Feb 11, 2022

Public workspaceIllumination Power and illumination stability V.1

DOI
dx.doi.org/10.17504/protocols.io.bzp8p5rw
Illumination Power and illumination stability
  • 1Allen Institute for Cell Science, Seattle, WA, USA;
  • 2Cancer Research UK Manchester Institute;
  • 3Scientifica;
  • 4PicoQuant GmbH;
  • 5Institute of Experimental Medicine, Budapest;
  • 6Newcastle University, BioImaging Unit, UK;
  • 7Carl Zeiss AG, Oberkochen, Germany;
  • 8Scientific and Technological Centers, Universitat de Barcelona (CCiTUB);
  • 9University of Calgary;
  • 10INL - International Iberian Nanotechnology Laboratory;
  • 11Live Cell Imaging Laboratory, Snyder Institute of Chronic Diseases, University of Calgary;
  • 12New York State Dept of Health;
  • 13Institute of Science and Technology Austria;
  • 14Advanced Optical Microscopy Centre;
  • 15Carl Zeiss Microscopy GmbH;
  • 16MRI, Biocampus, University of Montpellier, CNRS, INSERM, Montpellier, France;
  • 17Coherent GmbH & Co.KG;
  • 18Thorlabs GmbH;
  • 19UMass Chan Medical School;
  • 20Max Planck Institute for Biology of Ageing;
  • 21University Hospital Jena, Biomolecular Photonics Group;
  • 22University of British Columbia;
  • 23Department of Biomedicine, University of Basel;
  • 24Max Planck Institute for Multidisciplinary Sciences;
  • 25Department of Biomedical Engineering, University of Wisconsin - Madison;
  • 26Max Planck Insitute of Molecular Cell Biology and Genetics, Dresden, Germany;
  • 27ISO International Standards Organization TC172 SC5;
  • 28Oregon National Primate Research Center (ONPRC, OHSU West Campus);
  • 29Center for Structural Systems Biology (CSSB) Hamburg;
  • 30Cold Spring Harbor Laboratory;
  • 31Nikon Europe BV;
  • 32Cell Biology and Image Acquisition Core Facility, University of Ottawa;
  • 33Life Imaging Center, University of Freiburg, Germany;
  • 34Friedrich Miescher Institute for Biomedical Research
Nathalie Gaudreault
Open access
DOI: dx.doi.org/10.17504/protocols.io.bzp8p5rw
External link: https://quarep.org/
Protocol Citation: Nathalie Gaudreault, Steve Bagley, Rodrigo R Bammann, Fabio Barachati, Laszlo Barna, Veronika Boczonadi, Ulrike Boehm, Manel Bosch, Craig Brideau, Mariana T Carvalho, Pina Colarusso, Richard Cole, Nasser Darwish-Miranda, Sam Duwé, Frank Eismann, Orestis Faklaris, Andreas Felscher, Manfred Gonnert, David Grunwald, Marcel Kirchner, Birgit Hoffmann, Gabriel Krens, Alex J Laude, Jeffrey M LeDue, Pascal Lorentz, Miso Mitkovski, Michael S Nelson, Britta Schroth-Diez, Stanley Schwartz, Sathya Srinivasan, Roland Thuenauer, Tse-Luen Wee, Kees van der Oord, Chloë van Oostende-Triplet, Roland Nitschke, Laurent Gelman 2022. Illumination Power and illumination stability. protocols.io https://dx.doi.org/10.17504/protocols.io.bzp8p5rw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 02, 2021
Last Modified: March 16, 2023
Protocol Integer ID: 54752
Keywords: Illumination Power, Illumination stability, Laser Power, Laser stability, Laser linearity, Illumination linearity
Disclaimer
This protocol was developed by the members of QUAREP-LiMi Working Group 1. The member list can be found here: (https://quarep.org/working-groups/wg-1-illumination-power/wg-1-members/)

