Fluorescence and Holotomographic Imaging with the 3D Cell Explorer

The 3D Cell Explorer from Nanolive is designed to generate powerful 3D images and 4D time lapse images of living cells while achieving high resolutions, both spatial and temporal, at x,y: 180 nm; z: 400 nm; t: 1.7 sec. Label-free imaging is possible because the refractive index (RI) of the various components of the cell is measured directly by the microscope. But when it comes to investigating molecular structure or cellular biology, fluorescent markers can be used in combination with the RI to verify these findings or to find how fluorescent signals change with the different states of the cell.

The 3D Cell Explorer-fluo meets this need, being a platform that allows collection of accurate tomographic data with good-quality epifluorescence in three or four channels, using the CoolLed module.

This article describes how time-lapse images were acquired from mouse embryonic stem cells (mESCs) that underwent genetic modification to express the fluorescence ubiquitination cell cycle indicator (FUCCI). This is a double-color indicator using red and green to show the phases of the cell cycle. The use of the 3D Cell Explorer-fluo to make movies involving fluorescence as well as 3D refractive index imaging is discussed, and a solution is set forth for the analysis of the resulting time lapse experiment.

Live FUCCI mouse Embryonic Stem Cells from Nanolive on Vimeo.

The greatest difficulty in current cell biology research is with imaging fine cellular processes over the long term (Frechin et al., 2015; Kruse & Jülicher, 2005; Kueh, Champhekhar, Nutt, Elowitz, & Rothenberg, 2013; Skylaki, Hilsenbeck, & Schroeder, 2016). The aim is not just getting images of biological systems as they appear at one moment, but to visualize the processes as they occur (Muzzey, Gómez-Uribe, Mettetal, & van Oudenaarden, 2009).

Fluorescence microscopy is preferentially used for live imaging when there is high content, but the problem is the phototoxicity it causes to the living sample when different wavelengths are applied. The cell damage that results restricts the utility of this approach, particularly when mammalian embryonic stem cells and other sensitive cell lines are to be imaged.

The advantage of the 3D Cell Explorer is the laser irradiation using one hundred times lower energy than the least energetic fluorescent imaging technique used today. This makes it possible to continue live imaging endlessly, to obtain a 3D RI map of a cell.

On the other hand, sophisticated molecular testing or cellular biology investigations are dependent upon fluorescent imaging to visualize the development of specific cell structures, relate fluorescent reporters with each other or follow up various cell processes and states. It is also needed to trace signaling pathway changes using specific fluorescent reporters. This is why the 3D Cell Explorer-fluo is ideal for this purpose, offering the chance to perform fluorescent imaging as well as 3D RI mapping.

The combination of unlabeled holotomography with fluorescence imaging reduces phototoxicity due to fluorescence still more but allows the experiment to benefit from its use, especially with regard to tracing the path of fluorescent proteins. Using both these techniques avoids the use of multiple photostimulation on the live sample because of the availability of structural and dynamic holotomographic data. This can be used in place of fluorescence imaging if coupled with appropriate correlative analysis and image processing, or can seamlessly join two fluorescent images acquired successively.

This article discusses the monitoring of mESCs over the whole cell cycle using a combination of holotomography and epifluorescence. First describing the equipment and knowledge required, it then goes over the 3D Cell Explorer-fluo interface. Lastly, it suggests how to analyze an mESC movie with the expression of fluorescence ubiquitination cell cycle indicator (FUCCI) construct, so that cells can be observed in both red and green fluorescent channels.

Prerequisites

The first step is to obtain mESCs which express the FUCCI construct (Sakaue-Sawano et al., 2008) (mESCs-FUCCI), that are cultured in a glass bottom dish suitable for the 3D Cell Explorer inside a standard cell culture incubator. The sensor FUCCI contains both red and green fluorescent proteins, red (RFP) and a green (GFP), fused to cdt1 and geminin, molecular cell cycle regulators.

During the G1 phase of the cell cycle, degradation of geminin-GFP occurs. This means that only cdt1-RFP is left to express red nuclear fluorescence. During the S, G2, and M phases of the cell cycle, cdt1-RFP is degraded which leaves only geminin-GFP remains, causing green nuclear fluorescence.

As the cell transitions from G1 to S, there is a decrease in cdt1-RFP and increase in geminin-GFP. However, both remain in the cell, causing yellow nuclear fluorescence to result from the overlay of green and red. In short, the cell nucleus goes through colors from red through yellow to green during the cell cycle, but no nuclear color is seen when the cell divides.

Notes:

  1. This article does not deal with mESCs cell culture which is a specialized area of research. Details on this process should be sought from an expert to find the right type of dish coating and composition for growth medium in order to grow this particular type of cell line.
  2. The details in this article can be used for any fluorescent cell line by adjusting the details for specific fluorescent markers or reporters.

Secondly, you will need the top stage incubator setup from Nanolive. This includes the top stage incubation chamber, a controller pad, and a humidity controlling system. Nanolive carbon dioxide mixer and air pump are recommended to provide carbon dioxide in the right ratio, which means less money needs to be spent on compressed air.

The last item required is the current version of STEVE (v. 1.6 or above) so that the most advanced export functionalities and image post-processing features can be taken advantage of. The Cell Profiler 3 and FIJI software programs are also required.

To accomplish the complex task of plotting and presenting data, which needs a separate discussion, Matlab or Python IDE, or R, is recommended.

