lateral resolution

[Jan Grawe]

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Dear all,

I find that there is someting I have not
understood regarding lateral resolution in our confocal (Zeiss 510 meta).
As I understand it, lateral resolution should be
dependent on excitation wavelength and NA solely.
But what is the case if we open the pinhole a bit
above 1 AU? For some (admittedly special) samples, we use up to about 4AU.
As far as I understand it, on a 510, using a 40x
1.3 objective, 532ex/575em, zoom 1, the pinhole
is 300um, and the object-side projected pinhole
diameter is then about 2,2um. As the PMT detector
itself is not position-sensitive any light within
this 2.2um circle in the focal plane would be
detected. The practical resolution would then be
determined by the laser spot size (?).
Using the zeiss "optimal frame size" calculator
for nyqvist sampling on the above setting gives
me 2048*2048 pixels, which is the max available,
maybe it would calculate an even higher nunber of
pixels if the option was available. 2048*2048
gives me a pixel size of 0.11*0.11 um , which to
me seems small in relation to the spot size of the laser.
Is this reasoning correct, or where am I wrong?

Thanks in advance,

Jan Grawé
Cell Analysis Core Facility
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
[hidden email]
www.rudbeck.uu.se/cellanalys

[Guy Cox]

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The resolution, assuming the BFP of the objective is
filled (not always the case) will be given in fluorescence
by the Rayleigh formula r = 0.5lambda/NA.  This applies
to widefield or scanned microscopy.  With a very small
pinhole there is an additional 'confocal' resolution
improvement of 1.414 (root 2) (r confocal = r wf / 1.414).
The full improvement will only be given by an infinitely
small pinhole ... For some examples of finite sized
pinholes see Guy Cox & Colin Sheppard, 2004.  Practical
limits of resolution in confocal and non-linear microscopy.  
Microscopy Research & Technique, 63, 18-22 .

In practice you can get some lateral resolution improvement
in reflection mode but you won't get much, if any, in
fluorescence.  So when you open your pinhole you are not
really losing lateral resolution.  You are losing depth
resolution quite substantially.  There really shouldn't be
any reason to open the pinhole above 1 Airy unit since (unless
the system is misaligned) that will let through all the in
focus light.  After that you're just letting in out of
focus light.

                                              Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jan Grawé
Sent: Tuesday, 1 April 2008 6:01 PM
To: [hidden email]
Subject: lateral resolution

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

Dear all,

I find that there is someting I have not understood regarding lateral resolution in our confocal (Zeiss 510 meta).
As I understand it, lateral resolution should be dependent on excitation wavelength and NA solely.
But what is the case if we open the pinhole a bit above 1 AU? For some (admittedly special) samples, we use up to about 4AU.
As far as I understand it, on a 510, using a 40x
1.3 objective, 532ex/575em, zoom 1, the pinhole is 300um, and the object-side projected pinhole diameter is then about 2,2um. As the PMT detector itself is not position-sensitive any light within this 2.2um circle in the focal plane would be detected. The practical resolution would then be determined by the laser spot size (?).
Using the zeiss "optimal frame size" calculator for nyqvist sampling on the above setting gives me 2048*2048 pixels, which is the max available, maybe it would calculate an even higher nunber of pixels if the option was available. 2048*2048 gives me a pixel size of 0.11*0.11 um , which to me seems small in relation to the spot size of the laser.
Is this reasoning correct, or where am I wrong?

Thanks in advance,

Jan Grawé
Cell Analysis Core Facility
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
[hidden email]
www.rudbeck.uu.se/cellanalys

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[Kevin Braeckmans]

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

Dear Jan,

As you have indicated, when opening up the pinhole the resolution is
determined by the laser beam only. Laterally, your focused laser beam will
have a resolution of approx. 250nm. Following Nyquist, the pixel size should
indeed be about 100 to 110 nm. So, everything seems to be fine.

Best regards,

Kevin

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens Jan Grawé
> Verzonden: dinsdag 1 april 2008 10:01
> Aan: [hidden email]
> Onderwerp: lateral resolution
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear all,
>
> I find that there is someting I have not
> understood regarding lateral resolution in our confocal (Zeiss 510
> meta).
> As I understand it, lateral resolution should be
> dependent on excitation wavelength and NA solely.
> But what is the case if we open the pinhole a bit
> above 1 AU? For some (admittedly special) samples, we use up to about
> 4AU.
> As far as I understand it, on a 510, using a 40x
> 1.3 objective, 532ex/575em, zoom 1, the pinhole
> is 300um, and the object-side projected pinhole
> diameter is then about 2,2um. As the PMT detector
> itself is not position-sensitive any light within
> this 2.2um circle in the focal plane would be
> detected. The practical resolution would then be
> determined by the laser spot size (?).
> Using the zeiss "optimal frame size" calculator
> for nyqvist sampling on the above setting gives
> me 2048*2048 pixels, which is the max available,
> maybe it would calculate an even higher nunber of
> pixels if the option was available. 2048*2048
> gives me a pixel size of 0.11*0.11 um , which to
> me seems small in relation to the spot size of the laser.
> Is this reasoning correct, or where am I wrong?
>
> Thanks in advance,
>
> Jan Grawé
> Cell Analysis Core Facility
> Rudbecklaboratoriet/C5
> Dag hammarskjölds väg 20
> SE-75185 Uppsala
> SWEDEN
>
> Phone: +(0)18-4714657
> Cell:   +(0)70-2577874
> [hidden email]
> www.rudbeck.uu.se/cellanalys

[Michael Weber-4]

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

The correct pixel size is just correct by chance, since the Zeiss software
doesn't consider the pinhole diameter in the calculation of XY scaling.