The QUAREP-LiMi is a group of scientists interested in improving quality assessment (QA) and quality control (QC) in light microscopy. We first came together on April, 2020 and as of February 2022 the group has grown to 395 people from 34 countries spread around the world. We have members from academia, microscopy communities, companies, organizations or institutions related to standardization, scientific publishers, and observers from funding agencies.
Abstract
To obtain accurate, reproducible, and interpretable data when conducting imaging experiments, it is critical to consider external factors affecting data acquisition at various steps of the experimental workflow. Illumination power and stability represent two critical factors, especially when comparing fluorescence intensities between images during a time-lapse experiment or experiments performed at different times or on other microscopes.
The fluorescence signal can be generated by different types of light sources. These light sources and their coupling elements (e.g., fibers) can display varying performances over time as they age, move, or as environmental conditions change.
Unfortunately, microscope users can often only set illumination power as a percentage of its maximal output and, may therefore, not be aware of potential performance changes. It is important to recognize that a set percentage will not always yield the same illumination power in Watts at the objective over the course of an experiment, not to mention between days or systems. This means that selecting for example 10% output may lead to different experimental results over time and will not necessarily be comparable to outputs obtained from other lasers or microscopes, even those of the very same model.
If you are responsible for system maintenance, routinely measuring the illumination power, stability, and linearity over time can help you detect issues that affect the integrity of the system and thus the reproducibility of an experiment.
This protocol describes how to measure the stability and linearity of the illumination power using calibrated external power sensors. This protocol is at the moment intended for confocal systems (raster scanning and spinning disks), but will be extended later to other imaging modalities. It represents the collective experience of 60 imaging scientists. Measurements made by our working group with this protocol are available in a public database, which will be updated with further contributions from the community.
Image Attribution
Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy logo designed by Thao Do.
Guidelines
Laser safety and regulations
  • Please refer to the documentation provided by the manufacturer for additional warnings and preventive protective equipment (PPE) requirements (e.g. laser safety goggles). Always consult with your local Laser Safety Officer or Radiation Safety Officer and refer to your laboratory safety documentation for more information.
  • You can also consult your Laser Safety Standards ANSI Z136 in North America, SUVA 66049.D in Europe, and BS EN 60825-1 in the UK. Additionally, laser safety standards and regulations are covered by IEC norm 60825-1 and LED eye safety standards and regulations are covered by IEC norm 62471 in Europe.
Materials
  1. Raster scanning or spinning disk confocal microscope.
  2. Objective (e.g. 10x)
  3. Power meter and sensor (e.g Thorlabs power controller and slide sensor)
Safety warnings
Attention
Hazardous, visible or invisible radiation from lasers, lamps, and other light sources used for microscopy can cause permanent damage to the retina, skin burns and fire. Always follow proper laser safety protocols for your equipment and situation.

  • Hazardous, visible or invisible radiation from lasers, lamps, and other light sources used for microscopy can cause permanent damage to the retina, skin burns and fire. Always follow proper laser safety protocols for your equipment and situation.
Before start
Screen Shot 2021-11-02 at 10.57.37 AM.png
Safety information
Before you take any measurements, it is important to familiarize yourself with all laser safety rules and requirements to avoid eye and skin exposure to scattered or direct radiation.



Illumination power measurement
Illumination power measurement
Warm up the entire system, including the illumination source (i.e.lasers).
Note
Recommended standard waiting time is one hour. Gas lasers typically need more time than solid state or diode lasers to warm-up. For laser-specific recommended warm-up time refer to the product documentation. The necessary warm-up time for a given system can also be determined empirically by measuring how much time is required for illumination power to reach stability (See Figure 1). Monitoring warming-up time should be performed during installation and regularly afterward, as changes to the warm-up time are also an indicator of laser aging. Also, make sure the environment meets the operating specifications of the equipment (especially air renewal or cooling around the lasers and the scope, consult Appendix 3- STEP16).

Expected result
Figure 1. Examples of data collected for laser warm-up and short-term illumination power stability. The confocal microscope was not pre-warmed to illustrate the fluctuation of illumination power during this crucial preparation step. All laser intensities were controlled with an AOTF (acousto-optic tunable filter) and the laser power measured at the objective with a PM100A power meter and a S170C sensor (Thorlabs). The inset (greyed out) shows the first 25 min with greater details.
Figure 1. Examples of data collected for laser warm-up and short-term illumination power stability. The confocal microscope was not pre-warmed to illustrate the fluctuation of illumination power during this crucial preparation step. All laser intensities were controlled with an AOTF (acousto-optic tunable filter) and the laser power measured at the objective with a PM100A power meter and a S170C sensor (Thorlabs). The inset (greyed out) shows the first 25 min with greater details.


