In or Out, that's the question

[Konstantín Levitskiy]

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Dear all,

Lately several of our researchers want to know if some particles are able to
enter into the cells (by phagocytosis, etc.) or if some structures are
inside the cell or on the cell surface. Normally they have monolayer growth
cell type, like a HeLa-line and such. My advise usually is based on
consideration to make the cells round after tripsinisation and afterwards to
do a confocal study in a media without Ca iones to avoid quick adherence to
the plaque surface. One of the researcher had red-coloured particles and we
used calcein-AM to make a cell form visible. But the emission spectra of
calcein

https://www.thermofisher.com/order/catalog/product/C1430

is a bit wide and tends to saturate some compartments inside the cell. What
do you recommend in this case? What kind of dye would you use for cell
visualisation to cover different spectra range (405, 488, 561, 633)? Could
it be better to try maintaining the cells alive?

Also, take into consideration that particles diameter can be up to 1.5 mm
+-.

Thanks in advance,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email:  <mailto:[hidden email]> [hidden email]

Web:  <http://www.ibis-sevilla.es/> www.ibis-sevilla.es

 

[Martin Wessendorf-2]

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Dear Dr. Levitskiy--

1)  Are you exciting both the calcein and the particle simultaneously?  
If so, that may be part of the problem.  --Based on its excitation
spectrum, you should get no calcein fluorescence at all if you excite at
a wavelength higher than 540 nm.  Thus I'd use sequential excitation:
first excite the calcein (say, at 488 nm), and then excite the red
fluorophore, (making certain to use a wavelength higher than 540 nm).  
Unless the excitation spectrum of calcein changes qualitatively in the
cellular environment, you should be able to distinguish the two clearly.

2)  With regard to whether or not the particles are inside the cell--I
don't think that that question can necessarily be answered conclusively
by light microscopy.  If the particles are resolvably inside the cells,
then you're fine.  However, if they're close to the membrane, it may be
difficult to prove whether they're in or out.  If the fluorescence of
the particle can be quenched, you might try adding the quenching agent
to the culture medium.

3)  Could DIC be used to image the cell surface, rather than fluorescence?

(--The particles' sizes are up to 1.5 micrometers, not 1.5 millimeters,
correct?)

Good luck!

Martin Wessendorf





On 2/8/2017 7:01 AM, Konstantín Levitskiy 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 all,
>
> Lately several of our researchers want to know if some particles are able to
> enter into the cells (by phagocytosis, etc.) or if some structures are
> inside the cell or on the cell surface. Normally they have monolayer growth
> cell type, like a HeLa-line and such. My advise usually is based on
> consideration to make the cells round after tripsinisation and afterwards to
> do a confocal study in a media without Ca iones to avoid quick adherence to
> the plaque surface. One of the researcher had red-coloured particles and we
> used calcein-AM to make a cell form visible. But the emission spectra of
> calcein
>
> https://www.thermofisher.com/order/catalog/product/C1430
>
> is a bit wide and tends to saturate some compartments inside the cell. What
> do you recommend in this case? What kind of dye would you use for cell
> visualisation to cover different spectra range (405, 488, 561, 633)? Could
> it be better to try maintaining the cells alive?
>
> Also, take into consideration that particles diameter can be up to 1.5 mm
> +-.
>
> Thanks in advance,
>
> Dr. Konstantín Levitskiy
>
> Servicio de Microscopía
>
> InstitutodeBiomedicinadeSevilla - IBiS
>
> Campus del Hospital Universitario Virgen del Rocío
>
> Avda. Manuel Siurot s/nº
>
> 41013 Sevilla
>
> Tlfno: 955 92 3030
>
> Email:  <mailto:[hidden email]> [hidden email]
>
> Web:  <http://www.ibis-sevilla.es/> www.ibis-sevilla.es
>
>  

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Konstantín Levitskiy]

*****
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.
*****

Dear Martin,

 

Please, visit the images on

http://imgur.com/a/6566G

 

Ø  Dear Dr. Levitskiy--

Ø  1)  Are you exciting both the calcein and the particle simultaneously?  

 

Not, of course. Definitely it was a sequential scan.

