Strange Photo conversion

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Cameron Nowell-3 Cameron Nowell-3
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Strange Photo conversion

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Hi List,

We are trying to get some experiments up and running and have hit a bizarre
artifact with Cy5 getting photo converted and becoming very very bright.

The experimental desing is as follows
- fluorodish or ibidi channel slide
- coated with Poly-D-Lysine in PBS
- Washed PBS
- Washed Bi-carbonate pH 8.6
- Couple Cy 5 succinamide ester bi-carb pH 8.6
- Wash Bi-carb 8.6
- Wash PBS

IMaging on either a TIRF or confocal system we get a rapid and robust
increase in fluorescene in the Cy5 channel. Exited at any wavelegth (405,
488 or 647).

Any ideas on what could be going on?

Cheers

Cam


--

*Cameron J. Nowell*

Head



Imaging, FACS and Analysis Core

Monash Institute of Pharmaceutical Sciences

Monash University

399 Royal Parade

Parkville, VIC, 3052

Australia



*Email:* [hidden email]

*Phone: *+61 422882700



*LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>

*Research Gate: * Profile
<http://www.researchgate.net/profile/Cameron_Nowell>

*Google Scholar:* Profile
<https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>

*PubMed Bibliography: *Profile
<http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibliography/47922177/public/?sort=date&direction=ascending>
Cameron Nowell-3 Cameron Nowell-3
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Re: Strange Photo conversion

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

The plan is to use the poly d to anchor our cy5 that will be coupled to a
protein. Then come in with a fluorescent ligand in a different channel and
look at binding.

So if it's a quenching type thing would lowering the concentration of dye
help?


On 5 May 2017 11:27 am, <[hidden email]> wrote:

Are you trying to label the polylysine? In principle, heavily labeled stuff
can start out quenched and then with illumination to photobleach a few
molecules the system gets brighter. I've seen this a few times in the past,
though I have no way to know if you are in this strongly quenched regime.

> On May 4, 2017, at 6:05 PM, Cameron Nowell <[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 List,
>
> We are trying to get some experiments up and running and have hit a
bizarre

> artifact with Cy5 getting photo converted and becoming very very bright.
>
> The experimental desing is as follows
> - fluorodish or ibidi channel slide
> - coated with Poly-D-Lysine in PBS
> - Washed PBS
> - Washed Bi-carbonate pH 8.6
> - Couple Cy 5 succinamide ester bi-carb pH 8.6
> - Wash Bi-carb 8.6
> - Wash PBS
>
> IMaging on either a TIRF or confocal system we get a rapid and robust
> increase in fluorescene in the Cy5 channel. Exited at any wavelegth (405,
> 488 or 647).
>
> Any ideas on what could be going on?
>
> Cheers
>
> Cam
>
>
> --
>
> *Cameron J. Nowell*
>
> Head
>
>
>
> Imaging, FACS and Analysis Core
>
> Monash Institute of Pharmaceutical Sciences
>
> Monash University
>
> 399 Royal Parade
>
> Parkville, VIC, 3052
>
> Australia
>
>
>
> *Email:* [hidden email]
>
> *Phone: *+61 422882700
>
>
>
> *LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>
>
> *Research Gate: * Profile
> <http://www.researchgate.net/profile/Cameron_Nowell>
>
> *Google Scholar:* Profile
> <https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>
>
> *PubMed Bibliography: *Profile
> <http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/
bibliography/47922177/public/?sort=date&direction=ascending>
Cameron Nowell-3 Cameron Nowell-3
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Re: Strange Photo conversion

*****
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.
*****

The final experiment is to couple O6-benzyl-guanine via succinamide ester
to then allow attachment of a protien via a genetically encoded SNAP tag.

The Cy5 and poly d was just a proof of concept to see how stable the
labelling would be as  we tried the other way and couldn't see any signals.



On 5 May 2017 at 12:09, <[hidden email]> wrote:

> Sorry I still don't understand. Do you plan to label protein with Cy5
> separately first and then stick it to the polylysine in the channel after
> purification? Right now it sounds like you are just covalently attaching
> dye to lysine in the channel and I don't get why you would want that. Yes,
> lower concentrations of dye can reduce quenching if it is quenching, though
> as I said before, I am not certain.
>
> On May 4, 2017, at 6:32 PM, Cameron Nowell <[hidden email]>
> wrote:
>
> The plan is to use the poly d to anchor our cy5 that will be coupled to a
> protein. Then come in with a fluorescent ligand in a different channel and
> look at binding.
>
> So if it's a quenching type thing would lowering the concentration of dye
> help?
>
>
> On 5 May 2017 11:27 am, <[hidden email]> wrote:
>
> Are you trying to label the polylysine? In principle, heavily labeled
> stuff can start out quenched and then with illumination to photobleach a
> few molecules the system gets brighter. I've seen this a few times in the
> past, though I have no way to know if you are in this strongly quenched
> regime.
>
> > On May 4, 2017, at 6:05 PM, Cameron Nowell <[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 List,
> >
> > We are trying to get some experiments up and running and have hit a
> bizarre
> > artifact with Cy5 getting photo converted and becoming very very bright.
> >
> > The experimental desing is as follows
> > - fluorodish or ibidi channel slide
> > - coated with Poly-D-Lysine in PBS
> > - Washed PBS
> > - Washed Bi-carbonate pH 8.6
> > - Couple Cy 5 succinamide ester bi-carb pH 8.6
> > - Wash Bi-carb 8.6
> > - Wash PBS
> >
> > IMaging on either a TIRF or confocal system we get a rapid and robust
> > increase in fluorescene in the Cy5 channel. Exited at any wavelegth (405,
> > 488 or 647).
> >
> > Any ideas on what could be going on?
> >
> > Cheers
> >
> > Cam
> >
> >
> > --
> >
> > *Cameron J. Nowell*
> >
> > Head
> >
> >
> >
> > Imaging, FACS and Analysis Core
> >
> > Monash Institute of Pharmaceutical Sciences
> >
> > Monash University
> >
> > 399 Royal Parade
> >
> > Parkville, VIC, 3052
> >
> > Australia
> >
> >
> >
> > *Email:* [hidden email]
> >
> > *Phone: *+61 422882700
> >
> >
> >
> > *LinkedIn: *Profile <http://au.linkedin.com/pub/ca
> meron-nowell/23/57/884/>
> >
> > *Research Gate: * Profile
> > <http://www.researchgate.net/profile/Cameron_Nowell>
> >
> > *Google Scholar:* Profile
> > <https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>
> >
> > *PubMed Bibliography: *Profile
> > <http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibl
> iography/47922177/public/?sort=date&direction=ascending>
>
>
>


--

*Cameron J. Nowell*

Head



Imaging, FACS and Analysis Core

Monash Institute of Pharmaceutical Sciences

Monash University

399 Royal Parade

Parkville, VIC, 3052

Australia



*Email:* [hidden email]

*Phone: *+61 422882700



*LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>

*Research Gate: * Profile
<http://www.researchgate.net/profile/Cameron_Nowell>

*Google Scholar:* Profile
<https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>

*PubMed Bibliography: *Profile
<http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibliography/47922177/public/?sort=date&direction=ascending>
Kilgore, Jason A. Kilgore, Jason A.
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Re: Strange Photo conversion

*****
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, Cameron,

I don't know for certain, but I wonder if what you are seeing is a dye-dye quenching effect.

In other words, with some dyes (I'm not certain about Cy5, though), if over-labeled, will have FRET-based dye-dye quenching.  If you then photobleach the sample, it reduces the quenching, leading initially to a brighter signal.  (If the photobleaching continues, the signal will plateau at the point when completely unquenched, then will decrease as the dye molecules continue photobleaching).

If this is what is happening, then as you supposed, reducing the dye concentration should reduce the dye-dye quenching.

Cheers,

Jason


Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, then option 2.
Or dial direct at +1 541 335 0353
[hidden email]
www.thermofisher.com

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Thursday, May 04, 2017 8:55 PM
To: [hidden email]
Subject: Re: Strange Photo conversion

*****
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.
*****

The final experiment is to couple O6-benzyl-guanine via succinamide ester to then allow attachment of a protien via a genetically encoded SNAP tag.

The Cy5 and poly d was just a proof of concept to see how stable the labelling would be as  we tried the other way and couldn't see any signals.