Select a low NA, air objective, such as a 10x objective, for this measurement. Ensure it is clean and presents no signs of damage.
Note
This protocol can be used with other objective magnifications or performed without any objective, provided that the user operates the system in full awareness of laser safety and security standards. Be aware that different objective types may have different transmission efficiencies, thus compare results from the same objective only. Also, many sensors (with diffuser) show different power levels depending on the beam collimation, i.e., numerical aperture of the objective (see Power meter Q&A Appendix 1-Q1&2- STEP14). Ensure also that the objective used can withstand high laser powers at the required wavelength if the measurement needs to be made in such a regime.

Place the power meter sensor in front of the objective. Choose an appropriate power meter sensor for your laser (Consult our Power meter Q&A Appendix 1- STEP14). It is important and preferable that the sensor is fixed (in a stage holder) and stable during the measurement. The operator should not hold or touch the sensor during the measurement. Since the sensor can reflect laser light, avoid orientating the sensor in such a way that it could reflect laser light directly to your eyes and skin or any other reflecting surfaces.
Note
We recommend the use of a sensor with the shape and size of a slide, these are easier to fix on classical sample holders. For other sensor shapes, it might be possible to 3D print or have a workshop make a special holder. Examples of stage holders for different power sensors are shown in Figure 2.
Figure 2. Examples of power sensor holders. Computer Numerical Control (CNC) machined holders in multi-well size for use in upright or inverted microscope configuration for A) inverted and B) upright Coherent LM-2 VIS and C) inverted Newport 840 sensor model 818C, D) inverted and E) upright Coherent LM-10 HT power sensors and F) commercial slide holder (Oko Lab) for the Argo-Power (Argolight) power meter sensor.
Figure 2. Examples of power sensor holders. Computer Numerical Control (CNC) machined holders in multi-well size for use in upright or inverted microscope configuration for A) inverted and B) upright Coherent LM-2 VIS and C) inverted Newport 840 sensor model 818C, D) inverted and E) upright Coherent LM-10 HT power sensors and F) commercial slide holder (Oko Lab) for the Argo-Power (Argolight) power meter sensor.

Select the wavelength of interest on the power meter control panel (see examples in Figure 3).

Figure 3. Examples of control panels for power meters. Upper panel: Argo-Power (Argolight). Middle & lower panels: different Thorlabs software versions. A) Excitation wavelength selection, B) sampling period adjustment, C) power instruction in % selected from the imaging system software , D) power measurement in mW, E) auto range , F) averaging G) zeroing, and H) bandwidth. See Power meter Q&A Appendix 1- STEP14, for explanations.
Figure 3. Examples of control panels for power meters. Upper panel: Argo-Power (Argolight). Middle & lower panels: different Thorlabs software versions. A) Excitation wavelength selection, B) sampling period adjustment, C) power instruction in % selected from the imaging system software , D) power measurement in mW, E) auto range , F) averaging G) zeroing, and H) bandwidth. See Power meter Q&A Appendix 1- STEP14, for explanations.
Zero the power meter while the laser is off or the beam is blocked (Figure 3G). Ensure ambient light is kept to a minimum and remains consistent throughout the measurements (i.e. room light off, while a small light source on a desks can stay on to provide some visibility and avoid accidents).
Note
It is also better to cover the oculars and block any other stray light path that may reach the sensor. Try to perform measurements with stable, consistent and reproducible temperature, airflow and humidity, especially for open systems (see more about the impact of environmental parameters in Appendix 3- STEP16).

Center the illumination spot on the sensor. Avoid using the corners or the very periphery of the sensor for the measurement.


Note
The illuminated area on the sensor should be at least 1mm2 to avoid local variations within the sensor. If an objective is used, do not focus on the sensor as the focal spot might be too concentrated and damage the sensor and/or yield inaccurate readings, (See Power meter Q&A Appendix 1- STEP14).