 

Ø  If so, that may be part of the problem.  --Based on its excitation
spectrum, you should get no calcein fluorescence at all if you excite at a
wavelength higher than 540 nm.  Thus I'd use sequential excitation: first
excite the calcein (say, at 488 nm), and then excite the red fluorophore,
(making certain to use a wavelength higher than 540 nm).  Unless the
excitation spectrum of calcein changes qualitatively in the cellular
environment, you should be able to distinguish the two clearly.

 

I might be not explain well. The trouble of calcein that it is going to some
compartmentalisation and shine so bright that you can’t see the cell
dimension (limits), as in the post images for the first cell. For the next
cell it is more clear and as it is distributed more homogeneously.

It seems that the coloured particle has quite wide emission spectrum (or in
some way exciting spectrum), so it also appears in the green cannel.

 

Ø  2)  With regard to whether or not the particles are inside the cell--I
don't think that that question can necessarily be answered conclusively by
light microscopy.  If the particles are resolvably inside the cells, then
you're fine.  However, if they're close to the membrane, it may be difficult
to prove whether they're in or out.  If the fluorescence of the particle can
be quenched, you might try adding the quenching agent to the culture medium.

 

Ø  3)  Could DIC be used to image the cell surface, rather than
fluorescence?

 

I don’t know. We have to check it.

 

Ø  (--The particles' sizes are up to 1.5 micrometers, not 1.5 millimeters,
correct?)

 

Yes, you have to read microns ;-)

mkm = mm

 

Ø  Good luck!

Ø  Martin Wessendorf

 

Any advice for cell dye in blue, red or far red range?

 

 

 

On 2/8/2017 7:01 AM, Konstantín Levitskiy wrote:

> *****

> To join, leave or search the confocal microscopy listserv, go to:

>  <http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

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

> *****

>

> Dear all,

>

> Lately several of our researchers want to know if some particles are

> able to enter into the cells (by phagocytosis, etc.) or if some

> structures are inside the cell or on the cell surface. Normally they

> have monolayer growth cell type, like a HeLa-line and such. My advise

> usually is based on consideration to make the cells round after

> tripsinisation and afterwards to do a confocal study in a media

> without Ca iones to avoid quick adherence to the plaque surface. One

> of the researcher had red-coloured particles and we used calcein-AM to

> make a cell form visible. But the emission spectra of calcein

>

>  <https://www.thermofisher.com/order/catalog/product/C1430>
https://www.thermofisher.com/order/catalog/product/C1430

>

> is a bit wide and tends to saturate some compartments inside the cell.

> What do you recommend in this case? What kind of dye would you use for

> cell visualisation to cover different spectra range (405, 488, 561,

> 633)? Could it be better to try maintaining the cells alive?

>

> Also, take into consideration that particles diameter can be up to 1.5

> mm

> +-.

>

> Thanks in advance,

>

> Dr. Konstantín Levitskiy

>

> Servicio de Microscopía

>

> InstitutodeBiomedicinadeSevilla - IBiS

>

> Campus del Hospital Universitario Virgen del Rocío

>

> Avda. Manuel Siurot s/nº

>

> 41013 Sevilla

>

> Tlfno: 955 92 3030

>

> Email:  < <mailto:[hidden email]> mailto:[hidden email]>
<mailto:[hidden email]> [hidden email]

>

> Web:  < <http://www.ibis-sevilla.es/> http://www.ibis-sevilla.es/>
<http://www.ibis-sevilla.es> www.ibis-sevilla.es

>

>  

 

--

Martin Wessendorf, Ph.D.                   office: [phone]

Assoc Prof, Dept Neuroscience                 lab: [phone]

University of Minnesota             Preferred FAX: [phone]

6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]

Minneapolis, MN  55455                    e-mail:  <mailto:[hidden email]>
[hidden email]

[mcammer]

*****
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.
*****

I don't know if the images you posted are the raw data or whether a LUT has been applied.  The images show a lot of saturated voxels.
If light microscopy is going to be able to resolve this, it will only do so if there are no saturated voxels in the raw data.
And how about an additional label like celltracker violet or a membrane label?
_________________________________________
Michael Cammer, Optical Microscopy Specialist
http://ocs.med.nyu.edu/microscopy
http://microscopynotes.com/
Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Konstantín Levitskiy [[hidden email]]
Sent: Thursday, February 09, 2017 6:56 AM
To: [hidden email]
Subject: Re: In or Out, that's the question

