Laser module issues and support

General
1

Moin, @benedictdied !
I’ve tried testing the single wavelength laser with some cells with Apo15 (GFP) and had some questions/issues.

  1. The alignment of the laser doesn’t affect the image quality much, right? The laser module itself does not “aim” at the center of the LED Matrix which was already centered for phase contrast. We would like to adjust the position of the module with the pins that hold/mount the module to the cube, but they are not accesible without removing the ESP32’s PCB. Might be nice to re-design that for easier calibration :slight_smile:
  2. Does the dual-wavelength module (that’s on its way acording to DHL) use two PWM pins? Do we treat them as 2 different lasers for the UC2Client? Is there any code consideration to have to adapt/integrate this new laser?
  3. We were able to see some fluorescense signals on our other microscope (LIONHEART), so we have some positive control. We thought we might have the module upside down, but with both ways we don’t see any cell signal, but a different background noise intensity (see the following link with the pictures of the LIONHEART and these UC2I pictures: I created a google photos album with the photos taken, as the post doesn’t support tif files)

Could we have some support on this issue?

Mit freundlichen Grüßen
Matías Stingl

2

Hey @mstingl, thanks for reaching out. I’ll be out of office for the next 1 - 2 weeks. @wanghaoran1207 can help you in the meantime eventually. I’ll respond when I’ll have some more time :slight_smile:

3

Hi @wanghaoran1207 !
Please get in touch with me as soon as you can, as we’ve just tried the dual-wavelength module and only one wavelength is working.

Mit freundlichen Grüßen

4

hi @mstingl,

i will try to answer your question in following:

  1. The aligment of the laser does affcet the image quality, as we are doing widefield imaging with the laser, we assume the laser come out from the objective to be collimated. So the laser should be straight up and heading to the ceil. Which is more important, is laser is focus into the middle of the objective and come out you can check with a far distance, and see if it is go straight and still positioned on the center postion of the objective. If not then you should try to adjust the pins.
    Yeah for the mounting and alignment system, we should definitely rethink about it.

  2. yes, it has 2 PWM to tune the two lasers. You can try to change the configuration files. I think there you only used one laser you can add another one to control both of them. here is an example:
    “lasers”: {
    “488 Laser”: {
    “analogChannel”: null,
    “digitalLine”: null,
    “managerName”: “ESP32LEDLaserManager”,
    “managerProperties”: {
    “rs232device”: “ESP32”,
    “channel_index”: 1
    },
    “wavelength”: 488,
    “valueRangeMin”: 0,
    “valueRangeMax”: 32767
    },
    “635 Laser”: {
    “analogChannel”: null,
    “digitalLine”: null,
    “managerName”: “ESP32LEDLaserManager”,
    “managerProperties”: {
    “rs232device”: “ESP32”,
    “channel_index”: 2
    },
    “wavelength”: 635,
    “valueRangeMin”: 0,
    “valueRangeMax”: 32767
    }
    },

  3. can i ask which camera you have on your lionheart one? From the last image, i would like to say the laser is not focusing into the objective, could you check if you remove the objective, and see if the laser is focusing at the position where the objective supposed to be? It looks like the laser beam is focusing onto the sample plane, which should not be the case.