume by negative staining

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Hi Sylvie,

I am not sure this is the paper you are looking for, but in 2018 Valentin Nägerl's group published a paper on "super-resolution shadow imaging" (SUSHI), in which they measured the extracellular space in brain tissue. Very nice paper, with lots of different applications, but maybe not what you had in mind.
The reference is here:
https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29474910&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=IQeuqKb215p409D%2BDkCaFdQjSz7n4G2gkB%2FrcFPaYS4%3D&reserved=0
DOI: 10.1016/j.cell.2018.02.007

Best,
Nicolai

>>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
Dr. Nicolai T. Urban
Imaging Center @ Max Planck Florida Institute Jupiter, 33458 FL, USA

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: Thursday, 10 October 2019 10:31
To: [hidden email]
Subject: Measuring cell volume by negative staining

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Dear list

I remember reading a paper where the authors wanted to precisely measure the volume of cells . They added a fluorophore to the medium, acquired a z stack and segmented the negative areas in the image to find the cells.
I cannot find this paper. Would anyone have a reference?

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: +46 (0) 73 733 5008
LCI website<https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fki.se%2Fen%2Fbionut%2Fwelcome-to-the-lci-facility&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=5qX2cf8isbESdUEYjPs3OXdaM2JIL6JPlW%2BLdGSk2cA%3D&reserved=0>
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Tremblay, Jessy

RE : Measuring cell volume by negative staining

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Hi,

for that purpose I like to stain the surface of cells with WGA-alexa just before fixation, 5min (not too long or it starts internalise) in the media at 5-10ug/ml, wash then fix and follow your protocols.

my 2 cents.

regards

Jessy Tremblay
Agent de recherche
Responsable du Laboratoire de microscopie confocale et cytométrie en flux
INRS-Institut Armand-Frappier
531, boulevard des Prairies
Laval (Québec) H7V 1B7
Tel.: 450-687-5010 #4314
Fax.: 450-686-5501

________________________________________
De : Confocal Microscopy List [[hidden email]] de la part de Jeremy Adler [[hidden email]]
Envoyé : 11 octobre 2019 05:38
À : [hidden email]
Objet : Re: Measuring cell volume by negative staining

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You could try fluorescent microspheres - I would expect 0.5um to be excluded from fixed cells.
Your fixed cells are likely to shrink in the Z axis - understating their volume.

Jeremy Adler
BioVis
Uppsala U

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: den 10 oktober 2019 17:15
To: [hidden email]
Subject: Re: Measuring cell volume by negative staining

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But I forgot to mention that our cells are fixed and permeabilized which makes it more complicated. We need to find a dye that is large enough to stay outside. :-/

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: +46 (0) 73 733 5008
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of [hidden email]
Sent: 10 October 2019 16:53
To: [hidden email]
Subject: Re: Measuring cell volume by negative staining

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Hi Sylvie,

I am not sure this is the paper you are looking for, but in 2018 Valentin Nägerl's group published a paper on "super-resolution shadow imaging" (SUSHI), in which they measured the extracellular space in brain tissue. Very nice paper, with lots of different applications, but maybe not what you had in mind.
The reference is here:
https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29474910&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=IQeuqKb215p409D%2BDkCaFdQjSz7n4G2gkB%2FrcFPaYS4%3D&reserved=0
DOI: 10.1016/j.cell.2018.02.007

Best,
Nicolai

>>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
Dr. Nicolai T. Urban
Imaging Center @ Max Planck Florida Institute Jupiter, 33458 FL, USA

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: Thursday, 10 October 2019 10:31
To: [hidden email]
Subject: Measuring cell volume by negative staining

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Dear list

I remember reading a paper where the authors wanted to precisely measure the volume of cells . They added a fluorophore to the medium, acquired a z stack and segmented the negative areas in the image to find the cells.
I cannot find this paper. Would anyone have a reference?

