What is the window setting for Nikon A1 confocol

[Diego Zhu]

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Hi
I got a comment from a reviewer asking the setting for our spectral analyses:

"For spectral analyses, images were scanned using an excitation wavelength of 488 nm,
using an emission range of 500-740 nm, with a wavelength interval of x nm, and a window
of y nm..." This tells the reader how you sampled the spectrum and how precise each
sampling was. Of course, the narrower the window, the less fluorescence is available to
sample, but as I understand it, the Nikon confocal has fixed windows, and therefore also has
fixed wavelength intervals. Were some of the windows binned for each data point? You need
to explain this because each confocal does this a different way.

We set the wavelength interval at 6 nm, but I have no idea about the window. I cannot find
any setting on our Nikon confocal. Wondering anyone can tell me what is this window?

Thanking you in advance!

[Joshua Zachary Rappoport]

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Maybe they are asking how many spectral bins/channels
The A1R SI can do up to 32
I think if you say 6nm bins X n bins you are fine


Joshua Z. Rappoport PhD



Director of the Center for Advanced Microscopy and the Nikon Imaging Center at Northwestern University

Northwestern University Feinberg School of Medicine
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Chicago, IL 60611<x-apple-data-detectors://10/0>

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http://cam.facilities.northwestern.edu/

http://nic.feinberg.northwestern.edu/

www.rappoportlab.com<http://www.rappoportlab.com/>

On Mar 15, 2016, at 7:42 PM, Diego Zhu <[hidden email]<mailto:[hidden email]>> wrote:

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Hi
I got a comment from a reviewer asking the setting for our spectral analyses:

"For spectral analyses, images were scanned using an excitation wavelength of 488 nm,
using an emission range of 500-740 nm, with a wavelength interval of x nm, and a window
of y nm..." This tells the reader how you sampled the spectrum and how precise each
sampling was. Of course, the narrower the window, the less fluorescence is available to
sample, but as I understand it, the Nikon confocal has fixed windows, and therefore also has
fixed wavelength intervals. Were some of the windows binned for each data point? You need
to explain this because each confocal does this a different way.

We set the wavelength interval at 6 nm, but I have no idea about the window. I cannot find
any setting on our Nikon confocal. Wondering anyone can tell me what is this window?

Thanking you in advance!

[Paul Rigby-2]

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Hi Diego,
The spectral window for each channel is the spectral width of each detector element. If you set the spectral width to 6nm, then the window is 6nm, unless you do binning (combining adjacent detector elements into single "detector" with greater bandwidth).

One comment/question - the Nikon spectral detector has 32 channels, where each channel width can be set to 10nm, 6nm or 2.5nm. With a spectral window of 6nm, 32 channels will only cover the spectral range of 192nm. However, you stated that the range was 240nm (500 to 740nm). Did you do multiple spectral scans to cover that range? Perhaps this is why the reviewer may be confused and require more information.

I hope this helps.
Paul

Assoc. Prof. Paul Rigby
Optical Microscopy Specialist
Centre for Microscopy, Characterisation & Analysis (M519)
The University of Western Australia
35 Stirling Highway
Perth  WA  6009
Australia

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Diego Zhu
Sent: Wednesday, 16 March 2016 8:31 AM
To: [hidden email]
Subject: What is the window setting for Nikon A1 confocol

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Hi
I got a comment from a reviewer asking the setting for our spectral analyses:

"For spectral analyses, images were scanned using an excitation wavelength of 488 nm, using an emission range of 500-740 nm, with a wavelength interval of x nm, and a window of y nm..." This tells the reader how you sampled the spectrum and how precise each sampling was. Of course, the narrower the window, the less fluorescence is available to sample, but as I understand it, the Nikon confocal has fixed windows, and therefore also has fixed wavelength intervals. Were some of the windows binned for each data point? You need to explain this because each confocal does this a different way.

We set the wavelength interval at 6 nm, but I have no idea about the window. I cannot find any setting on our Nikon confocal. Wondering anyone can tell me what is this window?

Thanking you in advance!

[Rusty Nicovich]

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Diego,

At least on a Zeiss system, the actual windows used for each channel are
embedded in the metadata of the image file as XML or a Java hashtable.  I
imagine that Nikon does the same.  It might take a little digging but you
can probably get at this using the Bioformats MATLAB package and
interrogating a representative image file.

Thanks,
Rusty

On Wed, Mar 16, 2016 at 11:31 AM, Diego Zhu <[hidden email]> 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.
> *****
>
> Hi
> I got a comment from a reviewer asking the setting for our spectral
> analyses:
>
> "For spectral analyses, images were scanned using an excitation wavelength
> of 488 nm,
> using an emission range of 500-740 nm, with a wavelength interval of x nm,
> and a window
> of y nm..." This tells the reader how you sampled the spectrum and how
> precise each
> sampling was. Of course, the narrower the window, the less fluorescence is
> available to
> sample, but as I understand it, the Nikon confocal has fixed windows, and
> therefore also has
> fixed wavelength intervals. Were some of the windows binned for each data
> point? You need
> to explain this because each confocal does this a different way.
>
> We set the wavelength interval at 6 nm, but I have no idea about the
> window. I cannot find
> any setting on our Nikon confocal. Wondering anyone can tell me what is
> this window?
>
> Thanking you in advance!
>

[Diego Zhu]

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Many thanks Paul, Rusty and Joshua for your help! That is really helpful!