focused laser beam size on stage

[dbstongy]

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Dear list,

We have one Leica TCS SP5 MP confocal system and I would like to find out how the size of the laser beam on the stage is controlled?  When we change the pixel size to meet Nyquist criteria, does the laser focal point on the specimen change its size accordingly? Will the focused beam has same size to the pixels? What is the smallest focus size of the laser beam on the stage we can achieve? Does two photon excitation has the same scenarios?
Thank you very much in advance for your kind advice.
Best regards,

TONG Yan (Ms) :: National University of Singapore :: 14 Science Drive 4, Singapore 117543 :: (65) 6516 7202 (DID) :: (65) 6779 2486 (Fax) ::) :: [hidden email] (E) :: www.nus.edu.sg (W)Company Registration No: 200604346E

[Arne Seitz]

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Dear Yan,

the size of the excitation beam in confocal microscopy is usually diffraction limited. In the easiest case  the minimum size depends on the numerical aperture of the objectives and the used wavelength. Under the following link you can find a PSF generator with which you can calculate the size of your excitation spot (even with rather complicated models).

http://bigwww.epfl.ch/algorithms/psfgenerator/

In practice changing the size is only possible if you under fill the back focal aperture of your objective. In Leica SP2 models this was possible via the so called beam expander. But I'm not sure whether this is also true for the SP5 microscopes (I do not think so). The effective size of your excitation spot in MP microscopy is even more difficult to calculate. I suggest reading the following articles in order to get an idea about it.

Nat Biotechnol. 2003 Nov;21(11):1369-77.
Nonlinear magic: multiphoton microscopy in the biosciences.
Zipfel WR, Williams RM, Webb WW.

Deep tissue two-photon microscopy.
Helmchen F, Denk W.
Nat Methods. 2005 Dec;2(12):932-40. Review.

But you can easily find numerous other articles dealing with this topic.

By no means the size of the excitation beam will change if you change your sampling frequency (pixel size).

I hope that helps.

Best 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 Tong Yan
> Sent: jeudi 23 août 2012 15:58
> To: [hidden email]
> Subject: focused laser beam size on stage
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> We have one Leica TCS SP5 MP confocal system and I would like to find out
> how the size of the laser beam on the stage is controlled?  When we change
> the pixel size to meet Nyquist criteria, does the laser focal point on the
> specimen change its size accordingly? Will the focused beam has same size to
> the pixels? What is the smallest focus size of the laser beam on the stage we
> can achieve? Does two photon excitation has the same scenarios?
> Thank you very much in advance for your kind advice.
> Best regards,
>
> TONG Yan (Ms) :: National University of Singapore :: 14 Science Drive 4,
> Singapore 117543 :: (65) 6516 7202 (DID) :: (65) 6779 2486 (Fax) ::) ::
> [hidden email] (E) :: www.nus.edu.sg (W)Company Registration No:
> 200604346E

[George McNamara]

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Hi Tong,

I strongly recommend you contact your local Leica confocal microscope
representative and/or whoever is in charge of your instrument to get
training. In particular, you should not be messing with the multiphoton
laser until you understand the basics - and beyond - of using your
confocal microscopy. As I've mentioned previously on the listserv: "Do
not look at laser with remaining eye".

Brief answers:

size of the laser beam on the stage is controlled?

Size of the confocal laser excitation beam is defined by the numerical
aperture of the objective lens and the wavelength of light. An
assumption is the excitation beam "just fills" the back aperture of the
objective lens (underfilling results in a lower effective NA,
overfilling results in less excitation reaching the specimen and
possibility of excitation light scattering to unwanted places).  In
1-photon excitation, this is a double cone of light, with most of the
fluorescence being at the point of focus.

In the case of  multiphoton excitation, fluorescence (MPEF) emission
depends on the square (or cube) of excitation power. That is, double the
laser power, get four times the output of a molecule at the focus point
(until it photobleaches). At some distance from the focus, the photon
flux will be too low to produce MPEF - hence the intrinsic optical
sectioning. Since there is a volume that is being excited

When we change the pixel size to meet Nyquist criteria, does the laser focal point on the specimen change its size accordingly?

No.
When you change Pixel size on the Leica SP5, you are changing the image
format and/or zoom. These are both controls for the X and Y scanning
mirrors. The mirrors point the excitation beam to somewhere on the
specimen - have nothing to do with the focal point size.

Nyquist criteria - you can meet the Nyquist criteria with ANY objective
lens. However, a 0.3 NA objective lens is not going to give you the same
resolution as a 1.4 NA lens (the 1.4 NA lens might not give you anything
if you are imaging say 400 um deep into a specimen).

