The 3D Cell Explorer is designed to allow powerful 3D and 4D time-lapse imaging of living cells achieving excellent spatial and temporal resolution at x,y: 180 nm; z: 400 nm; t: 1.7 sec. To utilize this sophisticated technology to its utmost, Nanolive’s top stage incubator must be set up properly, particularly when stem cells are to be used, since these are known to be sensitive to experimental stresses.
This article describes some of the most essential and useful tips required to image mammalian stem cells over the long term up to weeks, with very good resolution and acquiring 3D images. The mode of recording unique stem cell movies is explained, as well as how to export them using STEVE, the 3D Cell Explorer software. Some important issues discussed here include: keeping the humidity, carbon dioxide and temperature at perfect levels, and following the right routine for imaging.
To view wonderful stem cell movies: https://vimeo.com/262374462.
The ability to achieve long-term imaging of fine dynamics of cellular processes is among the greatest obstacles to further advances in cell biology (Frechin et al., 2015; Kruse & Jülicher, 2005; Kueh, Champhekhar, Nutt, Elowitz, & Rothenberg, 2013; Skylaki, Hilsenbeck, & Schroeder, 2016). The aim of such research includes image acquisition of dynamic biological processes within living systems in snapshot mode, but more importantly it aims at looking at the whole process as it is happening (Muzzey, Gómez-Uribe, Mettetal, & van Oudenaarden, 2009).
The method preferred at present in live imaging work which involves acquisition of high content; this, however, produces phototoxicity when different wavelengths are used to stimulate the sample. The stress induced by light toxicity causes the production of free radicals in the cell which cause the cell to undergo damage by structural alteration.
As a result, strong cells like cancer cell lines must be imaged using a technique which balances the advantages of acquiring images at high frequency with those of live cell imaging long-term using low-frequency acquisition. This has not yet been achieved with sensitive cells such as stem cells derived from mammalian embryos which have no acceptable long-term movies as of now.
The 3D Cell Explorer uses laser illumination which transfers a hundred times less energy to the sample than even the lowest-powered fluorescent approaches used for imaging currently. Due to this, and if the data management is well arranged, along with careful environmental regulation, live image could be carried out without end, technically, at the highest speed of acquisition, namely, 1 image per 1.7 seconds. This is indisputably the greatest speed so far attained among all live cell imaging technologies using microscopes, and has turned around the field of live cell imaging of stem cells.
Figure 1. Live imaging of mouse embryonic stem cells for ~3 days. One image per minute. The 3D Cell Explorer generates no phototoxicity.
The first requirement for this experiment is the need for mouse Embryonic Stem Cells or mESCs, which are cultured in a glass dish suitable for use with the 3D Cell Explorer (for more details, see http://nanolive.ch/wp-content/uploads/nanolive-ibidi-labware.pdf) placed in a standard incubator for cell cultures. Other details to do with mESC culture in the laboratory should be obtained from a specialist who will be able to describe the required coating of the glass dish and the composition of the medium which is needed to keep this particular cell type alive, as this article does not deal with this topic.
The second necessity is the Nanolive top stage incubator setup, comprising not only the top stage incubator chamber and controller pad but a humidity system. Nanolive also recommends the use of their carbon dioxide mixer and air pump to make sure the proportion of carbon dioxide is correct, which can avoid the need to buy compressed air. The following link shows how a Nanolive top stage incubator should be set up: http://nanolive.ch/supporting-material/.
The last needed item is the most current version of the powerful software STEVE, or at least version 1.5, to take advantage of the advanced data export capability.
Figure 2. 3D Cell Explorer set up with its top stage incubator
An important precaution to note here is the recommendation to use a medium free of phenol red, to achieve the best live cell imaging. Such media are probably available at all standard suppliers, since these are optimized buffers designed for live cell imaging.
Preparation of Environment
It is important to give the system time to reach equilibrium before it is used experimentally. This will make sure that the parameters remain within acceptable limits. The most serious piece of advice here is to first of all make observations on the interaction of the 3D Cell Explorer and the room in which imaging is performed, with the top stage incubator.
This is because of the variation in temperature and humidity between rooms. During the live cell imaging experiment, people should not enter or leave the room, nor should air conditioners be set to automatic program changes during this time.
The first thing to do is to start the microscope and the computer. The chamber is then positioned on the microscope stage. In some cases the blue stickers attached to the removable part of the chamber have to be removed first to achieve a flat field of view. The chamber is connected properly and the lid is closed. For more details on setup see: http://nanolive.ch/supporting-material/#setup-incubator. The controller is turned on and the temperature set to 38 oC.
Figure 3. Top Stage Incubator Temperature Controller
The humidity bottle is then filled using distilled water. Humid air is injected at 1 liter per minute, at which point the small metal ball will be at the top of the mixer column. The carbon dioxide is not yet switched on as it is not needed. The system is left to achieve temperature equilibrium for about two hours, especially the stage of the microscope.
