"Should I focus when measuring laser power?" microscope experts questions

After our webinar “How to display live illumination power on your computer“, based on the Argo-POWER^HM device, we discussed some questions with microscopists and experts.
How do you correlate sampling period and noise? How critical is the focus of the incident light for power measurement reproducibility over time? Is it possible to estimate the surface for spinning disk systems in Daybook software?

1. How do you correlate sampling period and noise?

2. How critical is the focus of the incident light for power measurement reproducibility over time?

3. Can Daybook estimate irradiance for spinning disk systems?

4. Widefield irradiance requires the objective’s Field Number. Where to find it?

5. Is there a difference in illumination when measured with and without immersion medium?

6. Can the power measurements be saved into the database of Daybook Data Manager?

7. What is the expected lifetime of the device?

2. How critical is the focus of the incident light for power measurement reproducibility over time?

• In a widefield configuration, the light is focused onto the objective and more or less collimated (depending on the objective NA) after onto the sample.
• In a confocal configuration, the light is collimated onto the objective and more or less focused (depending on the objective NA) after onto the sample.

For that reason, in widefield mode, not being at the focal plane (to some extent) has little effect on the illumination power measurement.
On the contrary, in confocal mode, being far from the focal plane has an impact, as a non-negligible fraction of the beam may not reach the sensor, due to its strong divergence.

Another aspect to keep in mind when measuring the power at the focal plane of a confocal microscope is the possible saturation of the sensor when high NA objectives and/or high power lasers are used. A good compromise in confocal mode would be to measure the power not exactly at the focal plane, but close to it. For reproducible measurements, a lot of care must therefore be brought in confocal mode, much less in widefield mode.

To find the best focus, we recommend adjusting the focus until the highest optical power is measured. This indicates that the sensor is at the focal plane of the microscope objective.

3. Can Daybook estimate irradiance for spinning disk systems?

For the sake of reproducing experimental images and data independently from the used imaging system, it is important to know how much light a biological sample receives on a given surface, through the calculation of the surface power density, i.e. the irradiance.

• The irradiance (in W/cm²) is defined as the ratio of the optical power (in W) divided by the illuminated surface (in cm²).
• The optical power can be measured with a power meter at the focal plane of the objective.
• The surface, however, is difficult to measure but can be evaluated from calculation, depending on the microscope illumination mode.

Daybook software has three illumination modes pre-setup: wide-field, confocal laser-scanning and confocal spinning disk illuminations. They define the irradiance in live, based on the measured optical power and the theoretical calculation of the area.

4. Widefield irradiance requires the objective’s Field Number. Where to find it?

The Field Number is only required when you want to try to estimate the irradiance of a Widefield. For confocal systems, the Numerical Aperture would be required instead.

The Field Number can be found within the specs of the objectives.
Pay attention: sometimes, the Field Number (FN) and the Field Of View (FOV) can be mixed-up. They are related through the following equation: 𝐹𝑂𝑉=𝐹𝑁/𝑀𝑎𝑔.

You can find your objective specs on the website of the manufacturers:

• OLYMPUS objectives
• ZEISS objectives (confusion between FN and FOV)
• LEICA objectives  (confusion between FN and FOV)
• NIKON objectives

“The field diameter in an optical microscope is expressed by the field-of-view number or simply field number, which is the diameter of the viewfield expressed in millimeters and measured at the intermediate image plane.”   [1]

5. Is there a difference in illumination when measured with and without immersion medium?

For Widefield microscopes, the presence or absence of an immersion medium does not change the measure. Indeed, the incident angle is too low to create a total internal reflection.

For confocal microscopes, the beam is focalized. When used without an immersion medium, this would produce total internal reflection (TIR). TIR appears when the light gets out from a more refringent (refractive) medium (here the objective lens with index 1.52) to a less refringent one (here, the air with index 1) and when the angle of incidence is equal or greater than the limit angle.

In this case, of light beam flux is reflected back and therefore not accounted by the power sensor. The absolute power measured would therefore be underestimated, but it would still be possible to see the trends of power variation.

6. Can the power measurements be saved into the database of Daybook?

The results obtained from the Argo-POWER^HM can be stored in a database when Daybook Data Manager software is activated. When getting results in Daybook Analysis, click on the “Save in Data Manager” button.

The key metrics (power average and power stability) are displayed within Daybook Data Manager, showing their evolution through a color-coded timeline. 

Power average and power stability metrics displayed within Daybook Data Manager timeline

“For confocal imaging, the stability of the illumination power is of high importance.”

ISO standard 21073:2019

7. What is the expected lifetime of the device?

All our products are designed to be used for many years. Their lifetime is longer than the warranty period. We offer a warranty that their condition will not change, or we will be able to repair it.

 For the Argo-POWER^HM

* the Argo-HM patterns are warranted for life. The fluorescence will ever be present and the spatial dimensions of the patterns will not change – their intensity will fluctuate though (more here). If you break the glass, we can repair it, so you don’t have to buy a completely new solution (information: since Argolight creation in 2012, only 0.5 % of our customers claimed they broke the glass).

* The optical power meter is an electronic device so it is warranted for 1 year. If necessary, we can replace it, so you don’t have to buy a complete solution.

Enjoy imaging! For more details on other cool ways to operate your Argo-POWER^HM,  reach out to the Support Team and we’ll be happy to assist! 

Sources:

[1] Nikon, Microscopy U, “Properties of Microscope Objectives” 

Related terms: illumination power, live irradiance, power fluctuations, power-meter, power for biological applications, power instructions