3D Cell Explorer Experiments Best Practices

The 3D Cell Explorer can be used to provide powerful 3D images and 4D time-lapse images of living cells, while achieving excellent spatial and temporal resolution of (x,y: 180 nm; z: 400 nm; t: 1.7 sec). However, the setup determines how well the Nanolive top stage incubator can perform. This article aims to help set up the system for greatest stability during the experiment and to prevent the cells from undergoing stress within the incubator.

Here the right way to set up the top stage incubator is discussed, so that mammalian cells can be imaged for long periods, even over weeks if required. This means the right steps must be followed to maintain the optimum levels of humidity, temperature and carbon dioxide, and the appropriate imaging regime must be followed. This should enable experimenters to make full use of the feature that 3D Cell Explorer boasts of in unmatched measure: long term live cell imaging at high frequency.

Introduction

The most difficult issue in current cell biology is acquiring dynamic cellular images over the long term (Frechin et al., 2015; Kruse & Jülicher, 2005; Kueh, Champhekhar, Nutt, Elowitz, & Rothenberg, 2013; Skylaki, Hilsenbeck, & Schroeder, 2016). This is done to see how biological systems appear at the moment of imaging, but more importantly, to actually visualize the dynamic operation (Muzzey, Gómez-Uribe, Mettetal, & van Oudenaarden, 2009).

The problem with fluorescent microscopy, which is the current method of choice in high-content live imaging approaches, is the toxicity it causes to the sample. Various wavelengths cause cell damage by the release of free radicals, which alter the cell structure, and this reduces the type of imaging possible. In short, the use of most current approaches to live imaging involves a tightrope walk between short live imaging time with high frequency of acquisition, or prolonged imaging of the cell with low frequency of acquisition.

The former causes increased phototoxic stress to the sample. This means that if the fortunate researcher does manage to observe a process at detailed dynamic level, it cannot be ensured that the observations were not disturbed by the very imaging process. Image acquisition at low frequency is possibly more friendly to the sample, but lacks the ability to show fine dynamic processes, without completely ruling out the occurrence of perturbations due to the photostress.

The solution is in fact to perform long-term imaging at high frequency of acquisition. The laser used by the 3D Cell Explorer passes on a hundred times less energy to the sample than the most gentle fluorescent imaging method. Thus the technology is now in place to take live images of dynamic processes endlessly, at the most rapid acquisition rate possible, of 1 image per 1.7 seconds, as long as the data management and environmental control tools are properly installed. This is a method that far surpasses any other live cell imaging microscopy technology microscope.

Live imaging of mouse pre-adipocytes for 48 hours. One image per minute. The 3D Cell Explorer generates no phototoxicity.

Figure 1. Live imaging of mouse pre-adipocytes for 48 hours. One image per minute. The 3D Cell Explorer generates no phototoxicity.

Prerequisites

For an experiment involving the 3D Cell Explorer, it is necessary to use live cells from mammalian species, and to culture them in a glass bottom dish that can be used with the device. The dish is incubated inside a standard cell culture incubator. When animal cells are cultured in this way, in monolayers as far as possible, in the dishes advised above, they can be watched in action live using the 3D Cell Explorer.

Here the parameters dealing with mammalian cell growth are described but this is not a limitation, as any type of cell culture, including yeast and bacteria, can be used with the help of this article with slight modifications. The second necessity is the top stage incubator setup from Nanolive, including the incubation chamber designed for the top stage, a controller pad and a system to regulate humidity.

Nanolive carbon dioxide mixers and air pumps are recommended, to supply the right ratio of carbon dioxide and thus avoid the expense of using compressed air. For details on how to install the incubator, see: http://nanolive.ch/supporting-material/#setup-incubator.

3D Cell Explorer set up with its top stage incubator

Figure 2. 3D Cell Explorer set up with its top stage incubator

Important note: the use of phenol-red free medium is preferred for best live cell imaging performances. Your favorite supplier certainly makes buffers optimized for live cell imaging.