On 5 May 2017 at 12:09, <[hidden email]> wrote:

> Sorry I still don't understand. Do you plan to label protein with Cy5
> separately first and then stick it to the polylysine in the channel
> after purification? Right now it sounds like you are just covalently
> attaching dye to lysine in the channel and I don't get why you would
> want that. Yes, lower concentrations of dye can reduce quenching if it
> is quenching, though as I said before, I am not certain.
>
> On May 4, 2017, at 6:32 PM, Cameron Nowell <[hidden email]>
> wrote:
>
> The plan is to use the poly d to anchor our cy5 that will be coupled
> to a protein. Then come in with a fluorescent ligand in a different
> channel and look at binding.
>
> So if it's a quenching type thing would lowering the concentration of
> dye help?
>
>
> On 5 May 2017 11:27 am, <[hidden email]> wrote:
>
> Are you trying to label the polylysine? In principle, heavily labeled
> stuff can start out quenched and then with illumination to photobleach
> a few molecules the system gets brighter. I've seen this a few times
> in the past, though I have no way to know if you are in this strongly
> quenched regime.
>
> > On May 4, 2017, at 6:05 PM, Cameron Nowell
> > <[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 List,
> >
> > We are trying to get some experiments up and running and have hit a
> bizarre
> > artifact with Cy5 getting photo converted and becoming very very bright.
> >
> > The experimental desing is as follows
> > - fluorodish or ibidi channel slide
> > - coated with Poly-D-Lysine in PBS
> > - Washed PBS
> > - Washed Bi-carbonate pH 8.6
> > - Couple Cy 5 succinamide ester bi-carb pH 8.6
> > - Wash Bi-carb 8.6
> > - Wash PBS
> >
> > IMaging on either a TIRF or confocal system we get a rapid and
> > robust increase in fluorescene in the Cy5 channel. Exited at any
> > wavelegth (405,
> > 488 or 647).
> >
> > Any ideas on what could be going on?
> >
> > Cheers
> >
> > Cam
> >
> >
> > --
> >
> > *Cameron J. Nowell*
> >
> > Head
> >
> >
> >
> > Imaging, FACS and Analysis Core
> >
> > Monash Institute of Pharmaceutical Sciences
> >
> > Monash University
> >
> > 399 Royal Parade
> >
> > Parkville, VIC, 3052
> >
> > Australia
> >
> >
> >
> > *Email:* [hidden email]
> >
> > *Phone: *+61 422882700
> >
> >
> >
> > *LinkedIn: *Profile <http://au.linkedin.com/pub/ca
> meron-nowell/23/57/884/>
> >
> > *Research Gate: * Profile
> > <http://www.researchgate.net/profile/Cameron_Nowell>
> >
> > *Google Scholar:* Profile
> > <https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>
> >
> > *PubMed Bibliography: *Profile
> > <http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibl
> iography/47922177/public/?sort=date&direction=ascending>
>
>
>


--

*Cameron J. Nowell*

Head



Imaging, FACS and Analysis Core

Monash Institute of Pharmaceutical Sciences

Monash University

399 Royal Parade

Parkville, VIC, 3052

Australia



*Email:* [hidden email]

*Phone: *+61 422882700



*LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>

*Research Gate: * Profile
<http://www.researchgate.net/profile/Cameron_Nowell>

*Google Scholar:* Profile
<https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>

*PubMed Bibliography: *Profile
<http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibliography/47922177/public/?sort=date&direction=ascending>
Cameron Nowell-3 Cameron Nowell-3
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Re: Strange Photo conversion

*****
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,

Yep it was self quenching. Once we took the dilution down it stopped
getting brighter. Actually got it down dilute enough to see textbook step
wise bleaching of single fluors :)

