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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
>
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>
> 41013 Sevilla
>
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>
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>
> Web:  <http://www.ibis-sevilla.es/> www.ibis-sevilla.es
>
>   

-- 
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
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