Questions about axial elongation

[Pedro Camello]

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I´m preparing samples for 3D confocal imaging of two different objects:

1-      A paraformaldehyde-fixed flat nervous plexus mounted in Citifluor
(glycerol-pbs based). Antibodies against different neurotransmitter-related
enzymes.

2-      Live cells (smooth muscle) loaded with acidic and mitochondrial
fluorescent dyes.

 

In both cases I would like to perform z stacks. I have a Nikon A1 confocal.

-          For the plexus my intent is to use a 40x, oil, 1.3 NA objective.
Here I want clear separation of neurons within the ganglia (these are rather
flat, monolayers, but some degree of overlapping is present), and
localization of the main projections (at most, a few micrometers wide)

-          For the cells I´ll use the 60x, oil, 1.49. Here I want to observe
the mitochondrial network (particles even below 1 micrometer) and possibly
some lysosomes.

 

I assumed that 1 microm would be enough for the nervous plexus, and around
300-500 nm for the isolated cells (thinking of Nyquist criteria for the
objects I want to resolve)

My problem is that, when I make a validation looking at 4 micrometers
fluorescent beads (slides from Mol Probes) what I get after 3D
reconstruction is a elongated cucumber instead a sphere if I set the
acquisition following the theoretical parameters advised by Nikon´s
software. For example,  I got a sphere when I set sampling with the 40x at
50 nm instead the 270 nm recommended by the software (iris was set at
1.2-1.5 Airy units).

 

My questions:

-What would be the best z spacing and optical slice?

- Am I right if I use iris and z step so that I get 2-3 slices/micrometer
for the 60x objective? (I would accept 1 micrometer resolution for the 60x
living cells observations)

- And finally, why this elongation is on/off depending on z step?

 

I´ve looked for these answers in the list archives without success (probably
my fault)

 

Thanks in advance

 

Pedro

 

De: Confocal Microscopy List [mailto:[hidden email]] En
nombre de CONFOCALMICROSCOPY automatic digest system
Enviado el: lunes, 08 de noviembre de 2010 7:08
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Asunto: [POSIBLE SPAM] CONFOCALMICROSCOPY Digest - 6 Nov 2010 to 7 Nov 2010
(#2010-65)

 

 

[mmodel]

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Hi Pedro - if your 4 um beads are made of a material very different from what they are immersed in, they will act like small lenses and that will affect the psf. You may try putting them in an oil with the same refractive index as the bead material. But even then you may get some elongation along z. (3D deconvolution of spherically aberrated images using commercial software. J. Microsc. 2010) Best -

Mike


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pedro J Camello
Sent: Monday, November 08, 2010 11:10 AM
To: [hidden email]
Subject: Questions about axial elongation

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I´m preparing samples for 3D confocal imaging of two different objects:

1-      A paraformaldehyde-fixed flat nervous plexus mounted in Citifluor
(glycerol-pbs based). Antibodies against different neurotransmitter-related
enzymes.

2-      Live cells (smooth muscle) loaded with acidic and mitochondrial
fluorescent dyes.

 

In both cases I would like to perform z stacks. I have a Nikon A1 confocal.

-          For the plexus my intent is to use a 40x, oil, 1.3 NA objective.
Here I want clear separation of neurons within the ganglia (these are rather
flat, monolayers, but some degree of overlapping is present), and
localization of the main projections (at most, a few micrometers wide)

-          For the cells I´ll use the 60x, oil, 1.49. Here I want to observe
the mitochondrial network (particles even below 1 micrometer) and possibly
some lysosomes.

 

I assumed that 1 microm would be enough for the nervous plexus, and around
300-500 nm for the isolated cells (thinking of Nyquist criteria for the
objects I want to resolve)

My problem is that, when I make a validation looking at 4 micrometers
fluorescent beads (slides from Mol Probes) what I get after 3D
reconstruction is a elongated cucumber instead a sphere if I set the
acquisition following the theoretical parameters advised by Nikon´s
software. For example,  I got a sphere when I set sampling with the 40x at
50 nm instead the 270 nm recommended by the software (iris was set at
1.2-1.5 Airy units).

