Laser power in STORM experiments

[lechristophe]

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I've a question about super-resolution microscopy: what is the standard way
of estimating how much power reaches the sample in PALM/STORM experiments?

Let's say I've measured my laser power at the tip of the coupling optical
fiber (entering the microscope stand) to be 100 mW. How do I estimate the
power on the sample (in kW/cm^2) if I use a 100X objective? Using the TIRF
arm field number (16 mm) and objective magnification (100X) I get an
illuminated area of 20,000 µm^2, so even with 100% efficiency (which is
unlikely given the objective non-perfect transmission), that only
translates into 2 kW/cm^2 on the sample, which is quite low.

Given than a lot of STORM papers report powers in the 5-50 kW/cm^2 range, I
wonder if something is wrong in my calculation? I don't want a precise
measurement (which would necessitate measuring the actual output downstream
of the objective), but more a reliable way to get an estimate.

Thanks for your help,

--
Christophe Leterrier
Chercheur
Equipe Architecture des Domaines Axonaux
CRN2M CNRS UMR 7286 - Aix Marseille Université

[Zdenek Svindrych]

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Hi Christophe,

repeating your calculations I arrived at 0.5 kW/cm^2. Still not too much...
Some samples can still blink, just try it!

Unfortunately I don't know of any "standard blinking sample" that could help
you to reliably estimate the illumination intensity. My (standard) way is to
measure the laser power at some convenient place in the setup (I prefer the
back of the objective lens), measure the illuminated area and make some
assumptions about other effects, such as lens efficiency and homogeneity of
illumination.

If you are able to control the "TIRF condition" (the position of the spot at
the back focal plane of your objective lens), you might be able to put the
beam near the center of the objective. Then a low divergence beam will exit
the objective and you can measure the power in the sample space. But
remember that less light gets through the objective when you approach the
TIRF condition...

Also homogeneity of illumination is important in STORM (and hard to achieve
in TIRF). Maybe most of the light is concentrated in the center of your FOV
leading to much higher intensity there.

The way to get high illumination intensity is to focus the beam to smaller
FOV. My camera can only see 80 um by 80 um and I usually illuminate even
smaller region... You can try to achieve this by using smaller fiber
diameter (if your TIRF arm uses critical illumination) or fiber with lower
NA (for Koehler illumination). Adjustable field stop will of course not
increase the intensity, but it may eliminate some unwanted effects of uneven
illumination.

Good luck!

zdenek svindrych
Prague




---------- Původní zpráva ----------
Od: Christophe Leterrier <[hidden email]>
Komu: [hidden email]
Datum: 21. 2. 2014 12:16:22
Předmět: Laser power in STORM experiments

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I've a question about super-resolution microscopy: what is the standard way
of estimating how much power reaches the sample in PALM/STORM experiments?

Let's say I've measured my laser power at the tip of the coupling optical
fiber (entering the microscope stand) to be 100 mW. How do I estimate the
power on the sample (in kW/cm^2) if I use a 100X objective? Using the TIRF
arm field number (16 mm) and objective magnification (100X) I get an
illuminated area of 20,000 µm^2, so even with 100% efficiency (which is
unlikely given the objective non-perfect transmission), that only
translates into 2 kW/cm^2 on the sample, which is quite low.

Given than a lot of STORM papers report powers in the 5-50 kW/cm^2 range, I
wonder if something is wrong in my calculation? I don't want a precise
measurement (which would necessitate measuring the actual output downstream
of the objective), but more a reliable way to get an estimate.

Thanks for your help,

--
Christophe Leterrier
Chercheur
Equipe Architecture des Domaines Axonaux
CRN2M CNRS UMR 7286 - Aix Marseille Université"