*****
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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=JhT4E6hIVKvUotngVhqnd36GpEftXwGIckLmErSOWyU&e=  and include the link in your posting.
*****

Dear Martin,



Please, visit the images on

https://urldefense.proofpoint.com/v2/url?u=http-3A__imgur.com_a_6566G&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=aSAm9ro4FPlYymgCd3-9Xn126l-jH_ZY7tg9NVCiNoI&e=



Ø  Dear Dr. Levitskiy--

Ø  1)  Are you exciting both the calcein and the particle simultaneously?



Not, of course. Definitely it was a sequential scan.



Ø  If so, that may be part of the problem.  --Based on its excitation
spectrum, you should get no calcein fluorescence at all if you excite at a
wavelength higher than 540 nm.  Thus I'd use sequential excitation: first
excite the calcein (say, at 488 nm), and then excite the red fluorophore,
(making certain to use a wavelength higher than 540 nm).  Unless the
excitation spectrum of calcein changes qualitatively in the cellular
environment, you should be able to distinguish the two clearly.



I might be not explain well. The trouble of calcein that it is going to some
compartmentalisation and shine so bright that you can’t see the cell
dimension (limits), as in the post images for the first cell. For the next
cell it is more clear and as it is distributed more homogeneously.

It seems that the coloured particle has quite wide emission spectrum (or in
some way exciting spectrum), so it also appears in the green cannel.



Ø  2)  With regard to whether or not the particles are inside the cell--I
don't think that that question can necessarily be answered conclusively by
light microscopy.  If the particles are resolvably inside the cells, then
you're fine.  However, if they're close to the membrane, it may be difficult
to prove whether they're in or out.  If the fluorescence of the particle can
be quenched, you might try adding the quenching agent to the culture medium.



Ø  3)  Could DIC be used to image the cell surface, rather than
fluorescence?



I don’t know. We have to check it.



Ø  (--The particles' sizes are up to 1.5 micrometers, not 1.5 millimeters,
correct?)



Yes, you have to read microns ;-)

mkm = mm



Ø  Good luck!

Ø  Martin Wessendorf



Any advice for cell dye in blue, red or far red range?







On 2/8/2017 7:01 AM, Konstantín Levitskiy wrote:

> *****

> To join, leave or search the confocal microscopy listserv, go to:

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> Post images on  <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=JhT4E6hIVKvUotngVhqnd36GpEftXwGIckLmErSOWyU&e= > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=JhT4E6hIVKvUotngVhqnd36GpEftXwGIckLmErSOWyU&e=  and include
the link in your posting.

> *****

>

> Dear all,

>

> Lately several of our researchers want to know if some particles are

> able to enter into the cells (by phagocytosis, etc.) or if some

> structures are inside the cell or on the cell surface. Normally they

> have monolayer growth cell type, like a HeLa-line and such. My advise

> usually is based on consideration to make the cells round after

> tripsinisation and afterwards to do a confocal study in a media

> without Ca iones to avoid quick adherence to the plaque surface. One

> of the researcher had red-coloured particles and we used calcein-AM to

> make a cell form visible. But the emission spectra of calcein

>

>  <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.thermofisher.com_order_catalog_product_C1430&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=z0O2ipSwcOpW7M3EnRBTktTohUUBEHtgAE7XFYaWaPg&e= >
https://urldefense.proofpoint.com/v2/url?u=https-3A__www.thermofisher.com_order_catalog_product_C1430&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=z0O2ipSwcOpW7M3EnRBTktTohUUBEHtgAE7XFYaWaPg&e=

>

> is a bit wide and tends to saturate some compartments inside the cell.

> What do you recommend in this case? What kind of dye would you use for

> cell visualisation to cover different spectra range (405, 488, 561,

> 633)? Could it be better to try maintaining the cells alive?

>

> Also, take into consideration that particles diameter can be up to 1.5

> mm

> +-.