Why is the temperature set to 38 degrees rather than the 37 degrees required for the growth of mammalian cells? This is because the chamber temperature is not the same as the effective temperature in the well. This has been observed under experimental conditions: it is necessary to set the chamber to 38 degrees to get a temperature of 37 degrees in the well. This should be verified for each experimenter’s local conditions as follows: after two hours of equilibration, with unchanged gas and temperature parameters, a test well is filled with culture medium and put into the chamber, ensuring the green probe dips into the liquid (it may be kept in position using tape if needed). The chamber is closed.
The growth medium temperature is then measured after 30 minutes. If it is not approximately 37 degrees (give or take a few tenths of a degree more or less, which does not impact the experiment [Watanabe & Okada, 1967]), the chamber temperature must be adjusted again and the process repeated. The chamber temperature required for a well temperature of 37 degrees depends also upon room temperature and humidity levels, the latter being very much affected by air conditioning cycle changes. However, the gas flux entering the chamber also plays a major role, and should be set at the recommended level of 1 liter per minute.
Live cell imaging over the long term, particularly when stem cells are involved, requires careful humidity control. The air humidity saturation is almost 100% at the point where air enters the chamber, but this is not enough due to evaporation, which causes a minute loss of medium in the well.
This can disturb the extremely sensitive cellular metabolism of stem cells. To compensate, Okolab, which manufactures these top stage incubators, advises putting a stack of Whatman paper soaked in water, about 1 cm thick, all around the inner outlines of the chamber.
Figure 4 shows how to make such a stack. About 2-6 sheets (depending on the thickness of the paper) are sufficient for this purpose as they become thicker once soaked.
Figure 4. Dimensions for cutting a Whatman paper sponge
The amount of moisture in the paper must be carefully monitored, and the paper should be re-wetted through an access port on one side of the chamber, reached simply by removing a tiny screw. This rehumidification of the paper should take only a second. This method keeps the medium at pristine level for a week at least.
When using stem cells in research, the carbon dioxide level should be set to 4-5%, which is suitable for carbonate-containing buffers.
Figure 5. Manual Air / CO2 Mixer
Beginning Live Imaging
With temperature, humidity levels and the carbon dioxide proportion being under control, the experiment should be set up with the utmost rapidity. This ensures that the cells are out of the regulated environment as briefly as possible and minimizes the time in which the chamber lid is open.
After introducing the cells into the chamber, a wait of 30 minutes at least is essential for the cells to adjust to the conditions and for making sure that all the settings are in order.
Now the time-lapse may be begun.
Some Useful Tips During Acquisition
Provided the procedure discussed previously was followed, the drift in the Z position should be minimal, and is usually due to improper equilibration of system temperature or sometimes, rapid fluctuations in room temperature. For this reason people should be kept from leaving or entering the room, and air conditioning should be kept stable as long as the experiment is running.
For more than two days of acquisition, the water bottle will need to be refilled. During this procedure nothing else should be touched. Just open the bottle and fill it while the gas is on. This is a fast procedure and should not cause any effect on the experiment. If the bottle is on the same table as the microscope care must be taken to avoid bumping into or leaning on the table. Any movement or vibration can alter the positioning of the sample.
Setting Up the System for Acquisition of Images
The microscope optical path can be automatically calibrated at any well position, and after calibration the field of view can still be changed. If this is required one refractive index map may be acquired per minute to begin with, for as long a period as required, to observe the reaction of the cell type used.
Nanolive experimenters succeeded in producing a three-day movie of mECS (shown in Figure 1) which was only stopped because of cell overcrowding within the well. The acquisition rate of 4 images per minute also yielded perfect images. Even higher frequencies can be used without fear, seeing that the 3D Cell Explorer does not produce phototoxic stress to the sample cells.
Exporting Timepoint Images and Movies Using STEVE
After complete image acquisition, the time point images can be exported in different formats. The whole movie, or parts of it, can also be exported as .avi files. The export tool in STEVE provides step-by-step instructions in doing this using the right format, as shown in Figure 6, part 1 and 3.
It enables selection of the part of the movie that is to be exported, as in Part 2 of Figure 6. The z-slices of interest can be chosen (Figure 6, part 4), as well as the file format the export is in (Figure 6, part 5), and the file naming format (Figure 6, part 6). This kind of adaptability allows data to be exported into any desired format, particularly if STEVE is to be integrated into an image analysis workflow.
Figure 6. Exporting movies with the STEVE export tool
General Hardware and Software Needed
The following tools are required for this setup:
3D Cell Explorer models:
- 3D Cell Explorer
- 3D Cell Explorer-fluo
- Nanolive Top Stage Incubator
- Normal 3D Cell Explorer stage
- High grade 3D Cell Explorer stage
- STEVE – version 1.5 and and higher
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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
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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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