Preparation of the Environment Control

Temperature

It is essential to allow the system to reach equilibrium before it is used. This will make sure that the parameters remain within the acceptable limits. Nanolive very strongly advises users to see the way the top stage incubator behaves with the 3D Cell and with the room used for imaging, since each environment has its own unique temperature and humidity. During the experiment, while live imaging is going on, the room should be kept closed, without anyone coming in or leaving. If any air conditioners are running, they should be checked to make sure no automatic changes occur in the cooling settings.

Once everything is checked, the microscope and the computer are started up. The incubation chamber is put on the stage of the microscope. Care must be taken to remove blue stickers from the removable portion of the incubation chamber, in case a brand-new chamber is being used, as they could affect the degree of flatness of the field of view.

The chamber is connected in compliance with the instructions available in the manual, which can also be viewed in the video: http://nanolive.ch/supporting-material/#setup-incubator. The chamber lid is put on and the controller is switched on, setting the temperature control to 38 oC.

Top Stage Incubator Temperature Controller

Figure 3. Top Stage Incubator Temperature Controller

The next step consists of pouring distilled water into the humidity bottle at a rate of one liter per minute, to make the small metal ball come up to the top of the mixer column. The system now rests for about two hours to reach equilibrium, especially at the microscope stage. The temperature in the chamber is different from the effective well temperature. The reason for the higher temperature setting compared to the 37 oC at which mammalian cells typically grow is that this increase is required to obtain a well temperature of 37 degrees.

This difference can be checked for each user as follows: once the system has rested for two hours, and with all parameters unchanged, including the gas composition and the temperature, a test well is prepared using the culture medium, and put into the chamber. The green probe should dip into the liquid, and taped in position if required. The chamber is then closed. After a gap of 30 minutes, the growth medium is tested for temperature. If it is significantly deviated from the required 37 degrees, the chamber temperature is adjusted and the system allowed to equilibrate, until the right chamber temperature is reached.

The final chamber temperature required to achieve a well temperature of 37 degrees is dependent on several parameters, such as the ambient temperature and humidity, and gas flux into the chamber. As noted, air conditioning cycles must always be checked before setting the system to equilibrate. The gas flux must be set at one liter per minute according to the manufacturer’s recommendations.

A slight deviation, such as a couple of tenths of a degree, from the required 37 degrees is acceptable (Watanabe & Okada, 1967).

Humidity

Maintaining the right humidity is vital for proper live cell imaging over the long term. If an experiment is designed to last less than two days, the following guidelines do not apply. Otherwise, the steps given in section I must be followed. The saturation of humidity in the air is almost 100% when it reaches the chamber. This undergoes evaporation, albeit slowly, which can be observed after two days as a minute loss of growth medium, which, small as it is, can disturb sensitive cells significantly.

To prevent this, Okolab, the manufacturer of the top stage incubator, advises that a stack of Whatman paper one centimeter thick be placed all around the inner profiles of the chamber, well soaked in water. The procedure to make such a stack is shown in Figure 4. Typically, 2-6 sheets are sufficient for such a stack, since they become thicker as they absorb water.

Dimensions for cutting a Whatman paper sponge

Figure 4. Dimensions for cutting a Whatman paper sponge

The amount of water inside the paper must be strictly monitored. If re-soaking is required, it is possible to do so using one of the access ports on one side of the chamber, avoiding the need to open the chamber. On testing, this solution has been found to prevent any loss of solution for one week, at least.

pH Control

The carbon dioxide should be at about 8-10% if the medium used is DMEM or other media containing carbonate buffers.

Manual Air / CO2 Mixer

Figure 5. Manual Air / CO2 Mixer

This is more than the usual carbon dioxide percentage of 5% used in a standard incubator. This is because a top stage incubator shows decreased efficiency of gas exchange, being non-hermetically sealed unlike the typical 37 degrees incubator. However, this does not pose any danger of asphyxiation to the cells since in any conditions cultured cells experience an oxygen partial pressure which is at least ten times more than that of normal tissue.