>

> Thanks in advance,

>

> Dr. Konstantín Levitskiy

>

> Servicio de Microscopía

>

> InstitutodeBiomedicinadeSevilla - IBiS

>

> Campus del Hospital Universitario Virgen del Rocío

>

> Avda. Manuel Siurot s/nº

>

> 41013 Sevilla

>

> Tlfno: 955 92 3030

>

> Email:  < <mailto:[hidden email]> mailto:[hidden email]>
<mailto:[hidden email]> [hidden email]

>

> Web:  < <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla.es_&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=NhQGX15W2uBJWJBf5tueJLRQ8lRER-A-eH2Wq1a1mF8&e= > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla.es_&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=NhQGX15W2uBJWJBf5tueJLRQ8lRER-A-eH2Wq1a1mF8&e= >
<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla.es&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=4Fww7Qq0eekaIlK2uAOe5DzKAHnydGb5r-baBFkQtLU&e= > www.ibis-sevilla.es

>

>



--

Martin Wessendorf, Ph.D.                   office: [phone]

Assoc Prof, Dept Neuroscience                 lab: [phone]

University of Minnesota             Preferred FAX: [phone]

6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]

Minneapolis, MN  55455                    e-mail:  <mailto:[hidden email]>
[hidden email]

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[Sylvie Le Guyader]

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*****



---- Original Message ----
Subject: RE: In or Out, that's the question
Sent: 10 Feb 2017 9:43 a.m.
From: Sylvie Le Guyader <[hidden email]>
To: Confocal Microscopy List <[hidden email]>
Cc:

Hi Konstantin

- Why do you advise your users to round up the cells? You can very reliably look for internalization with adherent cells.
- Are you imaging in 16 bits? If not I think changing to 16 bits will help you avoid saturation in bright areas while still allowing you to image low areas.
- If I understand well, the cells become super fluorescent because tons of Calcein is internalized so it sounds to me that you have answered the questions to whether it is internalized or not.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: den 9 februari 2017 22:39
To: [hidden email]
Subject: Re: In or Out, that's the question

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

I don't know if the images you posted are the raw data or whether a LUT has been applied.  The images show a lot of saturated voxels.
If light microscopy is going to be able to resolve this, it will only do so if there are no saturated voxels in the raw data.
And how about an additional label like celltracker violet or a membrane label?
_________________________________________
Michael Cammer, Optical Microscopy Specialist http://ocs.med.nyu.edu/microscopy http://microscopynotes.com/
Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Konstantín Levitskiy [[hidden email]]
Sent: Thursday, February 09, 2017 6:56 AM
To: [hidden email]
Subject: Re: In or Out, that's the question

*****
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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=JhT4E6hIVKvUotngVhqnd36GpEftXwGIckLmErSOWyU&e=  and include the link in your posting.
*****

Dear Martin,



Please, visit the images on

https://urldefense.proofpoint.com/v2/url?u=http-3A__imgur.com_a_6566G&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=aSAm9ro4FPlYymgCd3-9Xn126l-jH_ZY7tg9NVCiNoI&e=



Ø  Dear Dr. Levitskiy--

Ø  1)  Are you exciting both the calcein and the particle simultaneously?



Not, of course. Definitely it was a sequential scan.



Ø  If so, that may be part of the problem.  --Based on its excitation spectrum, you should get no calcein fluorescence at all if you excite at a wavelength higher than 540 nm.  Thus I'd use sequential excitation: first excite the calcein (say, at 488 nm), and then excite the red fluorophore, (making certain to use a wavelength higher than 540 nm).  Unless the excitation spectrum of calcein changes qualitatively in the cellular environment, you should be able to distinguish the two clearly.



I might be not explain well. The trouble of calcein that it is going to some compartmentalisation and shine so bright that you can’t see the cell dimension (limits), as in the post images for the first cell. For the next cell it is more clear and as it is distributed more homogeneously.

It seems that the coloured particle has quite wide emission spectrum (or in some way exciting spectrum), so it also appears in the green cannel.



Ø  2)  With regard to whether or not the particles are inside the cell--I don't think that that question can necessarily be answered conclusively by light microscopy.  If the particles are resolvably inside the cells, then you're fine.  However, if they're close to the membrane, it may be difficult to prove whether they're in or out.  If the fluorescence of the particle can be quenched, you might try adding the quenching agent to the culture medium.



Ø  3)  Could DIC be used to image the cell surface, rather than fluorescence?



