Z sections

[Matthew Pearson-3]

Hi all,

I have a question concerning Z sectioning.  We have a Leica SP2 and when set the top and bottom range for a Z stack, the software automatically calculates the optimised number of sections, which is nearly always over 100!  This is however not practical as the stacks take too long to acquire.  Can anyone shed some light on how the software decides how many slices are necessary and if I haven't got time to take 100, how can I work out what will be a sufficient number.

Thanks for your help in advance.

Matt Pearson.

[Peter Carroll]

 > how the software decides how many slices are necessary and if I
haven't got time to take 100, how can I work out what will be a
sufficient number.

I guess it really depends on your goal, or what you're looking for, as
I'm not certain there's any arbitrary "sufficient number"...

So what are you looking for/at?




Matthew Pearson wrote:

>
> Hi all,
>
> I have a question concerning Z sectioning.  We have a Leica SP2 and
> when set the top and bottom range for a Z stack, the software
> automatically calculates the optimised number of sections, which is
> nearly always over 100!  This is however not practical as the stacks
> take too long to acquire.  Can anyone shed some light on how the
> software decides how many slices are necessary and if I haven't got
> time to take 100, how can I work out what will be a sufficient number.
>
> Thanks for your help in advance.
>
> Matt Pearson.
>
>
>

[Csúcs Gábor]

Dear Matt,

Typically such calculations are done by calculating the maximal optical
resolution in Z-direction for the given objective (NA) and pinhole size
and taking a 2x oversampling. This is how it is done e.g. in the Zeiss
software. The Leica SP2 unfortunately doesn't do this automatically - as
far as I recall it takes the X-Y resolution values also for calculating
the Z-sections. You can kind of cheat the system by setting the
oversampling factor to 2. (Under Settings/Scan options/Sampling factor).

Cheers   Gabor

>
> Hi all,
>
> I have a question concerning Z sectioning.  We have a Leica SP2 and
> when set the top and bottom range for a Z stack, the software
> automatically calculates the optimised number of sections, which is
> nearly always over 100!  This is however not practical as the stacks
> take too long to acquire.  Can anyone shed some light on how the
> software decides how many slices are necessary and if I haven't got
> time to take 100, how can I work out what will be a sufficient number.
>
> Thanks for your help in advance.
>
> Matt Pearson.
>
>
>

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: [phone]
Fax: [phone]
e-mail: [hidden email]

[Fred Mast]

If the purpose of your z-sectioning is to 3D-reconstruct your image than the optimal z-section spacing is determined by the axial resolution of your objective and Nyquist's sampling theorem. As has been noted on this list in the past, there are a number of excellent websites and textbooks available that properly develop and explain these ideas further. I would recommend the Nyquist calculator on the SVI website:

http://support.svi.nl/wiki/NyquistCalculator

The Handbook of Biological Confocal Microscopy by James Pawley is also an excellent resource in this regards. However, if time is of the essence and you can achieve your goals using a stereological approach then design a random sampling protocol and use statistics to extrapolate your data. The best protocol will be tailored to your needs and prior knowledge of your sample will help. ie. are you searching for a regular or irregular object... Your sample will determine the types of assumptions that can be made.

Hope that helps,

Fred

On 3-Oct-08, at 8:40 AM, Matthew Pearson wrote:

Hi all,

I have a question concerning Z sectioning.  We have a Leica SP2 and when set the top and bottom range for a Z stack, the software automatically calculates the optimised number of sections, which is nearly always over 100!  This is however not practical as the stacks take too long to acquire.  Can anyone shed some light on how the software decides how many slices are necessary and if I haven't got time to take 100, how can I work out what will be a sufficient number.

Thanks for your help in advance.

Matt Pearson.

Fred D. Mast

Department of Cell Biology

Medical Sciences Building Room 5-14

University of Alberta

Edmonton, Alberta, T6G 2H7

Canada

Tel: [phone]

[hidden email]

[Carol Norris]

Hi Matt,

Just some advice for after you've decided how many sections to use. Don't try to use one of the other preset #sections that appear when you click on the "sect" button.  I found that this causes the coordinates for the top and/or bottom of the stack to change. I tested this by imaging the same stack three times in a row at different preset section numbers, and found that the top of the stack had changed by 3 microns.  Leica had no explanation for this, they just said to select "others,"  which brings up a window in which you can enter either a section spacing or the number of sections.  