Cheers

Cam

On 6 May 2017 at 02:22, Kilgore, Jason A. <[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, Cameron,
>
> I don't know for certain, but I wonder if what you are seeing is a dye-dye
> quenching effect.
>
> In other words, with some dyes (I'm not certain about Cy5, though), if
> over-labeled, will have FRET-based dye-dye quenching.  If you then
> photobleach the sample, it reduces the quenching, leading initially to a
> brighter signal.  (If the photobleaching continues, the signal will plateau
> at the point when completely unquenched, then will decrease as the dye
> molecules continue photobleaching).
>
> If this is what is happening, then as you supposed, reducing the dye
> concentration should reduce the dye-dye quenching.
>
> Cheers,
>
> Jason
>
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Life Sciences Solutions
>
> Thermo Fisher Scientific
> 29851 Willow Creek Rd.
> Eugene, OR  97402-9132
> 1-800-955-6288 then option 4, then option 6, then option 2.
> Or dial direct at +1 541 335 0353
> [hidden email]
> www.thermofisher.com
>
> This communication is intended solely for the individual/entity to whom it
> is addressed. It may contain confidential or legally privileged
> information.  Any unauthorized disclosure or copying is prohibited and may
> be unlawful. If you have received this communication in error, please
> notify the sender immediately and delete it from your system.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Cameron Nowell
> Sent: Thursday, May 04, 2017 8:55 PM
> To: [hidden email]
> Subject: Re: Strange Photo conversion
>
> *****
> 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.
> *****
>
> The final experiment is to couple O6-benzyl-guanine via succinamide ester
> to then allow attachment of a protien via a genetically encoded SNAP tag.
>
> The Cy5 and poly d was just a proof of concept to see how stable the
> labelling would be as  we tried the other way and couldn't see any signals.
>
>
>
> On 5 May 2017 at 12:09, <[hidden email]> wrote:
>
> > Sorry I still don't understand. Do you plan to label protein with Cy5
> > separately first and then stick it to the polylysine in the channel
> > after purification? Right now it sounds like you are just covalently
> > attaching dye to lysine in the channel and I don't get why you would
> > want that. Yes, lower concentrations of dye can reduce quenching if it
> > is quenching, though as I said before, I am not certain.
> >
> > On May 4, 2017, at 6:32 PM, Cameron Nowell <[hidden email]>
> > wrote:
> >
> > The plan is to use the poly d to anchor our cy5 that will be coupled
> > to a protein. Then come in with a fluorescent ligand in a different
> > channel and look at binding.
> >
> > So if it's a quenching type thing would lowering the concentration of
> > dye help?
> >
> >
> > On 5 May 2017 11:27 am, <[hidden email]> wrote:
> >
> > Are you trying to label the polylysine? In principle, heavily labeled
> > stuff can start out quenched and then with illumination to photobleach
> > a few molecules the system gets brighter. I've seen this a few times
> > in the past, though I have no way to know if you are in this strongly
> > quenched regime.
> >
> > > On May 4, 2017, at 6:05 PM, Cameron Nowell
> > > <[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 List,
> > >
> > > We are trying to get some experiments up and running and have hit a
> > bizarre
> > > artifact with Cy5 getting photo converted and becoming very very
> bright.
> > >
> > > The experimental desing is as follows
> > > - fluorodish or ibidi channel slide
> > > - coated with Poly-D-Lysine in PBS
> > > - Washed PBS
> > > - Washed Bi-carbonate pH 8.6
> > > - Couple Cy 5 succinamide ester bi-carb pH 8.6
> > > - Wash Bi-carb 8.6
> > > - Wash PBS
> > >
> > > IMaging on either a TIRF or confocal system we get a rapid and
> > > robust increase in fluorescene in the Cy5 channel. Exited at any
> > > wavelegth (405,
> > > 488 or 647).
> > >
> > > Any ideas on what could be going on?
> > >
> > > Cheers
> > >
> > > Cam
> > >
> > >
> > > --
> > >
> > > *Cameron J. Nowell*
> > >
> > > Head
> > >
> > >
> > >
> > > Imaging, FACS and Analysis Core
> > >
> > > Monash Institute of Pharmaceutical Sciences
> > >
> > > Monash University
> > >
> > > 399 Royal Parade
> > >
> > > Parkville, VIC, 3052
> > >
> > > Australia
> > >
> > >
> > >
> > > *Email:* [hidden email]
> > >
> > > *Phone: *+61 422882700
> > >
> > >
> > >
> > > *LinkedIn: *Profile <http://au.linkedin.com/pub/ca
> > meron-nowell/23/57/884/>
> > >
> > > *Research Gate: * Profile
> > > <http://www.researchgate.net/profile/Cameron_Nowell>
> > >
> > > *Google Scholar:* Profile
> > > <https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>
> > >
> > > *PubMed Bibliography: *Profile
> > > <http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibl
> > iography/47922177/public/?sort=date&direction=ascending>
> >
> >
> >
>
>
> --
>
> *Cameron J. Nowell*
>
> Head
>
>
>
> Imaging, FACS and Analysis Core
>
> Monash Institute of Pharmaceutical Sciences
>
> Monash University
>
> 399 Royal Parade
>
> Parkville, VIC, 3052
>
> Australia
>
>
>
> *Email:* [hidden email]
>
> *Phone: *+61 422882700
>
>
>
> *LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>
>
> *Research Gate: * Profile
> <http://www.researchgate.net/profile/Cameron_Nowell>
>
> *Google Scholar:* Profile
> <https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>
>
> *PubMed Bibliography: *Profile
> <http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/
> bibliography/47922177/public/?sort=date&direction=ascending>
>



--

*Cameron J. Nowell*

Head



Imaging, FACS and Analysis Core

Monash Institute of Pharmaceutical Sciences

Monash University

399 Royal Parade

Parkville, VIC, 3052

Australia



*Email:* [hidden email]