I don’t know. We have to check it.



Ø  (--The particles' sizes are up to 1.5 micrometers, not 1.5 millimeters,
correct?)



Yes, you have to read microns ;-)

mkm = mm



Ø  Good luck!

Ø  Martin Wessendorf



Any advice for cell dye in blue, red or far red range?







On 2/8/2017 7:01 AM, Konstantín Levitskiy wrote:

> *****

> To join, leave or search the confocal microscopy listserv, go to:

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> Post images on
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> 6I&s=JhT4E6hIVKvUotngVhqnd36GpEftXwGIckLmErSOWyU&e=  and include
the link in your posting.

> *****

>

> Dear all,

>

> Lately several of our researchers want to know if some particles are

> able to enter into the cells (by phagocytosis, etc.) or if some

> structures are inside the cell or on the cell surface. Normally they

> have monolayer growth cell type, like a HeLa-line and such. My advise

> usually is based on consideration to make the cells round after

> tripsinisation and afterwards to do a confocal study in a media

> without Ca iones to avoid quick adherence to the plaque surface. One

> of the researcher had red-coloured particles and we used calcein-AM to

> make a cell form visible. But the emission spectra of calcein

>

>
> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.thermofisher
> .com_order_catalog_product_C1430&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGm
> uw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNME
> Q0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=z0O2ipSwcOpW7M3EnRBTktTohUUB
> EHtgAE7XFYaWaPg&e= >
https://urldefense.proofpoint.com/v2/url?u=https-3A__www.thermofisher.com_order_catalog_product_C1430&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=z0O2ipSwcOpW7M3EnRBTktTohUUBEHtgAE7XFYaWaPg&e=

>

> is a bit wide and tends to saturate some compartments inside the cell.

> What do you recommend in this case? What kind of dye would you use for

> cell visualisation to cover different spectra range (405, 488, 561,

> 633)? Could it be better to try maintaining the cells alive?

>

> Also, take into consideration that particles diameter can be up to 1.5

> mm

> +-.

>

> Thanks in advance,

>

> Dr. Konstantín Levitskiy

>

> Servicio de Microscopía

>

> InstitutodeBiomedicinadeSevilla - IBiS

>

> Campus del Hospital Universitario Virgen del Rocío

>

> Avda. Manuel Siurot s/nº

>

> 41013 Sevilla

>

> Tlfno: 955 92 3030

>

> Email:  < <mailto:[hidden email]>
> mailto:[hidden email]>
<mailto:[hidden email]> [hidden email]

>

> Web:  <
> <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevill
> a.es_&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05Lz
> tNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2
> wl2i_hUuyc6I&s=NhQGX15W2uBJWJBf5tueJLRQ8lRER-A-eH2Wq1a1mF8&e= >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla
> .es_&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05Lzt
> NstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2w
> l2i_hUuyc6I&s=NhQGX15W2uBJWJBf5tueJLRQ8lRER-A-eH2Wq1a1mF8&e= >
<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla.es&d=DQIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=4Fww7Qq0eekaIlK2uAOe5DzKAHnydGb5r-baBFkQtLU&e= > www.ibis-sevilla.es<http://www.ibis-sevilla.es>

>

>



--

Martin Wessendorf, Ph.D.                   office: [phone]

Assoc Prof, Dept Neuroscience                 lab: [phone]

University of Minnesota             Preferred FAX: [phone]

6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]

Minneapolis, MN  55455                    e-mail:  <mailto:[hidden email]>
[hidden email]

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[Konstantín Levitskiy]

*****
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 Sylvie.

 

I'm glad to hear you again.

 

> - Why do you advise your users to round up the cells? You can very
reliably look for internalization with adherent cells.

 

First of all it depends on the particle size. The researcher has the
particles of 1,4 and 1,8 microns. I suppose that if the particle is near the
nucleus it will be easier to decide, but if quite adherent cell type has the
particle included in the very thin two layer membrane with practically
nothing of cytoplasm it will be very difficult to appreciate the inclusion.

 

When the cell dimensions are several times bigger that the particle size, no
doubts if the cell are round and the particle is clearly inside. It would be
more complicated if the particle would be in the cell membrane.