 

My questions:

-What would be the best z spacing and optical slice?

- Am I right if I use iris and z step so that I get 2-3 slices/micrometer
for the 60x objective? (I would accept 1 micrometer resolution for the 60x
living cells observations)

- And finally, why this elongation is on/off depending on z step?

 

I´ve looked for these answers in the list archives without success (probably
my fault)

 

Thanks in advance

 

Pedro

 

De: Confocal Microscopy List [mailto:[hidden email]] En
nombre de CONFOCALMICROSCOPY automatic digest system
Enviado el: lunes, 08 de noviembre de 2010 7:08
Para: [hidden email]
Asunto: [POSIBLE SPAM] CONFOCALMICROSCOPY Digest - 6 Nov 2010 to 7 Nov 2010
(#2010-65)

 

 

[Mark Cannell]

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*****

Your problem lies in the non-spherical PSF and non-uniform (xyz)  
sampling while the software display presents cubic voxels...

Cheers


On 9/11/2010, at 5:10 AM, Pedro J Camello wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I´m preparing samples for 3D confocal imaging of two different  
> objects:
>
> 1-      A paraformaldehyde-fixed flat nervous plexus mounted in  
> Citifluor
> (glycerol-pbs based). Antibodies against different neurotransmitter-
> related
> enzymes.
>
> 2-      Live cells (smooth muscle) loaded with acidic and  
> mitochondrial
> fluorescent dyes.
>
>
>
> In both cases I would like to perform z stacks. I have a Nikon A1  
> confocal.
>
> -          For the plexus my intent is to use a 40x, oil, 1.3 NA  
> objective.
> Here I want clear separation of neurons within the ganglia (these  
> are rather
> flat, monolayers, but some degree of overlapping is present), and
> localization of the main projections (at most, a few micrometers wide)
>
> -          For the cells I´ll use the 60x, oil, 1.49. Here I want to  
> observe
> the mitochondrial network (particles even below 1 micrometer) and  
> possibly
> some lysosomes.
>
>
>
> I assumed that 1 microm would be enough for the nervous plexus, and  
> around
> 300-500 nm for the isolated cells (thinking of Nyquist criteria for  
> the
> objects I want to resolve)
>
> My problem is that, when I make a validation looking at 4 micrometers
> fluorescent beads (slides from Mol Probes) what I get after 3D
> reconstruction is a elongated cucumber instead a sphere if I set the
> acquisition following the theoretical parameters advised by Nikon´s
> software. For example,  I got a sphere when I set sampling with the  
> 40x at
> 50 nm instead the 270 nm recommended by the software (iris was set at
> 1.2-1.5 Airy units).
>
>
>
> My questions:
>
> -What would be the best z spacing and optical slice?
>
> - Am I right if I use iris and z step so that I get 2-3 slices/
> micrometer
> for the 60x objective? (I would accept 1 micrometer resolution for  
> the 60x
> living cells observations)
>
> - And finally, why this elongation is on/off depending on z step?
>
>
>
> I´ve looked for these answers in the list archives without success  
> (probably
> my fault)
>
>
>
> Thanks in advance
>
>
>
> Pedro
>
>
>
> De: Confocal Microscopy List  
> [mailto:[hidden email]] En
> nombre de CONFOCALMICROSCOPY automatic digest system
> Enviado el: lunes, 08 de noviembre de 2010 7:08
> Para: [hidden email]
> Asunto: [POSIBLE SPAM] CONFOCALMICROSCOPY Digest - 6 Nov 2010 to 7  
> Nov 2010
> (#2010-65)
>
>
>
>

[Guy Cox-2]

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*****

This is never going to work properly.  For your live cells you need to use a water-immersion objective.  That way there is (a) no apparent depth problem and (b) no spherical aberration (so long as you set your correction collar carefully).  What's more you'll then no longer need to set your pinhole above 1 Airy (which SA is forcing you to do with this set-up), so you'll get decent optical sectioning.  Resolution in X, Y and Z will be way better.

