A collaborative board game to teach co-localization analysis, in which the players, a.k.a The Scientists, team up against the game master , a.k.a Reviewer 3.

A.Game mode(s) :

In The Scientists have to prepare an Experiment Plan that will be reviewed by Reviewer 3. The Experiment Plan, is made of cards describing the microscope settings, the images settings and finally the analysis workflow.

You can play in two different modes:

• Science First
• Time is of the essence.

A.1. Science First

This is the recommended game mode for courses or with new gamers.

In , The Scientists goals(s) are to :

• fulfil all Reviewer 3's demands so The Scientists get a valid experiment
• try to get as few clocks () as possible at the end of the experiment

We'll just stress out one more time, the most important is to have a pleased Reviewer 3 (and only then try to limit the amount of )

A.2. Time is of the essence

The Scientists have a limited amount of at their disposal. The Scientists can only use 20 (25 or 30 for decreasing difficulty)

B. Start :

The Scientists receive an experiment box, containing :

Cards of categories :

Microscopy	Image	Analysis
		, ,

One of the six available experiments

Nbr	Experiment Board	Description	Stainings
1		The Protein-X translocates between the cytoplasm and the nuclei. The Scientists want to quantify to which extent.	Protein-X, Nuclei (DAPI)
2		The Protein-X looks like an organelle, The Scientists want to quantify if The Protein-X co-localizes more with the Golgi or the Mitochondria.	Protein-X , Nuclei (DAPI) and Organelle (either Golgi or Mitochondria)
3		The Protein-X looks like blobs, The Scientists want to quantify if The Protein-X co-localizes more or less with some Vesicles (Lysosomes) with , or without, a Drug-Y added to the media.	Protein-X , Vesicles
4		The Protein-X looks like it's following cytoskeletal component, The Scientists want to quantify if The Protein-X co-localizes more with the F-actin or the Microtubules.	Protein-X , F-actin (Phalloidin) the Microtubules
5		The Protein-X looks like blobs, The Scientists want to quantify if The Protein-X co-localizes more or less with the Golgi with , or without, a Drug-Y added to the media.	Protein-X , Golgi
6		The Protein-X looks like blobs, The Scientists want to quantify if The Protein-X co-localizes more or less with the Golgi with , or without, a Drug-Y added to the media.	Protein-X , Golgi , Whole Cell Staining

Because The Scientists can be a group of 4 to 5, some helpers boards (LINKS HERE) are available so all the players of team can have a look to the available cards.

C. Microscope Acquisition :

C.1 Microscope Settings :

The Scientists will pick up cards to define the microscopy settings they'll be using:

Card(s)	Description	Notes
, ,	The objective	...
, ,	The image modality you'll be using for your acquisition	Widefield ,or Spinning Disk, requires use of the Camera AND can't be used with PMT. Point Scanning Confocal. Point Scanning Confocal can only be used with PMT.
,	The detector	Camera requires either Widefield or Spinning Disk and can't be used with Point Scanning Confocal. PMT requires Point Scanning Confocal and can't be used with Spinning Disk or Widefield
	Super Resolution techniques are also available	Requires to be used in combination with a modality either Widefield or Point Scanning Confocal. During the Review step, The Scientists should detail which method they used, STED, SIM, STORM ...

C.2 Images Settings :

The Scientists will pick up cards to define the images settings they'll be using:

Card(s)	Description	Notes
,	Acquistion mode either sequential or simultaneous	Simultaneous is not compatible with camera based acquisition ( the "Dual Camera" card does not exist in the game yet 😁)
	Perform a tiled acquisition	Sum the on your and multiply this number by 4!
, ,	The xy dimension of your pixel	"10 nm" requires the "Super Resolution" card to be played
, ,	The Signal to Noise Ratio of your image	...
, ,	The z-step between your z acquisitions	Not necessary if you acquire a single plane.
, ,	The Volume coverage , either a single plane, a few planes or enough plane to cover the whole specimen.	...
...	...	...

D. Analysis Settings :

The Scientists will pick up cards to define the images settings they'll be using:

Card(s)	Description	Notes	Sub-category
	Perform deconvolution	Deconvolution will improve resolution and SNR of your image
	Perform a z-projection	Sum the of your and divide this number by 4
,	Perform the measurements on the whole image, or cell by cell.	"Cell by Cell" requires a segmentation of the cells to be used
, ,	Segment the Nuclei, or the cell, or both.	Make sure to have appropriate staining(s) to do so!	Segmentation
, ,	Segment a point, a blob like signal, or a complexe structure (banana-like or even more complex).	-------------	Segmentation
	Analysis of the overlap of the signals, combination of Area and Intensities of signals	Equivalent to Manders' cofficients	Metric , Pixel Based
	Analysis of the intensities correlation of the signals	Equivalent to Pearson's Correlation Cofficient	Metric , Pixel Based
	Analysis of the neighbourhood of the objects	Equivalent to Ripley's K function	Metric , Object Based , requires prior segmentation(s)
	Analysis of the distances between the centers of the objects	-------------	Metric , Object Based , requires prior segmentation(s)
	Analysis of the distances between the edges of the objects	-------------	Metric , Object Based , requires prior segmentation(s)
	Analysis relies on object classes, based on pixel intensities	-------------	Metric , Object Based , requires prior segmentation(s)
	Randomize the signal in the Image	In combination with the card "Intensity Correlation" it's equivalent to the Costes' randomization.	Metric

E. Review :

During the review:

• the Reviewer 3 will :

  • comments the Experiment Plan made by The Scientists
  • play card(s)

• The Scientists

  • answer to the Reviewer 3
  • can ask to play some cards
  • throw a dice (once for each card they want to play) , if The Scientists get :
    • 4,5,6 they can use the desired card
    • 1,2,3 they add the number of found on the card

Review Card	Description	"Details" Card(s)	Description
	...		You did Single Stain controls, it's time to use them!
	...		You checked that your signals have similar areas
	...		You didn't saturate your image by optimizing your acquisition. It was not so long afterall
	...		You checked that you were not bleaching your specimen while acquiring images
	...		You checked that protein expression is homogenous in the cell population
	...		You optimized your set of fluorophores
	...		You optimized your filter set / window of collection
	...		You checked that objective and mounting media are good choices to avoid Refractive Index Mismatch
	...		You checked the results of the deconvolution and optimized the parameters.
	...		...
	...		...
	...		...
	...		...
	...		...
	...

The game master can find some help to direct the game.

Thanks to

@lacan for first microscopy icons ! @leBIOP for first round of test and feedback !

TODOs

@TODO : add cards pinhole settings "1 Airy Unit per channel", "Match sampling thickness" @TODO : add card randomization object location @TODO : change artboard name for deconvolution optimized to fix name @TODO : add link to helper boards