 

I don't really think that tripsinization could affect the particle
inclusion, not favour for it (it is too quick step compared to the inclusion
process), on the contrary it's a normal step to collect cells for other
experiments.

 

> - Are you imaging in 16 bits? If not I think changing to 16 bits will help
you avoid saturation in bright areas while still allowing you to image low
areas.

 

In that case I wasn't. Next time I'll try it. Thanks for advice.

 

Nevertheless, I don't really need to see calcein itself when it can have so
different distribution inside the cell. We used it because we have it. We
only need some dye to see where the cell limits are, that the question was
for an advice. As you can see in the post images (http://imgur.com/a/6566G),
at least 2 types of cells can be distinguished with an internalization or
with an homogenous staining. Probably we´ll use only the second type for
statistics of the research.

 

> - If I understand well, the cells become super fluorescent because tons of
Calcein is internalized so it sounds to me that you have answered the
questions to whether it is internalized or not.

 

As I've mentioned above we used calcein only to see the cell, we need to
answer whether the fluorescent particles of 1,4 and 1,8 microns could be
included by the cell (they could have some medicine incorporated).

 

Best regards,

Konstantin

 

-----Mensaje original-----

De: Confocal Microscopy List [mailto:[hidden email]] En
nombre de Sylvie Le Guyader

Enviado el: sábado, 11 de febrero de 2017 14:15

Para: [hidden email]

Asunto: FW: RE: In or Out, that's the question

 

*****

 

---- Original Message ----

Subject: RE: In or Out, that's the question

Sent: 10 Feb 2017 9:43 a.m.

From: Sylvie Le Guyader <[hidden email]>

To: Confocal Microscopy List <[hidden email]>

Cc:

 

Hi Konstantin

 

- Why do you advise your users to round up the cells? You can very reliably
look for internalization with adherent cells.

- Are you imaging in 16 bits? If not I think changing to 16 bits will help
you avoid saturation in bright areas while still allowing you to image low
areas.

- If I understand well, the cells become super fluorescent because tons of
Calcein is internalized so it sounds to me that you have answered the
questions to whether it is internalized or not.

 

Med vänlig hälsning / Best regards

 

Sylvie

 

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

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Hälsovägen 7,

Novum, G lift, floor 6

14157 Huddinge

Sweden

mobile: [phone]

office: [phone]

LCI website

 

-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Cammer, Michael

Sent: den 9 februari 2017 22:39

To: [hidden email]

Subject: Re: In or Out, that's the question

 

*****

 

I don't know if the images you posted are the raw data or whether a LUT has
been applied.  The images show a lot of saturated voxels.

If light microscopy is going to be able to resolve this, it will only do so
if there are no saturated voxels in the raw data.

And how about an additional label like celltracker violet or a membrane
label?

_________________________________________

Michael Cammer, Optical Microscopy Specialist
http://ocs.med.nyu.edu/microscopy http://microscopynotes.com/

Cell: [phone]

 

________________________________________

From: Confocal Microscopy List [[hidden email]] on behalf
of Konstantín Levitskiy [[hidden email]]

Sent: Thursday, February 09, 2017 6:56 AM

To: [hidden email]

Subject: Re: In or Out, that's the question

 

*****

To join, leave or search the confocal microscopy listserv, go to:

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RTljOyaTWA2wl2i_hUuyc6I&s=SY_DhOrO9Io6AfaE8FTbdMB2qWY8X6tGx-xiQiKERJU&e=

Post images on
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFAw&c
=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjX
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tngVhqnd36GpEftXwGIckLmErSOWyU&e=  and include the link in your posting.

*****

 

Dear Martin,

Please, visit the images on

 

https://urldefense.proofpoint.com/v2/url?u=http-3A__imgur.com_a_6566G&d=DQIF
Aw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_v
AdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=aSAm9ro4F
PlYymgCd3-9Xn126l-jH_ZY7tg9NVCiNoI&e=

Ø  Dear Dr. Levitskiy--

Ø  1)  Are you exciting both the calcein and the particle simultaneously?

Not, of course. Definitely it was a sequential scan.