 

Glycerol is more problematic.  I've done some experiments on this and found that a water-immersion lens with a thin coverslip got me more or less within the correction range of the collar, and I got better results than with an oil lens.  (This was looking at FtsZ in bacteria where we needed all the resolution we could get).  In other words, I was setting the collar to a figure larger than the actual coverslip thickness  to compensate the glycerol.  However, this doesn't avoid the apparent depth problem (your lens is moving in water but your sample is in a higher index medium).  So your reconstruction will appear stretched in Z by an amount equal to the difference in RI between water and your Citifluor mountant.

 

There is a diagram of this at http://www.guycox.com/optical/correction.htm  (an embarrassing correction for a boo-boo in my book).

 

I guess you'll probably have to use some 3rd-party reconstruction software which allows you to enter the correction - I don't think the Nikon software will do it.  There must be a plug-in for Image J.

 

With oil you'll have the problem in reverse - the sample will appear squashed.  But it's a bit more complicated since SA will also affect the perceived depth - the circle of least confusion won't always match the geometrical focus.  So you'll have rather unpredictable results (well, unpredictable without some pretty complex maths).  

 

I guess the bottom line is, as Jim Pawley always tells us, the most important thing is to avoid SA.

 

                                                                                                      Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm <http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

 

Phone [phone]     Fax [phone]

             Mobile [phone]

______________________________________________

      http://www.guycox.net <http://www.guycox.net>

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pedro J Camello
Sent: Tuesday, 9 November 2010 3:10 AM
To: [hidden email]
Subject: Questions about axial elongation

 

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I´m preparing samples for 3D confocal imaging of two different objects:

1-      A paraformaldehyde-fixed flat nervous plexus mounted in Citifluor
(glycerol-pbs based). Antibodies against different neurotransmitter-related
enzymes.

2-      Live cells (smooth muscle) loaded with acidic and mitochondrial
fluorescent dyes.



In both cases I would like to perform z stacks. I have a Nikon A1 confocal.

-          For the plexus my intent is to use a 40x, oil, 1.3 NA objective.
Here I want clear separation of neurons within the ganglia (these are rather
flat, monolayers, but some degree of overlapping is present), and
localization of the main projections (at most, a few micrometers wide)

-          For the cells I´ll use the 60x, oil, 1.49. Here I want to observe
the mitochondrial network (particles even below 1 micrometer) and possibly
some lysosomes.



I assumed that 1 microm would be enough for the nervous plexus, and around
300-500 nm for the isolated cells (thinking of Nyquist criteria for the
objects I want to resolve)

My problem is that, when I make a validation looking at 4 micrometers
fluorescent beads (slides from Mol Probes) what I get after 3D
reconstruction is a elongated cucumber instead a sphere if I set the
acquisition following the theoretical parameters advised by Nikon´s
software. For example,  I got a sphere when I set sampling with the 40x at
50 nm instead the 270 nm recommended by the software (iris was set at
1.2-1.5 Airy units).



My questions:

-What would be the best z spacing and optical slice?

- Am I right if I use iris and z step so that I get 2-3 slices/micrometer
for the 60x objective? (I would accept 1 micrometer resolution for the 60x
living cells observations)

- And finally, why this elongation is on/off depending on z step?



I´ve looked for these answers in the list archives without success (probably
my fault)



Thanks in advance



Pedro



De: Confocal Microscopy List [mailto:[hidden email]] En
nombre de CONFOCALMICROSCOPY automatic digest system
Enviado el: lunes, 08 de noviembre de 2010 7:08
Para: [hidden email]
Asunto: [POSIBLE SPAM] CONFOCALMICROSCOPY Digest - 6 Nov 2010 to 7 Nov 2010
(#2010-65)





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