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FLIMinator edited this page Feb 15, 2023 · 70 revisions

Troubleshooting

See troubleshooting page if something is not working.

Summary document

This document will link to other pages with more detail on each topic. This is version 1. Theresa, I will add all the details requested so far :)

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Maintenance

  • keeping camera cooling unit sterile (and working). We need anti-microbial fluid.
  • cleaning stage insert
  • cleaning water immersion objective
  • checking omicron laser powers
  • bubbles in water immersion
  • 5% C02 humidifier water reservoir place piece of copper inside to sterilise

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Which water and when?

Example of a MilliQ machine for Type 1 water dispenser and types of bottles for storage.

  • type 1 ultra pure mq water in water immersion reservoir
  • type 1 ultra pure mq water in gas humidifier
  • distilled water for dOPM camera water cooling system

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Potential preliminary investigations

  • wide-field transillumination or epifluorescence single point or time-lapse
  • fixed samples
  • confocal microscopy
  • single view dOPM

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dOPM assay design

  • #1.5 coverslip bottom
  • organoid wells
  • beads in same gel type as sample
  • excel plate-map

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Setting up system checklist

  • NIS-elements configuration checklist

  • Once a month or less

    • calibrate the O2 heater
    • calibrate the okolab temperature control
    • calibrate flow rate of water immersion peristaltic pump

  • Week(s) in advance of time critical work

    • check stock of beads
    • check stock of suitable sample holder e.g. multi-well plates
    • check stock of gel for 3D assays e.g. matrigel
    • if using CO2 cylinders check if they need replacing
    • design dOPM experiment + preliminaries

  • The day before

    • turn on okolab enclosure environment control
    • turn on all hardware - except lasers & water immersion
    • check hard disk space
    • check water immersion reservoir
    • check gas humidifier reservoir
    • check camera cooling system reservoir

  • ON the day

    • turn on lasers, allocate an hour warm-up at least
    • water immersion, submerge objective so water at tip hour before imaging beads

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Acquisition checklist

before putting sample on:

  • wipe bottom of sample
  • clean objective
  • clean stage insert
  • no parafilm or tape on the base of the plate

Check sample is firmly mounted in stage insert and not loose or jarred.

  1. dry step with low mag, air objectives, 4x/10x/20x
    • prefind organoids JOBS script etc
    • collect widefield brightfield/epifluorescence JOBS script etc

Check sample is firmly mounted in stage insert and not loose or jarred. Switching between objectives with long vs short working distance i.e. 20x air and 60x water immersion makes chance of objective hitting sample and displacing it highly likely.

When moving across plate consider retracting objective by reasonable offset to avoid collision if using short working distance objective.

If confident explore using perfect focus when moving across sample with short working distance objective. See NIS-elements methods page.

  1. wet step with water immersion 60x
    • setting/checking up 60x water immersion correction collar
    • setting/checking up light-sheet co-alignment with dOPM image plane
    • setting/checking up dOPM view offsets
    • image bead volume with dOPM and co-register dOPM views in ImageJ
    • adjusting focus on prefind list JOBS script etc
    • collect widefield brightfield/epifluorescence JOBS script etc
    • acquisition JOBS script etc

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Starting-up/shutting down checklist

  • cameras, turn on dOPM from power brick outside dOPM enclosure
  • dOPM camera water cooling, check reservoir
  • lasers, turn on at Omicron software, check actually on before assuming they are warming up
  • water immersion pump, check reservoir
  • CO2, regulator at wall/cylinder, regulator at input to okolabs mixer
  • air pump for 5% CO2
  • microscope frame
  • CoolLED epifluorescence box
  • okolab enclosure environment control
  • sutter filter wheel
  • normally always left on:
    • remote refocus objective heater
    • picomotors for controlling light-sheet mirrors
    • pimag linear actuator for scanning dOPM remote refocus mirrors
    • microscope control box
    • XY stage

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Data checklist

  1. Before experiment

    • clear local storage disk space

  2. During experiment

    • follow a planned directory/file structure
    • plate-map
    • metadata
    • Omicron laser powers recorded
    • biology notes
    • experiment issue notes

  3. After experiment

    • backup data
    • start processing data

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Computer checklist

explore this stuff week before time critical experiments as software updates can be problematic (but are often required)

  • windows updates
  • nikon nis-elements updates
  • camera driver updates

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