Tip: When I train users who want "maximum resolution" on my Leica SP5 or
MP/SP5, I explain that the performance depends on their sample
preparation (thickness, refractive index, staining quality) as well as
the microscope. On the SP5 side, I give them the following recommendations:
* Leica plan apochromat 63x / 1.4NA lens
* Scan speed 600 Hz (not the LAS AF default 400 Hz), if zoom of 1.0 ...
if you are always using higher zoom, figure out what is the fastest scan
speed and use that. If you have the resonant scanner, this is
simplified: always 8000 Hz. My thanks to Jonathan Boyd, leica
applications, for pointing out that "faster is better". If you don't
believe me, compare (with standard scanner mode) brightness and
photobleaching at fastest vs slowest possible speed (1 Hz?).
* Pixel size: 60 nm (based on dxy = 0.6 * 500 nm / 1.4 NA = 214 nm, then
dxy/3.5 = 60 nm, since 2D image, not a 1D sine wave). Leica did a
horrible job with the user interface of LAS AF - they should have
enabled the user to specify the pixel size and field of view (zoom), and
adjust "format" as needed. Instead the default format list are all
powers of two.
* Averaging; whatever is needed to get a "nice" image. For fixed
specimens, I usually use sequential scan mode (or sequential stack mode
for Z-series), longest excitation laser line(s) first, and frame averaging.

I also leave the all the lasers on 24/5 (24/7 if there is a user on the
weekend ... ideally the room is at constant temperature all the time,
too). We have :eica "remote care" turned on so in theory Leica has the
data from us- and every other SP5 that has remote care turned on - to
determine if leaving the lasers 24/5(7) results in better laser
stability and lifetime than turning them off between sessions or every
night.
If anyone from Leica is reading this: how about publishing the data!


What is the smallest focus size of the laser beam on the stage we can achieve?

dxy = 0.6 * 500 nm / 1.4 NA = 214 nm ... assuming the laser light is
just filling the back aperture of the objective lens (get training from
your leica rep or instrument owner about this point).

Does two photon excitation has the same scenarios?

Yes, dxy = 0.6 * 800 nm / 1.4 NA = 343 nm, but effective excitation size
also depends on how much power you are using. I strongly recommend not
using the MP laser until you understand confocal operations and MP laser
safety. If your microscope does not have a blackout curtain around it, I
strongly recommend getting one installed (and cover as many LEDs and
other lights that are on the inside), both for any nondescanned
detectors (NDDs) and for laser safety (for the latter, all users will
need to abide by a policy of not scanning with the MP laser while the
curtain is open).


A nice (free) tutorial on confocal microscopy is the *Confocal Laser
Scanning Microscopy (Principle)*  at
http://www.zeiss.de/C1256D18002CC306/Order?OpenForm&lsm-bio

You can also order Jim Pawley's Handbook online at
http://www.amazon.com/Handbook-Biological-Confocal-Microscopy-Pawley/dp/038725921X

In the next few months P.O. Berggren of Karolinska Institute (and UMiami
and Korea) will be opening a lab at a new campus in Singapore. He will
be getting at least one Leica SP8 confocal microscope (maybe with STED).
So, someone from Leica is or will be around for installation and
hopefully training - get in depth training from your lab, PO's group
and/or Leica.

George


On 8/23/2012 9:57 AM, Tong Yan wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> We have one Leica TCS SP5 MP confocal system and I would like to find out how the size of the laser beam on the stage is controlled?  When we change the pixel size to meet Nyquist criteria, does the laser focal point on the specimen change its size accordingly? Will the focused beam has same size to the pixels? What is the smallest focus size of the laser beam on the stage we can achieve? Does two photon excitation has the same scenarios?
> Thank you very much in advance for your kind advice.
> Best regards,
>
> TONG Yan (Ms) :: National University of Singapore :: 14 Science Drive 4, Singapore 117543 :: (65) 6516 7202 (DID) :: (65) 6779 2486 (Fax) ::) :: [hidden email] (E) :: www.nus.edu.sg (W)Company Registration No: 200604346E
>    

[Pascal Weber]

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We have one Leica TCS SP5 MP confocal system and I would like to find out
how the size of the laser beam on the stage is controlled?

It is possible to controlled the size of the point in 2P. It's mentionned in one
answer you have to inderfill, but never over. To do that you have to add a
lense in the laser beam.

 When we change the pixel size to meet Nyquist criteria, does the laser focal
point on the specimen change its size accordingly?

It's only change with the NA.

 Will the focused beam has same size to the pixels? What is the smallest focus
size of the laser beam on the stage we can achieve?

I always obtain the same résolution with a 2P than a normal confocal but in
deep (over 1mm in tthe brain of over weeks mouse)


 Does two photon excitation has the same scenarios?

No because the pulse power is over a confocal and you can excite many fluo
at the same time. You have less bleaching due to the small excitaion point.
Infact may be the 'confocal' system if you use multi wave length 2P laser.
Thank you very much in advance for your kind advice.

[Guy Cox-2]

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This was already answered by Arne Seitz in the very first reply to this query!

The spot on the sample is a diffraction-limited spot - an Airy disk.  This is a function of the NA of the lens and has nothing to do with the pixel size you select on the scope.  The radius of the Airy disk is the resolution of that objective.

If you underfill the BFP (pupil) of the objective you are effectively reducing the NA and the spot will therefore get larger, making your resolution worse.  This is obviously a bad thing to do.  The Leica has a beam-expander so this should not happen (unless you start fiddling).

