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Disclaimer: these examples are mostly taken from my pre-Eikomagos career

Automated microscopy

The system described here is comprised of three parts:

  • Zeiss Axioplan 2 motorised microscope
  • Zeiss AxioCam MR5
  • Linkam custom-designed temperature controlled stage
Unfortunately there is no API (Application Programming Interface) for the Zeiss components, but as a fallback they can be controlled through the VBA module in its AxioVision software. The Linkam stage was controlled directly by sending commands over its RS232 interface.

Right half: contrast enhanced version of the left. Temperature estimated from freezing rate

The system was designed to run ice (re)crystallisation experiments in model (sucrose-solution) systems. The crystals in systems of interest are roughly rounded. However, naively freezing a sample results in a highly dendritic structure (video)

To obtain rounded crystals the sample is plunge frozen to -100°C. This puts the sample in a glassy state. On heating it up, at around -30°C as the sample leaves the glassy state, a lot of tiny crystals are formed which grow rapidly while the temperature rises further to the target temperature (e.g. -5°C).

Left: N₂ block; right: Peltier block; the sample holder is motorised

To support this procedure, the stage contains a liquid N₂ cooled block and a Peltier cooled block. The first is used to plunge freeze the sample; the second at the target temperature and is able to maintain its temperature very accurately (<0.01°C)

 

Apart from loading the sample and (dis)connecting N₂ gas at the appropriate time, the system is fully automated: the temperature, the sample's position, focus and neutral density filters, condenser aperture, field stop as appropriate (Köhler) for the objective. The output is a series of images and a log file that contains the images' name, microscope settings and temperature. These are taken for offline analysis.

Seeding the sample with crystals. Coming from the glassy state, the sample turns opaque due to scattering by the field of tiny crystals. As the crystals grow larger the sample becomes transparant again. Images taken using polarised light. The second part of the movie is greatly speeded up.

 

The video shows a typical recrystallisation experiment taken under polarised light. Note the mobility of the ice crystals and the recrystallisation process: smaller crystals melt and larger crystals grow larger.

The use of polarised light makes for prettier pictures and visualises changes in orientation of the crystals, but actual the experiment are run in standard transmitted light mode: crystals with their main axis parallel to the viewing direction have the same colour as the background so the colours cannot be used to identify (all) the crystals. The dynamic range needed is also greater.