spinning disk field of view and NA

[mfaretta]

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Dear all,
I was discussing with a friend about the  possibility to employ a large
area SCMOS to a spinning disk. In particular he made some considerations
regarding the spacing between disks to insert the dichroic mirrors,
microlenses dimension and the resulting real numerical aperture.
According to standard parameters (250 microns microlenses) he told me
there no way to reach  a high NA (let's say 1.4). I saw on the Yokogawa
website that they post an image of the new CSU W1 acquired with a
silicon immersion objective NA 1.00 (to employ a greater working
distance I suppose). Is this the case? That is the higher contrast
claimed is the result of a lower NA which cuts the low intensity
signals, i.e. background of scattered light?
Or this factor is someway corrected by changing the distance between
microlenses?
Thanks a lot for the help
Mario

--
Mario Faretta
Deapartment of Experimental Oncology
European Institute of Oncology
via Adamello 16
20139 Milan
Italy
Phone: [phone]
email: [hidden email]

[Pascal Weber]

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Obviously it is possible to use sCMOS with the W1. The problem is the size of the
field. As for using the high numerical aperture objectives: they will always be
better than others because you will see more weak signals. The drawback is that
Spinning optimize for a single wavelength and a goal. But it retains its
advantages: Fast scan and low toxic. I just bought one: A Zeiss with four lasers.
I had one with a head first generation CSU10 and used a water immersion 40X
objective. I adapted above DPSS laser and it worked very well.
For SCMOS cameras  is no need to use high magnification objectives, otherwise
you risk to exceed the optic resolution  it is the reasonwhy Yokogawa shows
images make a 40X objectiv.

[Gerhard Holst]

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As far as I know, Visitron has already equipped and realized a new Yokogawa system with 2 sCMOS cameras using full speed recording.

with best regards,

Gerhard Holst
_______________________________


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Pascal Weber
Gesendet: Montag, 8. April 2013 08:08
An: [hidden email]
Betreff: Re: spinning disk field of view and NA

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Obviously it is possible to use sCMOS with the W1. The problem is the size of the
field. As for using the high numerical aperture objectives: they will always be
better than others because you will see more weak signals. The drawback is that
Spinning optimize for a single wavelength and a goal. But it retains its
advantages: Fast scan and low toxic. I just bought one: A Zeiss with four lasers.
I had one with a head first generation CSU10 and used a water immersion 40X
objective. I adapted above DPSS laser and it worked very well.
For SCMOS cameras  is no need to use high magnification objectives, otherwise
you risk to exceed the optic resolution  it is the reasonwhy Yokogawa shows
images make a 40X objectiv.

[Arne Seitz]

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Dear Mario,

I suggest reading the following introduction to spinning disk microscopy:
http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html

It nicely summarizes the theory and explains what resolution can be achieved under which conditions. It is possible to reach a high NA with a spinning  disk system. You just have to choose your objective accordingly to the pinhole size of the disk (see table 1 in the link above).

A sCMOs camera can be used on a spinning disk (like every other camera) and it has no influence on the performance (=resolution ) of the system if operated in global-shutter mode. Using the rolling shutter mode would require a special synchronisation of camera and the disk. If you want to optimally use a sCMOS camera it is preferred to use a spinning disk head which supports the large camera chip. Yokogawa has realized this in the new W1 model, but there might be other vendors I'm currently not aware of, who are offering the same or planning to do so.

Changing the distances of the pinholes on the disk, decrease the probability of pinhole cross-talk and therefore will affect the z-resolution. To my understanding it has minor/no influence on the xy-resolution. But an increased distance will decrease the maximal acquisition speed.

Regards
Arne


---------------------------------------------------------------
Arne Seitz
Head of Bioimaging and Optics Platform (PT-BIOP)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Faculty of Life Sciences
Station 15, AI 0241
CH-1015 Lausanne

Phone: [phone]
Fax:      [phone]
http://biop.epfl.ch/
---------------------------------------------------------------

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of mfaretta
> Sent: lundi 8 avril 2013 07:05
> To: [hidden email]
> Subject: spinning disk field of view and NA
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
> I was discussing with a friend about the  possibility to employ a large area
> SCMOS to a spinning disk. In particular he made some considerations
> regarding the spacing between disks to insert the dichroic mirrors,
> microlenses dimension and the resulting real numerical aperture.
> According to standard parameters (250 microns microlenses) he told me
> there no way to reach  a high NA (let's say 1.4). I saw on the Yokogawa
> website that they post an image of the new CSU W1 acquired with a silicon
> immersion objective NA 1.00 (to employ a greater working distance I
> suppose). Is this the case? That is the higher contrast claimed is the result of
> a lower NA which cuts the low intensity signals, i.e. background of scattered
> light?
> Or this factor is someway corrected by changing the distance between
> microlenses?
> Thanks a lot for the help
> Mario
>
> --
> Mario Faretta
> Deapartment of Experimental Oncology
> European Institute of Oncology
> via Adamello 16
> 20139 Milan
> Italy
> Phone: [phone]
> email: [hidden email]

[WILDE Geraint]

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Dear Mario,

My intention is to quickly explain the Yokogawa W1 in the context of your email. Whilst I am from Andor and so have commercial interest in the product, I will try and remain neutral.