*Phone: *+61 422882700



*LinkedIn: *Profile <http://au.linkedin.com/pub/cameron-nowell/23/57/884/>

*Research Gate: * Profile
<http://www.researchgate.net/profile/Cameron_Nowell>

*Google Scholar:* Profile
<https://scholar.google.com/citations?user=qqQfgZIAAAAJ&hl=en>

*PubMed Bibliography: *Profile
<http://www.ncbi.nlm.nih.gov/sites/myncbi/1TK3WeparcvQr/bibliography/47922177/public/?sort=date&direction=ascending>
张莉莉 张莉莉
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measure vacuole and cell volume

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

Hi all,
I have hundreds of zess confocal z stack images. Now I need to measure the volume of the cell and volume of structures inside the cell. Are there softwares or quick way to do it? Thanks!
Best regards,
LILI ZHANG
mmodel mmodel
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Re: measure vacuole and cell volume

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Lili,

You can do that with Imaris (www.bitplane.com), Amira (https://www.fei.com/software/amira-3d-for-life-sciences/), Voloom (https://micro-dimensions.com/voloom/) and ImageJ plugins Volumest (http://lepo.it.da.ut.ee/~markkom/volumest/) and Volume Calculator (http://imagej.net/Volume_Calculator). You can also manual “integration” by summation of cell areas at each z-level multiplied by vertical step size. But remember to correct for vertical elongation or shortening

Mike


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of ???
Sent: Wednesday, May 10, 2017 9:38 AM
To: [hidden email]
Subject: measure vacuole and cell volume

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Hi all,
I have hundreds of zess confocal z stack images. Now I need to measure the volume of the cell and volume of structures inside the cell. Are there softwares or quick way to do it? Thanks!
Best regards,
LILI ZHANG
micro micro
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Re: measure vacuole and cell volume

In reply to this post by 张莉莉
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Hi Lili,

There's a lot of extra information that would be useful to us to be able to
help you with an answer. Perhaps you could upload an image of a slice to
imgur (see the header to this email), and that will provide us with some of
that (cell size, tissue/culture type, staining pattern, signal to noise
ratio, etc.)

Different tools will work with different efficacy depending on the
parameters I briefly listed above, but probably using FIJI (FIJI.sc) is the
way to go.

If you don't have any experience in programming or image processing, it's
not going to be quick this first time. If you have the intention to do a
similar experiment again and again, it is worth investing the time now to
learn how to automate the analysis, even if only partially, as it will save
you hours of mind-numbing analysis in the future.

Best wishes,
Chris
________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of 张莉莉 <[hidden email]>
Sent: Wednesday, May 10, 2017 9:37 AM
To: [hidden email]
Subject: measure vacuole and cell volume

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Hi all,
I have hundreds of zess confocal z stack images. Now I need to measure the volume of the cell and volume of structures inside the cell. Are there softwares or quick way to do it? Thanks!
Best regards,
LILI ZHANG

张莉莉 张莉莉
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Re: measure vacuole and cell volume

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Dear Mike and Chris,
Thank you for your suggestions. Sorry I cannot log in imgur. I do live zebrafish macrophages imaging, so the shape of cells is irregular shape.  Later, I also need to measure fluorescent intensity. Since I have a lot of images, so it is better for me to find a way to do automate analysis. So what can I start with if I want to learn how to automate the analysis?
Thanks!
Lili








At 2017-05-10 23:24:04, "micro" <[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 Lili,
>
>There's a lot of extra information that would be useful to us to be able to
>help you with an answer. Perhaps you could upload an image of a slice to
>imgur (see the header to this email), and that will provide us with some of
>that (cell size, tissue/culture type, staining pattern, signal to noise
>ratio, etc.)
>
>Different tools will work with different efficacy depending on the
>parameters I briefly listed above, but probably using FIJI (FIJI.sc) is the
>way to go.
>
>If you don't have any experience in programming or image processing, it's
>not going to be quick this first time. If you have the intention to do a
>similar experiment again and again, it is worth investing the time now to
>learn how to automate the analysis, even if only partially, as it will save
>you hours of mind-numbing analysis in the future.
>
>Best wishes,
>Chris
>________________________________________
>From: Confocal Microscopy List <[hidden email]> on behalf of 张莉莉 <[hidden email]>
>Sent: Wednesday, May 10, 2017 9:37 AM
>To: [hidden email]
>Subject: measure vacuole and cell volume
>
>*****
>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,
>I have hundreds of zess confocal z stack images. Now I need to measure the volume of the cell and volume of structures inside the cell. Are there softwares or quick way to do it? Thanks!
>Best regards,
>LILI ZHANG
>