No variation in measurements are expected on larger sensors over most of their surface, except at the very periphery and in corners. The illumination is sufficiently centered on the sensor when moving slightly the stage in XY or Z (focus) does not change the measured value. If no objective is used, the position of the power meter in the axial direction does not impact the measurement on raster scanning and spinning disk confocals, as long as the illumination does not spill outside of the sensor area.

Note
On inverted microscopes, when the sensor has a cross mark on its back indicating the center of the sensing area, transmitted light can be used to center the sensor on the illumination axis. (see Figure 4).
Figure 4. Slide power meter sensor mounted on the stage of an inverted microscope and aligned to the center of the sensing area using transmitted light and the cross mark located at the back of the sensor.
Figure 4. Slide power meter sensor mounted on the stage of an inverted microscope and aligned to the center of the sensing area using transmitted light and the cross mark located at the back of the sensor.

Switch on the laser and read the power output on the power meter (Figure 3D) using a suitable range if not automatically selected (Figure 3E).
At this stage and without logging the data yet, observe whether the power is stable, drifting or fluctuating over 10 seconds, as this can be an immediate indication of problems with laser control or certain optical elements.

Note
Raster scanning microscopes only: Ideally, the laser beam should be stationary. Indeed, when scanning, blanking of the laser between lines and frames, along with scanning speed, will affect the measured values. If it is not possible to keep the laser beam stationary, you may want to use the Fluorescence Correlation Spectroscopy (FCS) or single point stimulation option, if available. If no FCS or single point stimulation option are available, ensure you reuse the same settings each time you make the recordings, e.g. record pixel size, pixel dwell-time, and frame size (in pixel).

Note
Spinning Disk microscopes only: Rotation speed, disk position (confocal or wide-field mode), as well as the size of the field diaphragm may affect measurement. Ensure the very same microscope settings are used when measurements needs to be compared.

Illumination stability measurement
Illumination stability measurement
Proceed with the power measurement and measure illumination power in mW over time for each wavelength (excitation wavelength, Figure 3A) as follows: for short-term stability record every second for 5 minutes, for long-term stability record every 30 seconds, with 1 second integration time, over 120 minutes. Sampling, integration time and duration have to be set on the power meter before recording (Figure 3). See also Appendix 4: Tutorials-STEP17.
Note
These recommendations are mainly for routine maintenance. If the quality control is made to check the system's behavior for a specific experiment, use a frequency and duration that match your time-lapse conditions (see Figure 3B&C).

For routine maintenance, measure the illumination power stability at different laser intensities. We recommend starting with intensities corresponding roughly to 5%, 20% and 80% of maximum power. Record the powers measured in mW at the objective for these different percentages. When repeating the stability measurements at later times, set-up your system to get the same values in mW at the objective (i.e., not necessarily the same percentage). You need to measure the stability of a given illumination power (mW) at the objective, not of a given percentage.
Note
Assessing long-term stability for multiple wavelengths takes time. For some combinations of microscope and power meter brands, scripts allowing the automation of the measurements have been developed and are publicly available on our QUAREP Github repository. For more information, consult Appendix 2: Automation of measurements- STEP15.

Calculate illumination stability (as a % variation) using the following formula:
ΔPower (%) = 100x (1 - ((Pmax - Pmin) / (Pmax + Pmin)))
where Pmax and Pmin are the maximal and minimal powers recorded during the time interval measured.
Expected result
Figure 5. Examples of data collected for short and long term illumination power stability. A) Short term stability monitoring for a spinning disk equipped with 4 diode lasers. B&C) Long term monitoring for a spinning disk confocal and a raster scanning confocal microscope respectively. The spinning disk in (B) was not pre-warmed to show the warming up time of the lasers. The raster scanning in (C) was equipped with diode, DPSS and Argon - HeNe gas lasers. The Argon laser was particularly unstable due to aging and was later replaced. Measurements of the replacement laser are also shown (yellow triangles), displaying a better, but not perfect stability. Laser intensities for (B&C) were controlled with an AOTF. Power values of each laser line were normalized to the maximal value for each case.
Figure 5. Examples of data collected for short and long term illumination power stability. A) Short term stability monitoring for a spinning disk equipped with 4 diode lasers. B&C) Long term monitoring for a spinning disk confocal and a raster scanning confocal microscope respectively. The spinning disk in (B) was not pre-warmed to show the warming up time of the lasers. The raster scanning in (C) was equipped with diode, DPSS and Argon - HeNe gas lasers. The Argon laser was particularly unstable due to aging and was later replaced. Measurements of the replacement laser are also shown (yellow triangles), displaying a better, but not perfect stability. Laser intensities for (B&C) were controlled with an AOTF. Power values of each laser line were normalized to the maximal value for each case.


