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February 2017

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Subject:
Re: In or Out, that's the question
From:
"Cammer, Michael" <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Thu, 9 Feb 2017 21:39:26 +0000
Content-Type:
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*****
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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: (914) 309-3270

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

*****
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*****

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:[log in to unmask]> mailto:[log in to unmask]>
<mailto:[log in to unmask]> [log in to unmask]

>

> 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= >
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>

>



--

Martin Wessendorf, Ph.D.                   office: (612) 626-0145

Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991

University of Minnesota             Preferred FAX: (612) 624-8118

6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009

Minneapolis, MN  55455                    e-mail:  <mailto:[log in to unmask]>
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