Strange artifact in z series

[Pedro Camello]

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Hi all,

while performing z sections of mitochondria-stained living cells, I´m
finding that the bottom part of the cell displays a brighter mitochondria
fluoresecence, showing more contrast respect to cytosol than mitochondria in
the top part of the cell. My system scans z from bottom to top. If it were
simple bleaching due to laser light passing through the cell, I should
observe that the bottom mitochondria are also less stained in a second
series, but that´s not the case (the difference between top and bottom still
there)
Any idea to explain it?

Thanks

Pedro

[Ignatius, Mike]

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It is not uncommon to de-quench mitochondrial dyes with light.  As the dye bleaches, it is no longer is available to quenches others around it.  Eventually an optimum is reached, where the mitochondria are now brighter - or perfectly labeled.  Same thing can happen with dyes on antibodies.

One way to test is to reverse the stack order or to label with less dye.  But sounds like a feature, not a flaw here?

Mike Ignatius

Molecular Probes/Invitrogen

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pedro Camello
Sent: Tuesday, May 06, 2008 3:44 PM
To: [hidden email]
Subject: Strange artifact in z series

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi all,

while performing z sections of mitochondria-stained living cells, I´m
finding that the bottom part of the cell displays a brighter mitochondria
fluoresecence, showing more contrast respect to cytosol than mitochondria in
the top part of the cell. My system scans z from bottom to top. If it were
simple bleaching due to laser light passing through the cell, I should
observe that the bottom mitochondria are also less stained in a second
series, but that´s not the case (the difference between top and bottom still
there)
Any idea to explain it?

Thanks

Pedro

[Kurt Thorn]

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By bottom do you mean the part of the cell closest to the coverslip or
furthest from the coverslip?

It's not uncommon to see brighter fluorescence closer to the coverslip
than farther away from the coverslip as both scattering and spherical
aberration increase the further into a sample (i.e. from the coverslip)
one images.  Microscope objectives are corrected to give the best images
immediately adjacent to the coverslip and their optical performance
decreases the further into the sample you have to image.  Careful
matching of refractive indices of immersion media to the sample and
correction collars can minimize these effects.

Kurt

Pedro Camello wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi all,
>
> while performing z sections of mitochondria-stained living cells, I´m
> finding that the bottom part of the cell displays a brighter mitochondria
> fluoresecence, showing more contrast respect to cytosol than mitochondria in
> the top part of the cell. My system scans z from bottom to top. If it were
> simple bleaching due to laser light passing through the cell, I should
> observe that the bottom mitochondria are also less stained in a second
> series, but that´s not the case (the difference between top and bottom still
> there)
> Any idea to explain it?
>
> Thanks
>
> Pedro
>
>  

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

UCSF MC 2140
Genentech Hall Room S252
600 16th St.
San Francisco, CA 94158-2517

http://nic.ucsf.edu
phone [phone]
fax   [phone]

[mmodel]

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Hi Pedro:
 
If you are using an inverted scope then the areas close to the bottom are less affected by spherical aberration and scattering. That's why they should be brighter.
 
Mike
 

________________________________

From: Confocal Microscopy List on behalf of Pedro Camello
Sent: Tue 5/6/2008 6:44 PM
To: [hidden email]
Subject: Strange artifact in z series



Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi all,

while performing z sections of mitochondria-stained living cells, I´m
finding that the bottom part of the cell displays a brighter mitochondria
fluoresecence, showing more contrast respect to cytosol than mitochondria in
the top part of the cell. My system scans z from bottom to top. If it were
simple bleaching due to laser light passing through the cell, I should
observe that the bottom mitochondria are also less stained in a second
series, but that´s not the case (the difference between top and bottom still
there)
Any idea to explain it?

Thanks

Pedro

[Stephen Cody]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

G'day Pedro,

Are you using a water immersion lens?

A water immersion lens with a coverslip correction collar will be your best chance at minimising the effects of spherical aberration etc. These artefacts will probably still affect your imaging at depth, even with a water immersion lens, but the problem will be greatly reduced.  

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Victoria,      3050
Australia
Tel: [phone]    Fax: [phone]
email: [hidden email]
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Tip: Learn how to receive reminders about you microscope booking:
www.ludwig.edu.au/confocal/Local/Booking_Hint.htm  

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

From: Confocal Microscopy List on behalf of Pedro Camello
Sent: Tue 5/6/2008 6:44 PM
To: [hidden email]
Subject: Strange artifact in z series



Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi all,

while performing z sections of mitochondria-stained living cells, I´m
finding that the bottom part of the cell displays a brighter mitochondria
fluoresecence, showing more contrast respect to cytosol than mitochondria in
the top part of the cell. My system scans z from bottom to top. If it were
simple bleaching due to laser light passing through the cell, I should
observe that the bottom mitochondria are also less stained in a second
series, but that´s not the case (the difference between top and bottom still
there)
Any idea to explain it?

Thanks

Pedro


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[Pedro Camello]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Apologies for repeating the message. I hit the wrong button

Pedro

Pedro Camello escribió:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi all,
>
> while performing z sections of mitochondria-stained living cells, I´m
> finding that the bottom part of the cell displays a brighter mitochondria
> fluoresecence, showing more contrast respect to cytosol than mitochondria in
> the top part of the cell. My system scans z from bottom to top. If it were
> simple bleaching due to laser light passing through the cell, I should
> observe that the bottom mitochondria are also less stained in a second
> series, but that´s not the case (the difference between top and bottom still
> there)
> Any idea to explain it?
>
> Thanks
>
> Pedro
>
>