Illumination linearity measurement
Illumination linearity measurement

Note
Working within the linear range of the illumination power allows to adjust accurately the optical laser power absolute value (in mW) using fraction (or %) of its maximal value through the imaging software. If this relation is not linear, re-calibration of your system may be needed.
For laser power linearity measurement (or illumination linearity): record for 30 seconds with 1 second integration time, 10 incremental fractions of the total laser power in steps of 10%, from 0 to maximum optical laser power value.


Plot the average laser power value (mW) measured versus the optical laser power fraction (or %) and determine the goodness of fit of a simple linear regression by calculating the coefficient of determination (r2). A perfectly calibrated system should have an r2 value very close to 1, as shown below in Figure 6. We recommend making multiple measures (replicates) for accurate results.
Expected result
Figure 6. Example of illumination linearity assessment of laser power data acquired on a spinning disk confocal. Each plot shows the laser power in μW for a given power set in % from the software. This relationship can be tracked over time (dates of measurements shown in legend on the right of each graph) as shown here for A) the 488 nm laser line and B) the 640 nm laser line.
Figure 6. Example of illumination linearity assessment of laser power data acquired on a spinning disk confocal. Each plot shows the laser power in μW for a given power set in % from the software. This relationship can be tracked over time (dates of measurements shown in legend on the right of each graph) as shown here for A) the 488 nm laser line and B) the 640 nm laser line.


Note
If extremely low or extremely high laser powers are measured to match experimental settings, keep in mind that power modulating elements (e.g. AOTF) are more likely to behave non-linearly at the extreme ends of their power range. To catch issues on the low and high ends of the percentages settings you can measure with the following increments: 0, 1, 2, 3, 4, 8, 16 ,32 ,44, 56, 68, 84, 92, 96, 98, 99, 100 (%).


Results display and tracking
Results display and tracking

As of the 1st of December 2021, 17 operators from different institutions tested this protocol, and our group collected 292 measurements.

Stability measurements communicated so far by QUAREP members are displayed on the QUAREP WG1 dashboard for different illumination source types (see Figure 7 and online WG1 Dashboard). These results do not constitute a norm but may help microscope users to evaluate the performance of their system in comparison to other systems in the field. You are invited to add your own measurements through our online form or by filling and sending us the .csv file, which can be downloaded from the Dashboard.
Expected result
Figure 7. Screenshot of QUAREP WG1 Dashboard
Figure 7. Screenshot of QUAREP WG1 Dashboard


Note
Other recommendations and future directions

  • Recommended frequency of this measurement can vary, e.g. depending on the laser type. Manufacturers recommend monitoring every 2-4 weeks for a gas laser, every two months for a Diode-Pumped Solid-State (DPSS) laser and every 3-4 months for a diode laser. A higher frequency of this measurement may be needed if other factors influencing the sample illumination are at play, like for example the stability of the coupling system and the age and polarization of the laser fiber, the stability of the AOTF, etc.
  • Illumination power can be modulated in different manners (e.g., directly at the laser, or indirectly through an acousto-optic modulator (AOM) or AOTF, optical elements like neutral density (ND) filters or polarization filters, prisms or combinations thereof), and hence different components may be the source of instability. Consider potential source of error when interpreting results.
  • Some lasers (i.e., Argon lasers) can be operated in different operation modes (e.g., run/standby, constant power, or constant current mode), which may impact output power. The so called “constant power mode” seems to be related to fewer fluctuations, so its use is recommended.
  • Power measurements before starting and after having completed a sensitive experiment could be helpful to make sure that the illumination power or stability did not change.
  • This protocol will be updated to include other microscopy modalities (widefield, total internal reflection fluorescence - TIRF, light-sheet, super-resolution, multi-photon, etc.). It will also be extended to describe how to measure irradiance on the sample.