Setting up a Fluorescence Acquisition

Another article has already described the 3D Cell Explorer’s ability to provide unparalleled time-lapse imaging due to the absence of any phototoxicity. If data management is performed properly along with regulation of the environment, live imaging of even sensitive samples may be carried out endlessly, at high temporal resolution of 1 image/1.7 seconds.

Being capable of holotomography and fluorescence imaging at the same time gives the device used an edge, but means that the phototoxicity produced by the fluorescent imaging protocol must be controlled.

After bringing the cell to be studied into focus and prepared to be imaged in the top stage incubation chamber, the 4D acquisition button in the STEVE software is clicked. This raises a request to define the time-lapse base frequency, as well as the manner in which the refractive index (RI) as well as the separate fluorescent channels are to be programmed over this frequency. The aim of the procedure is to minimize phototoxicity by decreasing fluorescence acquisition while keeping the imaging frequency high using RI acquisition.

Set-up of fluorescence time lapse imaging

Figure 2. Set-up of fluorescence time lapse imaging

This article discusses this platform with a defined interval of 15 s between images, and with acquisition of an RI volume every 15 seconds, but of green and red fluorescent channels every 30 time points, or in other words every 7.5 minutes. This poses some degree of difficulty when stem cells are imaged in this way.

This is overcome by the powerful microscope camera facility as well as greatly advanced FUCCI construct, which makes the signal very bright. With these tools the light exposure is minimized as far as possible. All that has to be done is to click OK, set up the green and red fluorescence channels to 100 ms, with 1% power and 50% gain. Then the acquisition file location is to be defined so it can be stored, as shown in Figure 2.

mESCs-FUCCI time lapse imaging

Figure 3. mESCs-FUCCI time lapse imaging

Even if the resulting image is not up to expectations, and you want to stimulate the fluorescent dye further, it is recommended that the duration of exposure be increased first, before boosting the light power, so that the acute light exposure is kept at a low level but the fluorescent proteins GFP and RFP (in this experiment) are subjected to higher stimulation.

Data Export

The export tool integrated in this platform (for details, see “Growing and Filming Stem Cells with the 3D Cell Explorer” is designed to export individual time points and channels separately, while allowing the dataset to be organized correctly in .tif or .png format, using the green and red fluorescent channels.

Creating a Set of Nuclei Signal Images

It is to be noted that the solution proposed here is only one of many possible techniques. The purpose is to inspire other fluorescence-holotomography combinations which often comprise of quite distinct fluorescent signals, cellular structures and dynamic cellular processes. The use of FIJI software allows green and red image sequences to be opened (go to File>Import>Image sequence) in two separate stacks, by naming them correctly according to the image channel.

This done, the two stacks are merged to form a single additive signal from the green and red signals, which signals the nucleus (open Process>Calculator Plus...). The frames are saved as individual images with the same format that was used for the image sets of red and green signals, in the same folder, using a distinctive signal identifier.

Analysis of mESCs Cell Cycle

The following is a short description of the Cell Profiler 3, with the image pipeline that is handy for cell tracking and dynamic value extraction from the living cells imaged in the movie. This platform is a very useful tool but requires some familiarity to use properly, which is enabled with the detailed tutorial at http://cellprofiler.org/tutorials/.

With this tool three sets of images were created, the green channel, the red channel, and a sum of the two channels which provides the nuclear signal. The three sets are loaded and then used as the nuclear signal to feed the module, so that primary detection of the object can be done.

This is followed by nuclei segmentation with ease, as well as segmentation of individual nucleus objects. If required, individual z-slices of the RI signal from the imaged cell can even be taken for the detection of the cell boundaries at a defined height.

This is carried out by first creating the image sequences as needed in individual folders, that is, repeating the above procedure on the RI slices to be studied. These are then loaded and can be used for secondary detection of the objects. Here the segmented nuclei are the seed primary object.

Using the 3D Cell Explorer-fluo it is possible to add one more blue or far red channel to detect a dedicated nuclear stain, which makes object detection procedure more convenient. The problem is the need to use the right cell line to express this type of marker, as well as the possibility of exposing the cells to greater risk of phototoxicity.

The track object module is now used for the segmented nuclei, since they are well-defined and large. The “measure-“ modules of the Cell Profiler can be used to extract their features, which allows this kind of data representation to be generated using various cell parameters. For more details on the uses of the FUCCI tag, see Sakaue-Sawano et al., 2008.

Tracking FUCCI green and red fluorescent signals recorded with the 3D Cell Explorer-fluo

Figure 4. Tracking FUCCI green and red fluorescent signals recorded with the 3D Cell Explorer-fluo

General Hardware & Software Requirements

The following tools are required for this experiment:

3D Cell Explorer models:

  • 3D Cell Explorer-fluo

Incubation system:

  • Nanolive Top Stage Incubator

Microscope stage:

  • Normal 3D Cell Explorer stage
  • Hi-grade 3D Cell Explorer manual stage

Software:

  • STEVE – version 1.6 and higher.
  • FIJI
  • Cell Profiler 3

References

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 behaviour. Nature, 523(7558), 88–91. https://doi.org/10.1038/nature14429

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

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

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

Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., ... Miyawaki, A. (2008). Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression. Cell, 132(3), 487–498. https://doi.org/10.1016/j.cell.2007.12.033

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

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: May 6, 2019 at 10:32 AM

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