Michael

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Jan,
>
> As you have indicated, when opening up the pinhole the resolution is
> determined by the laser beam only. Laterally, your focused laser beam will
> have a resolution of approx. 250nm. Following Nyquist, the pixel size
> should
> indeed be about 100 to 110 nm. So, everything seems to be fine.
>
> Best regards,
>
> Kevin
>
>
>> -----Oorspronkelijk bericht-----
>> Van: Confocal Microscopy List [mailto:[hidden email]]
>> Namens Jan Grawé
>> Verzonden: dinsdag 1 april 2008 10:01
>> Aan: [hidden email]
>> Onderwerp: lateral resolution
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Dear all,
>>
>> I find that there is someting I have not
>> understood regarding lateral resolution in our confocal (Zeiss 510
>> meta).
>> As I understand it, lateral resolution should be
>> dependent on excitation wavelength and NA solely.
>> But what is the case if we open the pinhole a bit
>> above 1 AU? For some (admittedly special) samples, we use up to about
>> 4AU.
>> As far as I understand it, on a 510, using a 40x
>> 1.3 objective, 532ex/575em, zoom 1, the pinhole
>> is 300um, and the object-side projected pinhole
>> diameter is then about 2,2um. As the PMT detector
>> itself is not position-sensitive any light within
>> this 2.2um circle in the focal plane would be
>> detected. The practical resolution would then be
>> determined by the laser spot size (?).
>> Using the zeiss "optimal frame size" calculator
>> for nyqvist sampling on the above setting gives
>> me 2048*2048 pixels, which is the max available,
>> maybe it would calculate an even higher nunber of
>> pixels if the option was available. 2048*2048
>> gives me a pixel size of 0.11*0.11 um , which to
>> me seems small in relation to the spot size of the laser.
>> Is this reasoning correct, or where am I wrong?
>>
>> Thanks in advance,
>>
>> Jan Grawé
>> Cell Analysis Core Facility
>> Rudbecklaboratoriet/C5
>> Dag hammarskjölds väg 20
>> SE-75185 Uppsala
>> SWEDEN
>>
>> Phone: +(0)18-4714657
>> Cell:   +(0)70-2577874
>> [hidden email]
>> www.rudbeck.uu.se/cellanalys

[Ian Dobbie-2]

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

Michael Weber <[hidden email]> writes:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> The correct pixel size is just correct by chance, since the Zeiss software
> doesn't consider the pinhole diameter in the calculation of XY scaling.

There is some justification for this. Using the Rayleigh
criterion, the resolution is defined by the distance to the first
minimum of the Ariry disk. A confocal pinhole does not change this at
all, although it does reduce the FWHM of the peak (ie the shape
changes but the peak still has the same total width). Where a confocal
really wins is in Z resolution.

Ian

[Rietdorf, Jens]

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

Listers,

I strongly recommend to look back 10 years, there was a threat on the
confocal listserver mentioning this article:

Contrast, resolution, pixelation, dynamic range and signal-to-noise :
fundamental limits to resolution in fluorescence light microscopy -
E.H.K. Stelzer - J. Microscopy (1998) 189 : 15-24.

Basically, to meet the Rayleigh criterion under even moderate noise
conditions you need at least 8 samples to achieve the required contrast
to distinguish the peaks of 2 neighbouring Airy patterns. Translated for
the objective and lambda mentioned (1.3NA, 532nm ex.) this results in a
pixelsize of about 58nm(!). The Nyquist calculator from SVI even returns
51nm as the required pixel size.

Overall, in my opinion, the pinhole does not improve lateral resolution
at all under the conditions encountered in imaging of fluorescently
labeled biomaterials.

Cheers, jens

***Join the elmi meeting! Extended application deadline 14.4.08. More
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Ian Dobbie
Sent: Mittwoch, 2. April 2008 11:49
To: [hidden email]
Subject: Re: lateral resolution

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

Michael Weber <[hidden email]> writes:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> The correct pixel size is just correct by chance, since the Zeiss
> software doesn't consider the pinhole diameter in the calculation of
XY scaling.

There is some justification for this. Using the Rayleigh criterion, the
resolution is defined by the distance to the first minimum of the Ariry
disk. A confocal pinhole does not change this at all, although it does
reduce the FWHM of the peak (ie the shape changes but the peak still has
the same total width). Where a confocal really wins is in Z resolution.

Ian