Appendices
Appendices
Appendix 1: Power meter settings Q&A

Some of the answers below are specifics to Thorlabs' power meters
Note
1. How much of the sensor area needs to be illuminated for accurate measurement?
It is recommended to not focus on the sensor and to keep the diameter of the illuminated area within 1 to 10 mm.

2. Should I use immersion medium for my objective when measuring illumination power?
When measuring the power through a high NA objective (NA>1) it is important to check whether the sensor is designed to be used in air or whether an immersion medium should be used between the front objective lens and the sensor. Using appropriate immersion medium allows the sensor to detect the total laser power over a high NA, without losses arising from deflection or reflection. Most power meter sensors are not water-proof, and can be damaged by exposure to water or oil. However, some sensors were designed specifically for microscopy applications and can accept a drop of immersion media (for example the S170C sensor from Thorlabs).

There will be a difference in power measurements with and without immersion media when the NA of the objective is above 1.1. This is caused by:
  • Total internal reflection (which can be mitigated in the S170C via an index matching gel in between the silicon sensor and the cover window) and/or
  • Absorption of light from the immersion medium (i.e.10% less for water and 15-20% less for oil when compared to air).

3. Is the detection linear across the sensor?
The measured power response may not be linear near the edges of the sensor. Hence, it is important to center the beam to improve linearity but also to avoid clipping of the beam on the edges of the sensor.

4. How to prevent damage and saturation of the sensor?
Keeping the optical power below the maximum rating in the specification sheet prevents saturating the sensor. Higher powers than specified will saturate the detector or create zones of critical saturation on the sensor, leading to a non-linear measurement of the signal. You can use a neutral density filter to lower illumination power.

5. How does the integration and readout time work and what should be the preferred power meter bandwidth settings?
A photodiode has a response time of 1 µs, but its electronics behind it are slower depending on the readout device used. The rule of thumb is to set the bandwidth to "low". For very fast fluctuation measurements, it is best to use a very fast photodiode with an oscilloscope rather than a power meter, power meters being designed for slower changes (see Figure 3, Lower panel, H).
Very different speeds also require usage of different photodiodes. When a photodiode converts photons to electrons, the flow of electrons out of the sensor is governed by its capacitance. Thus, large photodiodes, with high capacitance, are poor at detecting fast dynamics, but their large area makes it easier to capture of the light beam, especially when it comes out a microscope objective with a low NA or when no objective at all is used. For fast dynamics, you will need a smaller photodiode, which has lower electrical capacitance and responds faster, and you will need electronics designed to sample quickly as well. For this, a high-speed oscilloscope is preferred.

6. Does the power meter need to be calibrated or just the sensor?
The power meter does not change over time, thus, only the sensor needs to be calibrated regularly. Calibration data is typically saved in a chip in the DB9 (red) connector of the sensor for Thorlabs power meters. Other vendors may store this information elsewhere. We recommend checking the calibration status of your sensor by comparing the results to those obtained with a different sensor.

7. How often does a power meter need to be calibrated?
It is different depending on the power meter you use, but typically the measurement accuracy may change by 2-3% over 3-4 years but could also deviate by up to 10% over a longer time. In general, it is recommended to calibrate a power meter yearly. However, if you are not exceeding the specified power rating, a calibration should be stable for 2-3 years. This is within the wavelength range of 300 to 1060 nm; shorter wavelengths may incur further deviation.


8. Is using a power meter safe?
For standard confocal microscopes, it is generally safe to take power meter measurements but before you do so, make sure you check with the vendor or the person who built your system. Also, check your organization’s regulations around laser safety, as they vary by country and jurisdiction. Precaution regarding the reflectivity of the coating of the sensor should be taken. Up to 0.5-1.5% reflection may occur when delivering high powers to the sensor. You can damage your eyes using a power meter through stray reflections from a significantly powerful lamp or laser, and you must be trained to carry out this procedure using eye protection, when necessary. Under no circumstances should you check high powered lasers such as those used in multi-photon systems, TIRF systems or point localization systems without training.Typically, less-reflective thermal sensors are used with higher power as they have higher maximum power ratings and are designed to measure such lasers.

