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Can we avoid medium evaporation when doing live cell imaging?

Evaporation as a major issue for long-term imaging

The majority of live-cell imaging experiments require long-term analysis. One main issue that can appear is related to the medium evaporation. This is particularly true when the medium volume of the biological sample is very small (few micro-litters). Evaporation can be a critical factor and highly impacts cells or even cell viability.

As it is mentioned in Ettinger and colleagues,(1) the main way to avoid media evaporation and ensuing changes in osmotic pressure is to have sealed chambers. Cherry Biotech offer the possibility to work with close and sealed chambers as explained above.

Other proposals,(2,3) mention the possibility of having a sandwich of cover slips where the sample is in the middle so it is very difficult for the liquid to evaporate.  The same research group mention the idea of using a seal chamber having the option of medium renewal.  

Some other options are explained in the following article (4) but the majority of them present the same limitation. Both stage incubators or a thermalization chamber around the microscope (the two main systems allowing thermalization and humidity control) face the same constrain of not allowing a direct access to the sample. For a review on these different systems, you can check out this review.


Experimentation showing the efficiency of our microscopy chamber at avoiding evaporation

evaporation-sealed-chamber-live-cell-imaging-setup

Fig. 1: Image of the setup of the evaporation experiment

Our CherryTemp temperature controller is delivered with a set of sample mounting elements. Our R&D team spent energy to provide a simple, user-friendly and easy-to-mount chamber which prevents from any evaporation.We developed a test using the CherryTemp sample mounting system that is used for thermalization biological samples.

The objective of this test is to check the capability of our closed chamber to avoid evaporation over a long period of time.


1/ Methods

evaporation-sealed-chamber

Fig. 2: Scheme of the different layers that provide a sealed chamber and avoid evaporation

Two sample mounting systems with two different spacers (A – white and B – transparent), both fully biocompatible, were used for this test. Spacer A is a standard silicon sheet commercially available whereas spacer B results from our developments.  To mimic a biological sample medium and ease the visualisation, we used a coloured liquid. Finally, the three pieces (cover slip + spacer + thermalization unit) are properly maintained into an insert fitting standards of microscope stages.


2/ Results

evaporation-sealed-chamber-live-cell-imaging-results-experiment

Fig. 3: table showing the evolution of the evaporation inside both chambers. Pictures took after 1h45, 16h30 and 65h. The A column corresponds to the A spacer which is a standard silicon sheet commercially available whereas the B one to the B spacer that results from our developments. The red shapes outline the liquid drop over time.

Figure 3 shows the evolution of the droplet after 1 h and 45 minute, then 16h and 30 minutes and finally after 65h. The main objective of these images is to make the evaporation process visual and easy to understand. The other main point is to show how important is to have a sealed chamber to keep in the medium and have no evaporation.

As it can be seen on the images the evolution of the medium inside chamber A and B is very different. Inside the blue circle is the initial medium inside each chamber.

  • On chamber A the medium evaporates more than the 90% after the 16h. At 65h there is no medium available, just the colorant concentrated on a droplet
  • The evolution in Chamber B is compatible with long-term imaging. Indeed, after 16h, the drop is though at the end of the test after 65 hours only half of the medium evaporated.

3/ Conclusion

This test shows the efficiency of this simple and user-friendly mounting system developed by the R&D department of Cherry Biotech.

The take-away observation is that spacer B provides a better sealing compared with the A one. This spacer B is the result of our focus to develop user-friendly devices. As mentioned by Ettinger, Dailey and Daum (1–3,5) perfect sealing is crucial to avoid all kind of evaporation as well as helping to keep the environment stable. The integration of this mounting system into our insert provides an ideal chamber to avoid evaporation while performing long-term imaging.

Bibliography/Sources

Figure 1: https://www.bioscience.org/2013/v5e/af/E594/fulltext.php?bframe=figures.htm

Nath, G.R. Devi / Pharmacology & Therapeutics 163 (2016) 94–108

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Written by Pablo Salaverria

Written by Pablo Salaverria

PhD student | Innovation Unit | H2020-MSCA-ITN-DivIDe

Pablo is part of the H2020-MSCA-ITN-ETN-DivIDe European network. LEARN MORE.
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