Ø  If so, that may be part of the problem.  --Based on its excitation
spectrum, you should get no calcein fluorescence at all if you excite at a
wavelength higher than 540 nm.  Thus I'd use sequential excitation: first
excite the calcein (say, at 488 nm), and then excite the red fluorophore,
(making certain to use a wavelength higher than 540 nm).  Unless the
excitation spectrum of calcein changes qualitatively in the cellular
environment, you should be able to distinguish the two clearly.

I might be not explain well. The trouble of calcein that it is going to some
compartmentalisation and shine so bright that you can’t see the cell
dimension (limits), as in the post images for the first cell. For the next
cell it is more clear and as it is distributed more homogeneously.

It seems that the coloured particle has quite wide emission spectrum (or in
some way exciting spectrum), so it also appears in the green cannel.

Ø  2)  With regard to whether or not the particles are inside the cell--I
don't think that that question can necessarily be answered conclusively by
light microscopy.  If the particles are resolvably inside the cells, then
you're fine.  However, if they're close to the membrane, it may be difficult
to prove whether they're in or out.  If the fluorescence of the particle can
be quenched, you might try adding the quenching agent to the culture medium.

Ø  3)  Could DIC be used to image the cell surface, rather than
fluorescence?

I don’t know. We have to check it.

Ø  (--The particles' sizes are up to 1.5 micrometers, not 1.5 millimeters,

correct?)

Yes, you have to read microns ;-)

mkm = mm (symbol type letter for html format)

Ø  Good luck!

Ø  Martin Wessendorf

 

Any advice for cell dye in blue, red or far red range?

 

On 2/8/2017 7:01 AM, Konstantín Levitskiy wrote:

 

> *****

 

> To join, leave or search the confocal microscopy listserv, go to:

>

> <https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi

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> BGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=o

> NMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc6I&s=SY_DhOrO9Io6AfaE8FTbdMB2q

> WY8X6tGx-xiQiKERJU&e= >

https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_
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RTljOyaTWA2wl2i_hUuyc6I&s=SY_DhOrO9Io6AfaE8FTbdMB2qWY8X6tGx-xiQiKERJU&e=

 

> Post images on

> <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=D

> QIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlb

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> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQ

> IFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm

> 5L5GDu_vAdjXk3frDLx_CqKkuo&m=oNMEQ0bSKojTnE4iRyZARTljOyaTWA2wl2i_hUuyc

> 6I&s=JhT4E6hIVKvUotngVhqnd36GpEftXwGIckLmErSOWyU&e=  and include

the link in your posting.

 

> *****

 

> Dear all,

 

> Lately several of our researchers want to know if some particles are able
to enter into the cells (by phagocytosis, etc.) or if some structures are
inside the cell or on the cell surface. Normally they have monolayer growth
cell type, like a HeLa-line and such. My advise usually is based on
consideration to make the cells round after tripsinisation and afterwards to
do a confocal study in a media without Ca iones to avoid quick adherence to
the plaque surface. One of the researcher had red-coloured particles and we
used calcein-AM to make a cell form visible. But the emission spectra of
calcein is a bit wide and tends to saturate some compartments inside the
cell.

 

> What do you recommend in this case? What kind of dye would you use for
cell visualisation to cover different spectra range (405, 488, 561,

633)? Could it be better to try maintaining the cells alive?

>

> Also, take into consideration that particles diameter can be up to 1.5
microns +-.

> Thanks in advance,

> Dr. Konstantín Levitskiy

> Servicio de Microscopía

> InstitutodeBiomedicinadeSevilla - IBiS

> Email:  < <mailto:[hidden email]>

> mailto:[hidden email]>

<mailto:[hidden email]> [hidden email]

>

> Web:  <

> <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevill

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> wl2i_hUuyc6I&s=NhQGX15W2uBJWJBf5tueJLRQ8lRER-A-eH2Wq1a1mF8&e= >

> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla

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<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla.es&d
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7Qq0eekaIlK2uAOe5DzKAHnydGb5r-baBFkQtLU&e= >
www.ibis-sevilla.es<http://www.ibis-sevilla.es>

 

Martin Wessendorf, Ph.D.                   office: [phone]

 

Assoc Prof, Dept Neuroscience                 lab: [phone]

 

University of Minnesota             Preferred FAX: [phone]

 

6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]

 

Minneapolis, MN  55455                    e-mail:  <mailto:[hidden email]>

[hidden email]

 

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