9. Can you capture the data and change settings on a computer?
If your power meter uses a standard Universal serial Bus (USB) to Commmon (COM ) terminal you can send Standard Commands for Programmable Instruments (SCPI) to the power meter and query data and other parameters. For example, the PM400 (Thorlabs) allows for different capture intervals to the internal memory via SCPI commands. Most common programming languages support serial terminal commands so it is often straight forward so writing a simple program to query the power meter from a personal computer is often straighforward. Some power meter consoles also support storage media such as a removable USB drive or Secure Digital (S.D.) card which can log data for later review.

10. How to calculate the photon flux from power measurements?
The number of photons (n) observed per unit time (t) is the photon flux (Φq).
Formula:
Photon Energy: Ep=hc/λ
Measured Power: Pmeas=n∙Ep/λ
Photon Flux: Φq=n/t=Pmeas/Ep=Pmeas∙λ/hc

(c =3∙108 ms and Planck constant h=6.62607015∙10−34 Js)

11. Are there different types of power meter sensors?
Yes, three different types: photodiode, thermal power, and pyroelectric energy sensors.
  • Photodiode sensors have a strong spectral sensitivity dependence and must be calibrated over the entire wavelength range. On the other hand, they have a high dynamic range (70dB), very low noise, and high response speed. Photodiodes are made of different semiconductor materials like silicon (190-1100 nm), germanium (400-1700nm), and indium gallium arsenide (800-2600nm). They are common for low-to-medium power measurements typical of visible light confocal microscopes.
  • Thermal power sensors use the Seebeck effect that turns heat flow into a power-proportional voltage. They can be used up to very high-power levels. Their useful dynamic range is much lower than the range of the photodiode sensors (30dB). Another drawback of the type is that they are very sensitive to ambient temperature or airflow (drafts). Hence, they should be shielded from moving air during measurements. In general, the speed of response is low. Therefore, a few seconds of settling time is often required
  • Pyroelectric energy sensors can only handle pulsed signals (no continuous wave). A pyroelectric crystal converts the heat impact of a laser pulse into its energy-proportional voltage. They are more commonly used for high energy, lower repetition rate pulse lasers which are not common in microscopy except for certain photo-activation and microsurgery applications.

12. Using the same type of sensor, will all power meters give me the same values?
Yes, most of the time. However, different values can be observed between different models of power meter sensors due to the sensor's calibration, electronics, and composition (a sensor without gel filling the gap between the filter glass and sensor surface will show lower power levels with high NA objectives).
Different values can also be obtained when measuring broadband light sources. Different detectors or filters have different spectral curves, which can run in opposite directions. Since you can only set one wavelength point from the entire incident light spectrum, the light outside the wavelength set point gets weighted differently and causes the difference in the reading.

13. Do different light sources (e.g., laser, LED, lamp) require different type of sensors?
Thermal sensors best measure broadband light sources, such as white lamps, as this type of sensor is not wavelength sensitive.
Narrower-band light sources, such as filtered lamps, lasers, and LEDs can be measured with photodiode sensors but the accuracy of the measurement will depend on the bandwidth of the source. Most lasers and LEDs are narrow band enough that the error will be negligible, but some broadband LEDs (phosphor type without a bandpass filter) may be more accurately measured with a thermal sensor if sufficiently powerful. If a photodiode is used for a broadband light source, the error may be compensated for by considering the wavelength response of the photodiode, as discussed above.

14. Is electric grounding of a power meter important?
Power meters typically filter interference from nearby electronics or power cords. For very sensitive measurements, or particularly electrically noisy environments (rare), additional measures may be necessary (like grounding your sensor). It is also preferable to use the power meter in battery-operation mode (unplugged from wall power) as this will isolate the meter electronics from any noise coming through the power lines.

