3D and 4D holotomographic imaging of unstained specimens

The rapid progression in fluorescence imaging for cell biology over the last 20 years has invariably led to challenges around toxicity of fluorophores, biological relevance of genetically modified cells and ethical dilemmas around genetic modification. The quest to find high-resolution intracellular imaging techniques that do not require any expression of artificial genes has led to the upsurge in development of unlabelled techniques.

One such field is that of quantitative phase imaging (QPI), which enables researchers to observe cells in real time and to perform long-term studies. This technique was developed to generate 2D or 3D optical density maps of cells in culture without the requirements for staining or genetic modification of the sample.

Several methods have been developed within the field of QPI to extract this quantitative phase data from unstained/untreated samples. These include techniques such as spatial light interference microscopy, gradient light interference microscopy, holotomography, digital holographic microscopy and ptychography among others.

Nanolive 3D Cell Explorer Holotomography Microscope

A standalone microscope, the Nanolive 3D Cell Explorer combines holography quantitative phase imaging (QPI) with tomography to provide high-resolution X-Y and Z data of cells and organelles. Like all QPI instruments, the optical path differences between an imaging beam and a reference beam are combined to extract refractive index data. By angling the imaging beam at 45°, further tomographic data is generated and the final image is a combination holotomographic image of the 3D structure of the cell. Also, the generation of large quantitative 4D datasets means you can precisely analyse cell dynamics over long periods with frequent observations in vitro.

Given the non-toxic nature of QPI, it is ideally suited to imaging sensitive cell populations. With extremely low phototoxicity, long-term live cell imaging of even primary cells is possible. This has applications in areas such as stem cell research, drug testing and cancer research.

Nanolive currently offers three different models: the 3D Cell Explorer, the 3D Cell Explorer-fluo and the CX-A.

Nanolive 3D Cell Explorer

The 3D Cell Explorer measures the quantitative refractive index of cell organelles in seconds. Hence, it is possible to carry out non-invasive in vitro imaging of almost any kind of cells with up to 30 μm depth of reconstruction. This allows for biological features to be segmented based on their physical characteristics. Researchers can study cell life cycle processes of growth, division and death in 3D and 4D.

This device also offers high-resolution and high-sensitivity characterisation of multiple cell organelles based on their refractive index. This makes it possible to explore and measure up to 8 cell organelles simultaneously with unprecedented detail and resolution; marker-free and preparation-free based on their own physical density. Moreover, thanks to a dedicated top-stage incubator you can monitor cell compartments and their kinetics and dynamics in real time at every second without interfering with their natural physiology.

Nanolive 3D Cell Explorer-fluo

The 3D Cell Explorer-fluo transforms 2D fluorescence into 3D cell tomography to identify cell organelles through fluorescence and to non-invasively monitor their structures and dynamics in 3D and marker-free. Users can explore fluorescence (three channels) and digital stains (eight channels) simultaneously and image live cells as long as they require.

Nanolive CX-A (automated live cell imaging)

Nanolive’s CX-A extends the exploration of living cells from single cells to cell populations. It enables scientists to investigate macro cellular dynamics like cell health, proliferation, movement and function, as well as micro organelle dynamics and interactions, eg, mitochondrial network characterisation.

The CX-A is designed to work with 96-well plates to multiply and parallelise experimental conditions, hence bringing undoubtable significance to each experiment and delivering solid biological insights to researchers. Furthermore, the system is equipped with multiple imaging modalities to correlate and compare physical and chemical information at each time-point.

Finally, 3D data sets of every single image at every single time-point are automatically acquired in real time: a fully integrated solution adapted to the most advanced professional needs.

The CX-A can image living cells for a virtually limitless amount of time. Thanks to its unique harm-free method of cell preparation and observation, hundreds of images can be collected each hour, transforming endpoint assays to continuous analysis, for days or weeks, while cells remain unperturbed in a physiologically controlled environment. Users can freely define the perfect imaging regimen for their cells without worrying about phototoxicity or photobleaching.

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