Another useful piece of advice is to use the media mentioned above that have been optimized for imaging live cells. The media that use HEPES buffer can maintain their pH at 7.4 without carbon dioxide. These are also highly recommended if premixed 5% carbon dioxide and air are used.

It should be noted that overconfluence typically occurs during live cell imaging over a long period, and increasing apoptosis rates are seen which will make the medium acidic over time, even if one or more of the above techniques are used to counter acidity.

Starting the Live Imaging Experiment

Now that the chamber’s humidity, temperature carbon dioxide are all properly regulated, the living cells may. Things should now go fast to prevent the cells from remaining out of this environment for any longer than necessary, and to close the chamber lid as fast as possible. After the cells in the chamber, a period of at least 30 minutes should be allowed for adaptation to the environment as well as to verify the experimental conditions. this you are now ready to start your time-lapse.

During Acquisition

Provided the experimental procedure is followed as described above, there should be very minor drift in the Z position of the sample. Any drift observed may be attributed in most cases to the improper equilibration of the temperature of the system.

It could also be the result of quick temperature fluctuations. To prevent this, it must be stressed that people should not leave or enter the room during the experiment, and the air-conditioning cycle should not change during this period.

If more than two days are required for image acquisition, the water bottle must be filled again. This requires just opening the bottle and pouring in water with the gas still on. Nothing else should be touched.

The procedure takes only seconds and should not affect the experiment. Precautions must be taken to avoid colliding with the bottle or leaning on the table on which the bottle is placed, if the microscope is on the same table. Any such vibration or movement can change the position in which the sample is imaged.

General Hardware & Software Requirements

The required equipment and software for live imaging with this system include:

3D Cell Explorer models:

  • 3D Cell Explorer
  • 3D Cell Explorer-fluo

Incubation system:

  • Nanolive Top Stage Incubator

Microscope stage:

  • Normal 3D Cell Explorer stage
  • High grade 3D Cell Explorer stage

Software:

  • STEVE – all versions

References

  1. Frechin, M., Stoeger, T., Daetwyler, S., Gehin, C., Battich, N., Damm, E.-M., … Pelkmans, L. (2015). Cell-intrinsic adaptation of lipid composition to local crowding drives social behavior. Nature, 523(7558), 88–91. https://doi.org/10.1038/nature14429
  2. Kruse, K., & Jülicher, F. (2005). Oscillations in cell biology. Current Opinion in Cell Biology, 17(1), 20–26. https://doi.org/10.1016/j.ceb.2004.12.007
  3. Kueh, H. Y., Champhekhar, A., Nutt, S. L., Elowitz, M. B., & Rothenberg, E. V. (2013). Positive Feedback Between PU.1 and the Cell Cycle Controls Myeloid Differentiation. Science (New York, N.Y.), 670. https://doi.org/10.1126/science.1240831
  4. Muzzey, D., Gómez-Uribe, C. a., Mettetal, J. T., & van Oudenaarden, A. (2009). A Systems-Level Analysis of Perfect Adaptation in Yeast Osmoregulation. Cell, 138(1), 160–171. https://doi.org/10.1016/j.cell.2009.04.047
  5. Skylaki, S., Hilsenbeck, O., & Schroeder, T. (2016). Challenges in long-term imaging and quantification of single-cell dynamics. Nature Biotechnology, 34(11), 1137–1144. https://doi.org/10.1038/nbt.3713
  6. Watanabe, I., & Okada, S. (1967). Effects of temperature on growth rate of cultured mammalian cells (L5178Y). The Journal of Cell Biology, 32(2), 309–23. https://doi.org/10.1083/jcb.32.2.309

About Nanolive SA

Nanolive SA are scientists, working for scientists.

Their belief is that each and every Biologist, Researcher and Physician should be able to explore and interact instantly with living cells without damaging them.

Nanolive want to support the study of how living cells and bacteria work, evolve and react, thus building a solid base for new drugs and therapies, in order to enable breakthrough researches.

This is the reason why they have developed the 3D Cell Explorer.


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Last updated: Mar 12, 2019 at 4:04 AM

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