Application of 3D Cell Explorer to Study of Interactions and Reactions

Nanoparticle internalization

On this page you can explore nanoparticles inside living cells as we see them: in 3‐D and stain‐free.

Nanoparticles within Cells as Visualized via 3D Cell Explorer

The unique behavior and properties of nanoparticles in a biological milieu allows us to address fundamental questions in Biology. Their applications in the Biological or Medical field are many, and include:

  • Procedures of cell manipulation (cell detection and separation);
  • Delivery agents (drug and gene delivery);
  • Cellular therapy (tumor destruction via heating, known as hyperthermia);
  • Phagokinetic studies (following internalization processes).

The 3D Cell Explorer allows for:

  • Label‐free, real‐time monitoring of Nanoparticles (“NP”) uptake;
  • Monitoring of living cell morphological changes due to NP interaction;
  • Kinetic and dynamic studies of NP uptake;
  • Intra‐ and extra‐cellular 3‐D localization of NP in living cell cultures;
  • Volume measurements of intra-cellular loading of nanoparticles.

Human Umbilical Vein Endothelial Cells incubated with gold nanoparticles

Human Umbilical Vein Endothelial Cells (“HUVECs”) grown to ˞40% confluency in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100) were fixed with 1% PFA for 10–15 minutes and then washed with PBS. The cells were treated with 40nm BPEI gold nanoparticles.

Sample courtesy of Dr. Parwathy Chandran and Dr. Nancy A. Monteiro-Riviere – Nanotechnology Innovation Center of Kansas State University.

Human cervical cancer cells treated with Iron oxide nanoparticles

HeLa cells grown to ˞30-40% confluency in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100) were fixed with 1%PFA for 10-15 minutes and then washed with PBS. The cells were treated with 1mg/ml of Iron oxide nanoparticles (7-20nm).

Sample courtesy of Débora Bonvin, LTP, Ecole Polytechnique Fédérale de Lausanne, Switzerland.

Drugs and Toxicity

On this page you can explore drugs and toxicity effects on cells as we see them: live, in 3‐D and stain‐free.

Nanolive’s technology allows for:

  • Label‐free, real‐time monitoring of cell morphological changes during drug exposure;
  • Following drug internalization process;
  • Quantitative measure of intra‐cellular drug loading (direct volume measurement);
  • Definition of drug loading cellular compartment;
  • Real‐time drug cellular toxicity evaluation.
Time-lapse exposure panel

Drug molecules can have multiple effects on cells. Depending on the dose or the type of molecules used, the cell response can vary. It can vary from the stopping of growth of cells, to stopping of division of cells, to their rapid death (necrosis), or controlled death (apoptosis).

Those effects can be measured in terms of toxicity to the cell. The most common assay to measure toxicity is to assess the membrane integrity. To this purpose, chemically staining the cells is necessary, which is a tedious and time‐consuming process.

Nanolive’s new technology offers the possibility of doing without these chemicals thanks to a digital staining done post‐acquisition through the STEVE software. In addition, one can observe — in real time — the response of cells to the different drugs, thanks to the 3D Cell Explorer.

Induced NaOH necrosis

ID8-ova cells (ID8 murine ovarian tumor cell line transduced with ovalbumin) were grown to ˞40% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium) in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100). The time‐lapse experiment was conducted at RT for 2 minutes. NaOh was added to the medium during the acquisition to trigger the necrosis of the cell.

Cell‐Cell Interaction

On this page you can explore interactions between cells as we see them: in 3‐D and stain‐free.

The 3D Cell Explorer allows for:

  • Label‐free 3‐D interaction monitoring;
  • Morphological and quantitative tracking of cells of interest;
  • Morphological assessment of interaction between cells.

There are various types of cell-cell interactions. In complex systems such as organs and tissues, cells are not isolated. They need to communicate with each other in order to adapt to and thus survive in their environment. This kind of interaction happens when cells are in physical contact with each other and communication is done via the membrane’s proteins.

Another type of interaction can happen through the excretion of a signal (autocrine, paracrine, or endocrine). In this case, molecules are taken up by cells and cause a specific reaction, depending on the stimulation.

In both cases, the need for a high spatial‐ and temporal‐resolution microscope is crucial in order to observe the interactions. Nanolive’s technology, the 3D Cell Explorer, allows us to explore those mechanisms in a non‐invasive, 3‐D and Live manner. On top of that, cells are not altered by fluorescence manipulation which canlead to the detection of false interactions; instead, the staining is done post‐acquisition in a digital manner thanks to STEVE software.

Living T-cell killing a living cancer cell

ID8-ova cells (ID8 murine ovarian tumor cell line transduced with ovalbumin) seeded (˞2×104 cells) in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100) and grown to 20-30% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium). The cells were incubated with ˞5×105 T-cells. Cells were imaged using imaging buffer (PBS + 25mM Glucose + 10mM HEPES). The time-lapse imaging experiment where conducted at RT for 41 minutes, capturing images every second.

Living T-cell interacting with a living antigen presenting cell

Fibroblast reticular cells seeded (˞2×104 cells) in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100) and grown to ˞20–30% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium). The cells were previously treated to express antigen specific receptors for T‐cells, and then incubated with ˞5×105 T‐cells. Cells were imaged using imaging buffer (PBS + 25mM Glucose + 10mM HEPES). The time‐lapse imaging experiment where conducted at RT for 45 minutes, capturing images every second.

Cancer cells and amoeba in co-culture

Mouse skin melanoma cancer cells (B16, p35) were grown to 40% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium) in 35mm glass‐bottom culture dishes (FluoroDishes™ WPI, #FD35-100). They were incubated overnight with dictyostelium amoebae cells (WT1, p14) grown in HL-5 medium. The time‐lapse imaging experiment was conducted with a standard top‐stage incubator set to 37°C and 5% CO2 for 8 hours capturing images every minute.

Cell‐Bacteria Interaction

On this page you can explore interactions between cells and bacteria as we see them: in 3‐D and stain‐free.

The 3D Cell Explorer allows for:

  • Label‐free 3‐D infection process monitoring;
  • 3‐D localization of bacteria inside living cells;
  • Detection of mammalian cell infections;
  • Volume measurements of intra‐cellular parasites;
  • Visualization of the process of microorganism internalization.

Not only do cells interact among themselves, but they interact with other types of micro‐organisms. Most of the time these interactions are harmful to the host. Bacteria try to interact with cells in order to invade them. They bind to the cell’s structures (including glycolipids and glycoproteins) and try to modify their cytoskeleton. In other cases, these interactions are good for the host, as is the case for the gut microbiota. In both cases, researchers are trying to examine these interactions in detail. Nanolive’s new technology, the 3D Cell Explorer, allows the visualization of these interactions at a high temporal and spatial resolution. Thanks to STEVE software, neither the cells nor the bacteria are harmed by a chemical staining, as this staining is done digitally based on their specific Refractive Index values.

Section of Bee Gut

Rehydrated paraffin embedded bee gut sections (5 µm) was imaged with the 3D Cell Explorer. The final “panoramic view” of the whole tissue section (more than 400 micrometers) was obtained by stitching multiple acquisitions by using other software. Like human intestine, bee gut is colonized by bacteria population. Most of the gut microbial community resides in the ileum.

Live HeLa Cells Infected with Chlamydia trachomatis

Living HeLa cells infected by Chlamydia trachomatis were imaged with the 3D Cell Explorer. The parasite inclusion bodies are displayed in green inside the cell cytoplasm.

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