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: +46 (0) 73 733 5008
LCI website<https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fki.se%2Fen%2Fbionut%2Fwelcome-to-the-lci-facility&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=5qX2cf8isbESdUEYjPs3OXdaM2JIL6JPlW%2BLdGSk2cA%3D&reserved=0>
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Mike Nelson

Re: Measuring cell volume by negative staining

In reply to this post by Jeremy Adler-4

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This reminded me of a thread on the microscopy forum where the effects of
various mounting media types on the structures of the fixed cells were
compared. Might be worth a look.

https://forum.microlist.org/t/hardening-mounting-media-that-doesnt-compress-sample/416

Mike

On Fri, Oct 11, 2019, 2:51 AM Jeremy Adler <[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.
> *****
>
> You could try fluorescent microspheres - I would expect 0.5um to be
> excluded from fixed cells.
> Your fixed cells are likely to shrink in the Z axis - understating their
> volume.
>
> Jeremy Adler
> BioVis
> Uppsala U
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Sylvie Le Guyader
> Sent: den 10 oktober 2019 17:15
> To: [hidden email]
> Subject: Re: Measuring cell volume by negative staining
>
> *****
> 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.
> *****
>
> But I forgot to mention that our cells are fixed and permeabilized which
> makes it more complicated. We need to find a dye that is large enough to
> stay outside. :-/
>
> 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: +46 (0) 73 733 5008
> LCI website
> Follow our microscopy blog!
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of [hidden email]
> Sent: 10 October 2019 16:53
> To: [hidden email]
> Subject: Re: Measuring cell volume by negative staining
>
> *****
> 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&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488393237&sdata=A%2BP115B%2FQXTCtI2Yt7YFjNQNTGiUpa5I3ISXJuMGTHc%3D&reserved=0
> Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488393237&sdata=F%2BVE%2FsUhVI%2BvKs0GGy02DkLDVU9g2vGOWVeX3VzUXO8%3D&reserved=0
> and include the link in your posting.
> *****
>
> Hi Sylvie,
>
> I am not sure this is the paper you are looking for, but in 2018 Valentin
> Nägerl's group published a paper on "super-resolution shadow imaging"
> (SUSHI), in which they measured the extracellular space in brain tissue.
> Very nice paper, with lots of different applications, but maybe not what
> you had in mind.
> The reference is here:
>
> https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29474910&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=IQeuqKb215p409D%2BDkCaFdQjSz7n4G2gkB%2FrcFPaYS4%3D&reserved=0
> DOI: 10.1016/j.cell.2018.02.007
>
> Best,
> Nicolai
>
> >>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
> Dr. Nicolai T. Urban
> Imaging Center @ Max Planck Florida Institute Jupiter, 33458 FL, USA
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Sylvie Le Guyader
> Sent: Thursday, 10 October 2019 10:31
> To: [hidden email]
> Subject: Measuring cell volume by negative staining
>
> *****
> 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&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=Dtu04mI5S5u2m1%2Fz6zhkI0EEyZUqvbLOERd3TsAVcgQ%3D&reserved=0
> Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=Y%2BbXagdTeGp%2FEuAGbkQrtK3PL9bG9Yq2yo2GQeRzPzA%3D&reserved=0
> and include the link in your posting.
> *****
>
> Dear list
>
> I remember reading a paper where the authors wanted to precisely measure
> the volume of cells . They added a fluorophore to the medium, acquired a z
> stack and segmented the negative areas in the image to find the cells.
> I cannot find this paper. Would anyone have a reference?
>
> 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: +46 (0) 73 733 5008
> LCI website<
> https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fki.se%2Fen%2Fbionut%2Fwelcome-to-the-lci-facility&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=5qX2cf8isbESdUEYjPs3OXdaM2JIL6JPlW%2BLdGSk2cA%3D&reserved=0
> >
> Follow our microscopy blog!<
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopykarolinska.se%2F&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=zWzTlrwva5SJ%2Blb1xxbP5BB7NiMhMi6T6afqWCo0giU%3D&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<
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> >.
>
>
> 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>.
>
>
>
>
>
>
>
>
> När du har kontakt med oss på Uppsala universitet med e-post så innebär
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Michelle Peckham

