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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) ::) :: [log in to unmask] (E) :: www.nus.edu.sg (W)Company Registration No: 200604346E
>