TIRF illumination is usually a bit stronger in the evanescent wave (I would say 20% more than in epi mode). One option would be to measure the laser power first in epi mode with a laser power measurement device (with the objective). Then, using fluorescent beads (200 nm in size = thickness of the evanescent wave) on coverglass you could measure their intensity in epi versus tirf mode.

That should give you a good estimation of the illumination power of the evanescent wave.

Indeed, it is important to try to play in the same range of power that others published, although this epi vs TIRF difference in intensity is probably the range within which you'll have to play to find your own settings.

Finally, when you want to measure the illumination power in kW/cm^2 do not make the mistake to use the size of your field of view for your maths. You'll have to estimate the size of your illumination spot (which is bigger than your field of view). To do so, fluorescent conjugates of dextran are quite useful. You adsorb a 1/1000 dilution on a coverglass and then you bleach one area at high power. Then at lower laser power you can scan your coverslip to measure the diameter of your illumination spot.

Hi Paul,

Close the field stop on your microscope so that the illumination is limited to the camera area you are reading out from (256x256 pixel central ROI for example). Turn up the laser to 100% and measure the power at the objective with a power meter.  This is assuming you are using 100% for STORM.

You know the pixel dimensions and pixel size and can use that to calculate area.  In my example, lets consider the 256x256 region with 160 nm/pixel (100X lens with 16 x 16 um physical pixel size of the commonly used Andor iXon DU 897).  This gives you an area of 1.68 X 10-5 cm2.  Divide the measured power by this.  For example, if you measure 50 mW out of the objective, and divide by the area above I calculate a power density of 3 kW/cm2.

Hi Paul,

on our dSTORM microscopes we measure the laser power in EPI at the objectives front lens. As Nicolas mentioned the power density in HILO or TIRF is increased by a factor you can measure as described above. Confining the field of illumination as described by Christopher is also very helpful for the measurement.

Many thanks for your comments and suggestions - the power of ResearchGate!

When our TIRF slider arrives next week (Zeiss, manual) I'll try out a combination of these recommendations.

I'm a little surprised that the power density in TIRF is higher than in epi. Could any of you point me in the direction of the theory behind this? Is it the confinement of the illumination energy within a thin section that 'concentrates' the power?

Hi Paul,

the image you will find following the link to nature might help to understand why power density is increasing in HILO illumination. As dz is decreasing when going to TIRF the power stays constant and therefore the power density is increasing.

http://www.nature.com/nmeth/journal/v5/n2/fig_tab/nmeth1171_F1.html