15. Is zeroing the power meter at each wavelength important?
The zeroing feature measures dark current (if the sensor is covered and is not detecting ambient light), and subtracts it from future measurements. It is not wavelength dependent. Therefore, you can simply do it once when you switch it on.
The zeroing feature can also be used to compensate for room lighting by performing the “zero” sampling with the sensor uncovered, but the light source to be measured shuttered or blocked.
After zeroing, keep the room light settings constant during your measurement.

16. How to measure the pulsed input and peak power of your light source?
You need a fast response/reacting power meter (like the new Thorlabs PM103) and sensor, or a pyroelectric energy sensor for these kinds of measurements. For microscopy applications, typically only some fast-flashing stimulation or uncaging light sources require these types of measurement. If the power meter response speed cannot follow the pulses, you will get an average measurement as the sensor will average the effect of the pulses over time in its reading.

17. How do circularity, shape and diameter of the beam, can affect the measurements?
The power reading (Watts) should not be affected by the shape and diameter of the illumination spot except when the illumination spot overfills the sensor, or is focused so tightly on the sensor that it creates local variation or saturates the sensor area (this can damage the sensor). It is best to have a defocused illumination spot, as mentioned in Question 1.
The power per unit area (usually W or mW per cm2) is impacted by the illumination spot's shape, intensity profile, and diameter. If the geometry and profile parameters of the illumination spot are known, they can be used to calculate irradiance (mW per cm2) once the overall power in Watts is measured.

18. What does "averaging" exactly do?
Depending on your chosen settings, it averages the results of multiple readings, reducing measurement noise. For example, if you set the power meter with an average of 100, it will take 100 readings and report their average. Note that averaging slows down the speed of measurement, as the meter must acquire all the readings to be used in the average before performing the average and displaying the result, although most meters are fast enough that 10’s or 100’s of readings for an average is not excessive. (Figure 3F)




Appendix 2: Automation of measurements


Note
The outlined illumination power and stability assessment protocol requires measurements repeated over different conditions for each microscope and its respective excitation light sources. Therefore, extensive time will be consumed, if the measurements are conducted manually in a sequential manner. However, most modern microscopes provide an automation programming interface capable of communicating with a calibrated external power sensor. The automated measurement has a two-fold aim: to make time-intensive measurements feasible through automatic interleaving of the different measurement conditions and to improve reproducibility by repeating measurements in an identical fashion. QUAREP-LiMi provides examples (scripts, documentation and videos) that can be adapted to a series of microscopes: Link to GitHub repo

Appendix 3: Impact of environmental factors
Note
The power of illumination delivered to a sample depends on environmental conditions such as temperature fluctuations of the equipment and the surrounding environment, airflow, humidity changes in the laboratory, and instrument vibrations. Hence, all these parameters should be inspected and measured routinely.

For temperature measurements: Ensure that the entire system is turned on and sufficient time has elapsed for the equipment to warm up (approx. 1 hour) to reach a steady-state.

Caution: The temperature measurements should be performed as close to the system as possible to account for local temperature fluctuations in the room.

Additional recommendation: If you routinely carry out live-cell imaging and/or require temperatures different from room temperature, it is advisable to carry out power measurements under these conditions since they may introduce fluctuations in the illumination power, as mentioned above.

Measuring relative humidity at the same interval is also recommended.

If power fluctuates, but the temperature is stable within +/- 1 degree Celsius/hour, and the relative humidity within 40 +/- 20 %, other environmental factors, such as airflow (including drafts) or vibrations, should be considered and measured.

Avoid direct airflow towards the equipment, as drafts and gusts will cause transient temperature changes, which will impact stability. If the air vents in the room are directly above the equipment or otherwise blowing air towards it, consider adding a deflector or metal or filter cloth diffuser to the outlet to divert the airflow.

Caution: Ensure that these adjustments do not create fire hazards or other safety issues when placed close to light fixtures or other heat sources. Check with your facilities administration before implementing ventilation modifications.

Additional recommendations:
  • If constructing a new space, a laminar flow ventilation scheme for the room is best, as this delivers regular air movement concentrated around the edges of the room, away from the equipment. Otherwise, it is good practice to avoid placing equipment directly underneath vents when planning your equipment layout.
  • Doors should be closed during an experiment or protected by an inside curtain to block drafts.