Re: Measuring cell volume by negative staining

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Perhaps this paper - just published - might have an interesting nugget regarding this? (possibly could be used in fixed cells as well)

Constitutive expression of a fluorescent protein reports the size of
live human cells

Daniel F. Berenson, Evgeny Zatulovskiy, Shicong Xie, and Jan M.
Skotheim
Molecular Biology of the Cell, Vol. 0, No. 0: mbc.E19-03-0171.
https://www.molbiolcell.org/doi/10.1091/mbc.E19-03-0171?ai=25zg&ui=2rn1&af=T

On 11/10/2019, 18:31, "Confocal Microscopy List on behalf of Mike Nelson" <[hidden email] on behalf of [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.
*****

This reminded me of a thread on the microscopy forum where the effects of
various mounting media types on the structures of the fixed cells were
compared. Might be worth a look.

https://forum.microlist.org/t/hardening-mounting-media-that-doesnt-compress-sample/416

Mike

On Fri, Oct 11, 2019, 2:51 AM Jeremy Adler <[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.
> *****
>
> You could try fluorescent microspheres - I would expect 0.5um to be
> excluded from fixed cells.
> Your fixed cells are likely to shrink in the Z axis - understating their
> volume.
>
> Jeremy Adler
> BioVis
> Uppsala U
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Sylvie Le Guyader
> Sent: den 10 oktober 2019 17:15
> To: [hidden email]
> Subject: Re: Measuring cell volume by negative staining
>
> *****
> 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.
> *****
>
> But I forgot to mention that our cells are fixed and permeabilized which
> makes it more complicated. We need to find a dye that is large enough to
> stay outside. :-/
>
> 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: +46 (0) 73 733 5008
> LCI website
> Follow our microscopy blog!
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of [hidden email]
> Sent: 10 October 2019 16:53
> To: [hidden email]
> Subject: Re: Measuring cell volume by negative staining
>
> *****
> 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&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488393237&sdata=A%2BP115B%2FQXTCtI2Yt7YFjNQNTGiUpa5I3ISXJuMGTHc%3D&reserved=0
> Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488393237&sdata=F%2BVE%2FsUhVI%2BvKs0GGy02DkLDVU9g2vGOWVeX3VzUXO8%3D&reserved=0
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> *****
>
> Hi Sylvie,
>
> I am not sure this is the paper you are looking for, but in 2018 Valentin
> Nägerl's group published a paper on "super-resolution shadow imaging"
> (SUSHI), in which they measured the extracellular space in brain tissue.
> Very nice paper, with lots of different applications, but maybe not what
> you had in mind.
> The reference is here:
>
> https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29474910&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C6aba2a5736224f1042f808d74d91b386%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063160488403234&sdata=IQeuqKb215p409D%2BDkCaFdQjSz7n4G2gkB%2FrcFPaYS4%3D&reserved=0
> DOI: 10.1016/j.cell.2018.02.007
>
> Best,
> Nicolai
>
> >>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
> Dr. Nicolai T. Urban
> Imaging Center @ Max Planck Florida Institute Jupiter, 33458 FL, USA
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Sylvie Le Guyader
> Sent: Thursday, 10 October 2019 10:31
> To: [hidden email]
> Subject: Measuring cell volume by negative staining
>
> *****
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> Post images on
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> *****
>
> Dear list
>
> I remember reading a paper where the authors wanted to precisely measure
> the volume of cells . They added a fluorophore to the medium, acquired a z
> stack and segmented the negative areas in the image to find the cells.
> I cannot find this paper. Would anyone have a reference?
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
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