On the original Yokogawa CSU 10, 22 and X models, the field of illumination was designed originally for traditional interline CCD's, with a window limited to 7x10mm.  The pinhole size (50 microns) optimized for 100x high NA (1.4) objectives.  Consequently the microlenses were designed to match accordingly.  This meant the unit was excellent for high NA objectives, and typically performed best at 60x and above. Resolution optimal, with nyquist sampling possible with a 6.45um pixel size.  40x oil was OK, but below this and confocality struggles.  In addition, the number of pinholes and spacing between was designed for optimal sensitivity, and therefore speed and low phototoxicity, on samples typically imaged at higher magnification.  The downside of this is that if you have thick samples you can get cross-talk between pinholes and so higher background.

With the new CSU W1 Yokogawa have addressed these points.

1. They now have a field of illumination/view that can match sCMOS (16.6x14mm).  Resolution has not been affected.  Therefore this is great for larger sampling area and larger samples.  Your light source now has to fill a larger area, but there is more than enough laser power in lasers these days to accommodate this, and the final power density at your sample can still be the same, so you do not have to worry about increased phototoxicity.
2. There are two pinhole sizes available.  25 micron and 50 micron (as above) for low and high NA/magnification objectives respectively. This therefore means you can now also perform high speed confocal imaging on large and thick samples at low magnifications.
3. The spacing between pinholes has increased.  This results in less pinhole crosstalk, and so much better performance (higher contrast) with thicker samples.  However, it does also mean a reduced light throughput compared to the CSU-X model requiring longer camera exposures.  Still a fast live cell imaging system though!  Our experience shows amazing performance with thick samples, imaging up to 200 microns and possibly even beyond.
4. There are other implementations such as
        a) standard brightfield bypass for light efficient, non-confocal imaging (e.g. brightfield, epi-fluorescence)
        b) option of dual camera port for dual camera simultaneous capture (identical cameras) or for switching between different cameras (e.g EMCCD for high   sensitivity, sCMOS for high resolution large field of view).
        c) Near-infra red port option (recently made available)

It is worth noting that there is still a place in the market for the CSU-X model of the Yokogawa unit.  This still offers the best solution for maximum speed and sensitivity if this is what your application requires.

Yes, the silicon immersion objectives were most probably chosen for the relatively high NA, but most importantly in combination with an excellent working distance for deep imaging.  They give great results.

Though not technically very detailed, I hope this helps clarify the W1 compared to other models.

Kind regards

Geraint

Geraint Wilde, Ph.D.
Product Specialist for Microscopy Systems

Andor Technology plc
7 Millennium Way
Springvale Business Park
Belfast
BT12 7AL
N. Ireland
United Kingdom.

http://www.andor.com/microscopy-systems/revolution/wd-system

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of mfaretta
Sent: 08 April 2013 06:05
To: [hidden email]
Subject: [CONFOCALMICROSCOPY] spinning disk field of view and NA

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear all,
I was discussing with a friend about the  possibility to employ a large area SCMOS to a spinning disk. In particular he made some considerations regarding the spacing between disks to insert the dichroic mirrors, microlenses dimension and the resulting real numerical aperture.
According to standard parameters (250 microns microlenses) he told me there no way to reach  a high NA (let's say 1.4). I saw on the Yokogawa website that they post an image of the new CSU W1 acquired with a silicon immersion objective NA 1.00 (to employ a greater working distance I suppose). Is this the case? That is the higher contrast claimed is the result of a lower NA which cuts the low intensity signals, i.e. background of scattered light?
Or this factor is someway corrected by changing the distance between microlenses?
Thanks a lot for the help
Mario

--
Mario Faretta
Deapartment of Experimental Oncology
European Institute of Oncology
via Adamello 16
20139 Milan
Italy
Phone: [phone]
email: [hidden email]


[http://www.andor.com/newsletter/footer/sig1.jpg]<http://www.andor.com/microscopy-systems/revolution/wd-system>

[Andrea Latini-4]

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