Re: More power and less exposure or vice versa – use of 100% laser

[Claire Brown]

In my experience for cell migration the cells definitely prefer low power
for a long time. I have never seemed to find the time to systematically
measure this but I should. Cell migration is very sensitive to light so it
is a good assay to test it with. You can also measure the migration rates
with brightfield imaging for comparisons with no fluorescence light applied.

I was once using a Fluoview 300 at 0.1% power to measure protrusion rates
with 20s intervals for imaging and when I went up to 0.2% the lamellipodia
immediately started to retract. I also know when using TIRF illumination
where on the lower 100 nm of the cell is exposed to light the cells are much
more dynamic and motile.

I would definitely go with 100 ms at 1%.

I let you know if I get some more concrete data on this.

Sincerely,

Claire
McGill Imaging Facility

[Seb Stephens]

Dear Claire,
 
I just skimmed through your recent paper on live cell miscroscopy. Finally someone has brought out a paper that anwers so many of my questions! It really is an expert review for which I cannot stress enough how important it is to me. Do you hold a course on live cell imaging?
 
I note in your paper you talk about supplementing 25mM HEPES, have you ever tried less? (We used to use 10mM and now not at all).
 
Thank you for your input as to my problem on laser power and exposure time. What you say makes sense and gives me a direction which is exactly what I wanted.
 
Alors forcement vous parlez Francais dans la belle ville de Montreal!
 
Je vous remerice pour votre aide et peut etre un jours je passerai a McGill.
 
Cordialement

 

 

Sébastien Stephens

Harvard University School of Dental Medicine
188 Longwood Ave REB 313
Boston, MA 02115
Ph: 617-4327328
Fax: [phone]
[hidden email]

 

> Date: Mon, 13 Jul 2009 09:23:04 -0500
> From: [hidden email]
> Subject: Re: More power and less exposure or vice versa – use of 100% laser
> To: [hidden email]
>
> In my experience for cell migration the cells definitely prefer low power
> for a long time. I have never seemed to find the time to systematically
> measure this but I should. Cell migration is very sensitive to light so it
> is a good assay to test it with. You can also measure the migration rates
> with brightfield imaging for comparisons with no fluorescence light applied.
>
> I was once using a Fluoview 300 at 0.1% power to measure protrusion rates
> with 20s intervals for imaging and when I went up to 0.2% the lamellipodia
> immediately started to retract. I also know when using TIRF illumination
> where on the lower 100 nm of the cell is exposed to light the cells are much
> more dynamic and motile.
>
> I would definitely go with 100 ms at 1%.
>
> I let you know if I get some more concrete data on this.
>
> Sincerely,
>
> Claire
> McGill Imaging Facility

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