If you overfill the BFP you will lose some light, that's all.  Resolution and spot-size is unchanged, and you can always dial in more laser.  

In multiphoton you get a resolution improvement of 1.414 (sqrt 2) which compensates for the longer wavelength.  (800nm in MP is equivalent to 565nm in confocal or widefield)  This explains why you see much the same resolution in both MP and confocal.   (In principle you can get the same benefit in confocal, but only with a very tiny pinhole, which is impracticable.  Normal confocal fluorescence has the same resolution as widefield fluorescence).  

                                                                                 Guy



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pascal Weber
Sent: Tuesday, 28 August 2012 4:07 PM
To: [hidden email]
Subject: Re: focused laser beam size on stage

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We have one Leica TCS SP5 MP confocal system and I would like to find out how the size of the laser beam on the stage is controlled?

It is possible to controlled the size of the point in 2P. It's mentionned in one answer you have to inderfill, but never over. To do that you have to add a lense in the laser beam.

 When we change the pixel size to meet Nyquist criteria, does the laser focal point on the specimen change its size accordingly?

It's only change with the NA.

 Will the focused beam has same size to the pixels? What is the smallest focus size of the laser beam on the stage we can achieve?

I always obtain the same résolution with a 2P than a normal confocal but in deep (over 1mm in tthe brain of over weeks mouse)


 Does two photon excitation has the same scenarios?

No because the pulse power is over a confocal and you can excite many fluo
at the same time. You have less bleaching due to the small excitaion point.
Infact may be the 'confocal' system if you use multi wave length 2P laser.
Thank you very much in advance for your kind advice.

[Michael Doube]

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> If you overfill the BFP you will lose some light, that's all.
Unless you are using a high transmission percentage of a two-photon
laser, at which point you might start melting the black plastic iris
which is mounted in the back of some objectives (from experience).

Michael

[Pascal Weber]

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The black ring is not normally done in plastic but metal (Zeiss objectiv).
Normally the stand contains a lens that compensates for filling the pupil for
each objectiv (except Nikon ?!!). The laser power is not concentrated on this
ring.

[Craig Brideau]

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The A1 has an automated beam expander for its Chameleon.  It's calibrated
for each lens.

Craig



On Wed, Aug 29, 2012 at 12:28 AM, Pascal Weber <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The black ring is not normally done in plastic but metal (Zeiss objectiv).
> Normally the stand contains a lens that compensates for filling the pupil
> for
> each objectiv (except Nikon ?!!). The laser power is not concentrated on
> this
> ring.
>

[Pascal Weber]

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Hi Craig, Where did they put the beam expander on Nikon A1. We have a Nikon
two photon (last generation) and i never saw it !
They have an automatic alignment behind the statif i think that's all !
Let me know. As i know only Olympus have a real beam expander (telescope)
in the beampath.

[Craig Brideau]

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There is a lens inside the giant A1 scan head which is on a motorized
translation mount.  My system is heavily hacked (it's what I do) so I have
manual control over it.  Normally the A1 software is configured to jog the
lens around somewhat.  You can use it to adjust the spot size at the back
aperture of the lens.  You'll have to ask Nikon what the software normally
does with that lens control in the unhacked version.

Craig


On Thu, Aug 30, 2012 at 12:18 AM, Pascal Weber <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Craig, Where did they put the beam expander on Nikon A1. We have a Nikon
> two photon (last generation) and i never saw it !
> They have an automatic alignment behind the statif i think that's all !
> Let me know. As i know only Olympus have a real beam expander (telescope)
> in the beampath.
>

[Pascal Weber]

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Ok Craig thank you for these precisions. I knew that Olympus uses a driven
telescope  for modulating the beam diameter before entering the scan head.
That does not change the filling of the rear back lens.

[Guy Cox-2]

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How is that possible?

                               Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox  2nd edition 2012  CRC Press
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
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 Pascal Weber
Sent: Friday, 31 August 2012 4:49 AM
To: [hidden email]
Subject: Re: focused laser beam size on stage

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Ok Craig thank you for these precisions. I knew that Olympus uses a driven
telescope  for modulating the beam diameter before entering the scan head.
That does not change the filling of the rear back lens.

[dbstongy]

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Dear All,
Thank you for all the help.
Final conclusion: We need the information about the laser focused volume on the stage for both two photon and one photon excitation laser, so we are going to measure the FWHM of objective PSF with the diffraction limited beads.
Best regards,


TONG Yan
Department of Biological Sciences
National University of Singapore


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Friday, August 31, 2012 6:48 AM
To: [hidden email]
Subject: Re: focused laser beam size on stage

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How is that possible?

                               Guy

Optical Imaging Techniques in Cell Biology by Guy Cox  2nd edition 2012  CRC Press
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for Microscopy & Microanalysis, 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 Pascal Weber
Sent: Friday, 31 August 2012 4:49 AM
To: [hidden email]
Subject: Re: focused laser beam size on stage

*****
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Ok Craig thank you for these precisions. I knew that Olympus uses a driven telescope  for modulating the beam diameter before entering the scan head.
That does not change the filling of the rear back lens.