Cell Cycle Analysis

 

 

 

Live FUCCI mESC imaged for over 48 hours with the 3D Cell Explorer-fluo

Cell cycle and cell death are basic cellular processes. Modifications in cell shape, cell volume and cell organelles are observed in each of the phases of the cell cycle and even during cell death.

Nanolive imaging allows for non-invasive long-term live cell imaging at very high spatio-temporal resolution. This breakthrough leads to the observation and characterization of continuous processes such as pre-mitotic nuclear rotation, mitosis, apoptosis, necrosis, autophagy, and pyroptosis in a new, holistic fashion.

Cell division analysis

Kinetics, dynamics and morphological changes unveiled during mitosis

The 3D Cell Explorer is able to discriminate different mitotic phases based on chromatin RI values and monitor changes in nuclear RI, shape and size during mitosis (e.g. DNA condensation, chromosomes alignment and segregation, mitotic spindle formation, metaphase plate, sister chromatids, contractile ring).

Furthermore, it is possible to follow changes in cellular shape and thickness during cell division and measure the processes of surface attachment/detachment (Figure 1).

Figure 1: Changes in cellular shape during mitosis in fibroblastic reticular cells.

Spectacular Mitosis in Mesenchymal Stem Cells

In the provided example of a spectacular cell division taking place in a living sample of human mesenchymal stem cells cultured with low-serum cell growth medium[2] and observed under the 3D Cell Explorer, the characterization of the different steps and structures of mitosis was possible. Further details and static images of the high-quality footage obtained are here described in Figure 2.

Read our detailed blog post here: https://nanolive.ch/mitosis-in-mesenchymal-stem-cells/

Figure 2: Phases of mitosis in human mesenchymal stem cells.

Mitosis in Human Umbilical Vein Endothelial Cells

During the live cell imaging of the sample we could observe chromatin condensation, chromosome alineation in the metaphase plate, chromatids migration and the formation of the contractile ring and further separation in two fully functional daughter cells.

Read our detailed blogpost here: https://nanolive.ch/failed-mitotic-exit-in-human-umbilical-vein-endothelial-cells/.

Mouse Fibroblastic Reticular Cell (FRC) mitosis

Fibroblastic Reticular Cells (FRCs) were grown to 60% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium) in 35mm glass bottom culture dishes (FluoroDishes™ WPI, #FD35-100). The time-lapse imaging experiment was conducted with a standard top-stage incubator set to 37°C and 5% CO2 for 45 minutes, capturing images every 30 seconds.

Observation of a mitosis in an umbilical cord blood isolated CD34+ HSC sample

A sample of human cord blood CD34+ stem/progenitor cells kindly provided by Lonza was cultured in X-VIVOTM 15 serum-free hematopoietic cell medium, supplemented with recombinant human thrombopoietin (25ng/mL), Rlt3 ligand (25ng/mL) and stem cell factor (13ng/mL). The culture was also coated with fibronectin.

A remarkably long live cell observation (3 images/min during 15hours) was performed with Nanolive’s 3D Cell Explorer, and a cell division was captured. To date and to our knowledge, this live cell movie featuring CD34+ HSC is the first one ever obtained.

One can distinguish characteristic steps of mitosis such as chromatin condensation inside the nucleus that lead to chromosome formation, the alignment of the chromosomes in the metaphase plate and the migration of sister chromatids to opposite ends of the cells. Eventually, two daughter cells were obtained as a result of the contractile ring effect during cytokinesis.

Read more here: https://nanolive.ch/cd34-mitosis/

Pre-mitotic nuclear rotation

Label-free live cell imaging of mouse breast cancer cells

Nanolive imaging with the 3D Cell Explorer allows for the visualization of the interactions of cellular organelles, by their refractive indices. This led to the observation of nuclear rotation implicated in cellular reorganization before mitosis[1].

The video shows mouse breast cancer cells imaged with the 3D Cell Explorer: Nanolive imaging reveals 3D (in all axes) nuclear and organelle rotations in mammalian cells.

With special thanks to  NBE therapeutics for the reagents used to obtain the video.

[1] P. A. Sandoz et al., “Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells,” bioRxiv, p. 407239, Sep. 2018.

Label-free live cell imaging of Pre-adipocytes

Nanolive microscopy reveals pre-mitotic nuclear and organelle rotations in mammalian cells.

See videos and the pre-publication here: https://nanolive.ch/publication-van-der-goot/

Cell Death

Cell death is the event of a cell ceasing to carry out its functions.

Cells can die due to several factors. It could be due to external factors such as mechanical injury or exposure to toxins. Cells can also be attacked by the immune system if they are infected or faulty. Generally, given that billions of new cells are produced daily, it is important for older ones to die in order to maintain homeostasis which is a physiological balance.

How do cells die?

There are different ways a cell can die:

Programmed cell deaths (PCDs), such as apoptosis, autophagy and pyroptosis, are controlled processes that confer advantages during an organism’s life cycle. They are involved in a variety of biological events, including morphogenesis, maintenance of tissue homeostasis, and elimination of harmful cells. Malfunctioning of PCD leads to various diseases in humans, including cancer and several degenerative diseases.
Nanolive imaging enables to observe those processes in 3D, in real-time and in a non-invasive manner. 

Apoptosis

This is a Programmed Cell Death of a mouse Preadipocyte cell. During real-time monitoring, we can observe the typical morphological cellular changes as blebbing, cell shrinkage and chromatin condensation. The cells were imaged for 1h30 at a frequency of 1 image every 30 seconds.

Autophagy

This cell death consists of the formation of a double membrane vesicle around targeted cellular components. This vesicle, known as an autophagosome, then migrates through the cytosol to merge with a lysosome filled with hydrolytic enzymes, resulting in the breakdown of the autophagosomes contents.

Autophagy itself is a fundamental process in the adaptation to starvation and an efficient survival strategy in times of physiological stress. So rather than committing suicide, the cell degrades and recycles its inner organelles and unused proteins to maintain homeostasis and metabolic functions and ensure its survival.  In this video, Cell death was induced by the addition of a drug (combination of imipramine and ticlopidine) to mouse glioblastoma cells (LN18). The cells were imaged every 60 seconds over a period of 9 hours. You can see the vacuolization of the cytoplasm followed by heavy lysosomal activity within these autophagic cells. The large autophagosomes fuse with lysosomes, which degrade the organelles and proteins from the cell’s cytoplasm.

Special thanks to the Laboratory of Translational Oncology – Prof. Douglas Hanahan from EPFL.

Necrosis

This is a very rapid process characterized by swelling of organelles, dilatation of the nuclear membrane, increased cell volume (oncosis), culminating in the disruption of the plasma membrane and subsequent loss of intracellular contents. ID8-ova cells (ID8 murine ovarian tumor cell line transduced with ovalbumin). NaOh was added to the medium during the acquisition to trigger this type of cell death. The cells were imaged for 2 minutes at a frequency of 1 image every 2 seconds.

Pyroptosis

In this video mouse macrophages were infected with Listeria. The immune cells recognize the foreign danger signals within themselves, release pro-inflammatory cytokines, swell, burst and die. These cells were imaged for 25 minutes at a frequency of 1 image every 15 seconds.

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