Regards,

Carol

On Oct 3, 2008, at 10:40 AM, Matthew Pearson wrote:

Hi all,

I have a question concerning Z sectioning.  We have a Leica SP2 and when set the top and bottom range for a Z stack, the software automatically calculates the optimised number of sections, which is nearly always over 100!  This is however not practical as the stacks take too long to acquire.  Can anyone shed some light on how the software decides how many slices are necessary and if I haven't got time to take 100, how can I work out what will be a sufficient number.

Thanks for your help in advance.

Matt Pearson.

Carol E. Norris, Ph.D

Facility Scientist

Flow Cytometry/Confocal Microscopy Facility

Biotechnology/Bioservices Center

University of Connecticut Unit 3149

91 N. Eagleville Rd

Storrs, CT 06269-3149

Phone [phone]

Fax [phone]

[Mario-2]

Matt,

Fred's reply is particularly relevant. I would make a suggestion,
though, which is that because of R.I. mismatch and spherical
aberration the actual z-axis resolutions is almost always worse than
the theoretical by a significant amount. For example, a 1.4 NA oil
objective at 10 um into a sample should have a z- res about 0.45 um
(FWHM) using 488 nm. In actual practise, it is usually closer to
0.6-0.8 um and it gets worse (much worse) the further into a sample
you go. In any event, the closer you stay near the coverslip boundary
the more justified it is to use a high sampling number. In the case
of a layer of cells 20 um thick, a 128 sections gives ~0.16 um
spacing. Much more than you need to satisfy Nyquist even if
everything is perfect for 488 nm excitation. Realistically, 64
sections should do the job in a real sample. Fewer than that starts
to become risky for a 1.4 NA lens.

Depending on what you are doing, 64 frames should be enough.

Good luck
Mario

>Hi all,
>
>I have a question concerning Z sectioning.  We have a Leica SP2 and
>when set the top and bottom range for a Z stack, the software
>automatically calculates the optimised number of sections, which is
>nearly always over 100!  This is however not practical as the stacks
>take too long to acquire.  Can anyone shed some light on how the
>software decides how many slices are necessary and if I haven't got
>time to take 100, how can I work out what will be a sufficient
>number.
>
>Thanks for your help in advance.
>
>Matt Pearson.

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]
[hidden email]

[Christian Liebig]

Hi Carol,

what the Leica software does (AFAIK):
- you choose the number of layers you want (dropdown menu or over the
"others" dialogue, doesn't matter)
- the software divides the stacksize chosen by you with this number and
gets a value for your stepsize (if you choose a stepsize from the
"others" dialogue start from here)
- the software adapts the stepsize value to the nearest stepsize the
z-motor can handle
- then the software multiplies the new stepsize with the number of
layers and there you have the new z range for your stack.

This is just 3rd party information, maybe it would be better if someone
from Leica would comment on this.

Kind regards,

Christian



Carol Norris wrote:

> Hi Matt,
>
> Just some advice for after you've decided how many sections to use.
> Don't try to use one of the other preset #sections that appear when
> you click on the "sect" button.  I found that this causes the
> coordinates for the top and/or bottom of the stack to change. I tested
> this by imaging the same stack three times in a row at different
> preset section numbers, and found that the top of the stack had
> changed by 3 microns.  Leica had no explanation for this, they just
> said to select "others,"  which brings up a window in which you can
> enter either a section spacing or the number of sections.  
>
> Regards,
> Carol
>
> On Oct 3, 2008, at 10:40 AM, Matthew Pearson wrote:
>
>>
>> Hi all,
>>
>> I have a question concerning Z sectioning.  We have a Leica SP2 and
>> when set the top and bottom range for a Z stack, the software
>> automatically calculates the optimised number of sections, which is
>> nearly always over 100!  This is however not practical as the stacks
>> take too long to acquire.  Can anyone shed some light on how the
>> software decides how many slices are necessary and if I haven't got
>> time to take 100, how can I work out what will be a sufficient number.
>>
>> Thanks for your help in advance.
>>
>> Matt Pearson.
>>
>>
>
>
>
> Carol E. Norris, Ph.D
>
> Facility Scientist
>
> Flow Cytometry/Confocal Microscopy Facility
>
> Biotechnology/Bioservices Center
>
> University of Connecticut Unit 3149
>
> 91 N. Eagleville Rd
>
> Storrs, CT 06269-3149
>
>
> Phone [phone]
>
> Fax [phone]
>
>

--



Christian Liebig, PhD

FILM - Facility for Imaging by Light Microscopy
- Facility Assistant -
Sir Alexander Fleming Building, desk 403
Imperial College London / South Kensington
Exhibition Road
London SW7 2AZ
UK

Tel. [phone]
Fax [phone]
E-mail [hidden email]
Website: http://imperial.ac.uk/imagingfacility