WG 1 – Illumination Power

The subgroup for automation has developed a new script for Zeiss instrument that makes the old one obsolete. It will be uploaded on the website soon. Other participants also inform that more scripts for Leica and Visitron are being developed. We also all agreed on the fact that the GitHub is a living repository and will evolve with time, even when the protocols are done. We then reviewed the latest comments on the protocol and made small improvements to it. We decide to keep the stability formula as it is, even though it might be very sensitive to one outlier and does not reflect always what is wrong. However, we agree that to know there is an outlier or something is wrong is already very important, and one has to plot the stability curve anyhow, and can thereby immediate see why the stability value might be low. Nathalie explains the process for co-authorship and endorsement. We will ask WG chairs to read and say is they want to co-sign or endorse “works for me” through protocol.io. QUAREP general assembly: Laurent will prepare a presentation. Survey: we will align with Roland.

Summary of last meeting by Nathalie.

Feed-back from subgroups:

• Glossary subgroup only worked on camera part, not the illumination part.
• CAD files for power meter sensor holders have been loaded on the GitHub.

Recently, a new holder for Thorlabs slides in a shape that fits into a multi-well plates holders has been made in Freiburg. Works only for inverted microscopes. Automation: Arne Fallish is working on a program analysing the data recorded with Thorlabs power meter, eventually feed data back to Zeiss Software. We should try to involve big companies more in the automation and encourage them to develop macros. Nathalie shows the new layout of the protocol, made by her and Laurent, to remove step cases and use collapsible notes: Britta discussed the issue with people from Protocol.io and they may investigate it, but right now step-cases are branching out and one cannot come back into the main common/root protocol. Roland indicates that the meeting with Emma has been recorded. One should be able to get in from our QUAREP Cloud. Britta suggests that appendices should be moved in an ad hoc section, if Protocol.io creates one, or we should have a dedicated box in the other sections, since the appendices are not steps and do not belong to the protocol. Another option is to create a pdf and add it to the description section. We will aslo ask protocols.io for an abbreviation box under the description tab. We will ask all WG1 participants, e.g., co-authors of version 1, to read the new protocol before we publish. If we do not here from them, it means that we can keep their names. Britta suggests asking feed-back from all QUAREP members to make sure we do not overlap and contradict part of their work. We agreed. Nathalie will investigate if in protocol.io we could have the endorsement separate from authorship. We screened the videos made by Laurent on power meter usage. We will add the logo of QUAREP on all slides. Also add the corresponding steps from the protocol. We will also consider a submission to PLOS ONE protocols. We finally made improvements to the protocol, based on WG1 members comments.

We recapitulated the last changes in steps 1 to 4 of the protocol, and reviewed the new Question 19 about broad-band illumination sources in Appendix 1. We then further adapted steps 5 to 8 to cover new illumination/microscopy modalities.

We briefly discussed progress made by our sub- groups and completed the review of the appendix 1b generated by Manfred on power meter settings for other light source. We all agreed to merge it to appendix 1. We also reviewed and discussed comments and suggestions posted in protocols.io. We now have updated step 1 through 4 of the protocols to include other illumination sources and wide-field systems.

We welcomed two new members of WG1: Katarzyna Cialowicz and Kurt Weiss.

News from the subgroups: Glossary: a first document with a mission statement is to be found in the new folder WG1-MetaDataModel. This is in coordination with WG7, as it has been also done in WG2. The subgroup will try to have as many companies on board as possible. Automation: no meeting since last time. Power meter: see below paragraph on “Appendix 1b”.

Appendix 1b: Measuring Broadband Light Sources. We rephrased the introductory sentence and the first paragraph on Lasers. We then discussed the relevant operations for a broadband illumination. We identified at least three important parameters that may influence the absolute power measurement: the spectrum of the light source, the specifications of the excitation filter set and of the dichroic, and the responsivity of the power meter sensor. However, to make the measurement easier and robust, we agreed on a convention: the wavelength to be set on the power meter should be in the middle of the bandpass of the excitation filter. Note that if the measurement is done within the linear range of the responsivity curve of the power meter, using the center of the bandpass is a way of averaging responsivity values. The specific spectrum of the light source within the excitation bandpass should not have any significant influence on a biological experiment, and is not getting further consideration here.

We had a smaller group of 14 participants to the 20^th meeting. Nevertheless, we had once again a productive discussion and made some progress reviewing our version 2 of the protocol and made additions and changes to step 2 and 3 to include widefield microscope illumination. We also heard progress made from our subgroups:

1. Extension of the automation section (Led by Miso mitkovski@em.mpg.de and Nasser ndarwish@ist.ac.at): new members joined (Chloe and Rodrigo) and the group is now testing and integrating the automation script (for both acquisition and analysis) with data management using AirTable.
2. Extension of Power Meter Q&A (Led by Manfred mgonnert@thorlabs.com ): a new appendix (1b) has been created on how to measure power of broadband light sources. Please review on Protocol.io here
3. Metadata/Definition specific to WG1 (Led by Britta schroth@mpi-cbg.de  & David david.grunwald@umassmed.edu): still working on the WG2 camera glossary (the plan is to get as many companies involved as possible).

During this meeting, we made good progress on our version 2 of the protocol, extending microscopy modalities to wide-field imaging. We also have a re-actualized AirTable form and fields. We also heard progress made in our subgroups:

1. Extension of the automation subgroup had a meeting. The recording can be found here. A new tutorial video on how to install and use the automation macro while using Nikon NIS element and the Thorlab power meter software has been created. You can find it here.
2. The power meter Q&A is being extended to include information about LED light source. Please contact Manfred mgonnert@thorlabs.com if you would like to contribute.

We also had an extensive discussions on pulsed lasers, on the technology behind LEDs, and the inherent lack of stability of arc lamps. Finally, we completed our recommendations for the warm-up step for all light sources and used the step case function of Protocols.io to highlight differences.

Quick reminder of WG1 next priorities, based on the votes of WG1 members:

1. Extend the protocol to other microscopy modalities (wide-field microscope – LED, Arc-Lamps, lasers)
2. Extend the protocol to different laser types (i.e. multiphoton)
3. Extend the protocol to other laser-based modalities : (TIRF, SIM, light sheet)

This month we reviewed the metadata fields needed for Wide-field microscope – LED, Arc-Lamps, and others light source in AirTable Dashboard and adjusted our Form. Report from our subgroups were minimal. The Automation subgroup is planning to meet, see below. Please contact the lead of the subgroup you would like to contribute to.

1. Extension of the automation section (led by Miso mitkovski@em.mpg.de & Nasser ndarwish@ist.ac.at). Next Monday (05/09) 17:00 CET, the automation subgroup will meet. Kees will present his latest macros. https://zoom.us/j/97497511867; Meeting ID: 974 9751 1867; Passcode: 445833
2. Extension of Power Meter Q&A (Led by Manfred mgonnert@thorlabs.com)
3. Metadata/Definition specific to WG1 (Led by Britta schroth@mpi-cbg.de  & David david.grunwald@umassmed.edu)

Finally, we briefly discussed the possibility of using the Step Care function in Protocols.io template to add specific recommendations for the different Microscopy modalities and light sources. See example already created in the new version 2 of the protocol on protocols.io. The private new version of the protocol can be found here. Our next meeting will be on Wednesday June 1^st, 5pm CEST. Looking forward to seeing you all there again!