Nanolive’s patented technology

The technology behind Nanolive’s microscopes is unique worldwide and is based on a fundamental patent (US 8,937,722 & EU WO 2011/121523).

The combination of holography and rotational scanning makes Nanolive imaging a revolutionary technology.

Holography offers a unique means to measure cells in their native environment: label-free, non-invasive, manipulation-free, and interference-free. Rotational scanning allows 3D reconstructions, noise robustness, and a resolution far beyond the accepted limit for light.

To achieve optimal results for every measurement, the optics of our technology are completely self-adjusting. It guarantees the best alignment conditions – whatever the environment is.

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How does the 3D Cell Explorer produce cell images with better than 200 nm lateral resolution?

Explaining the Resolution Jump

Rayleigh resolution for incoherent light (e.g. fluo) is 400nm=0.61*520nm/(0.8), for coherent light (e.g. holography) 533nm=0.82*520nm/(0.8) with the diffraction limit (e.g. any imaging by niquist) of 325nm=0.5*520nm/(0.8). In our case for the 3D Cell Explorer configuration, the maximal expected resolution is around 160nm=0.5*520nm/(2*0.8) and we estimate the real resolution (according to Rayleigh) to be about ~190nm. Therefore, taking into account the illuminating wavelength the experimental resolution is 190nm=/lambda/(3.5 NA), indicating a sub-diffraction resolution (d=325 nm at given wavelength and NA) imaging in far-field (non-fluorescent) transmission microscopy.

In conclusion, the 2 terminologies of (i) optical resolution (the real one) and (ii) sampling resolution (the one on the screen) are separated for 3D holotomographic microscopy. For the sake of computational cost-reduction, Nanolive chose to have the sampling optimal at the optical resolution, hence about 180 nm. Again, we can do this, thanks to our technology WITHOUT loosing any real resolution, but of course a user could over-sample the images and have double/triple/etc sampling on the screen with the same optical resolution (e.g. simple zero-padding in fft space to have a smoother image).

In life sciences, fixation refers to the process of preservation of histological, cytological and microbiological specimens such as cells and tissues at the status they were originally taken. A protocol of fixation usually precedes immunostaining, which is essential to immobilize biological structures and make them accessible to antibodies.

There are different mechanisms of fixation depending on the reagent used. As already shown in a previous publication from Sandoz et al (Fig.2A), dehydrating fixation methods severely affected organelles structure preservation and thereby their RI. A widely used alternative is paraformaldehyde (PFA), which acts creating covalent chemical cross-links between proteins. Even if PFA rapidly penetrates the sample, the process can take up to a few weeks in case of human biopsies for example.

Since fixation aims to provide a snapshot of a living situation, we visualized the process of fixing living samples with PFA using Nanolive’s 3D Cell Explorer. We conclude that cell structures are affected by the fixation process.

Two samples of living mouse pre-adipocytes were imaged for 30 min before initiating the fixation process. Images were taken every 15 seconds both before and after fixation. On the 0.2% PFA condition, PFA was directly diluted in medium with a final concentration of 0.2% PFA at 30min. On the 2% PFA condition, the medium was completely removed and replaced by 2% PFA in PBS after 30min. The process of fixation was recorded for up to 1 hour.

Highlights

All in all, these observations strongly question the use of fixed samples in research involving membrane dependent systems.

A concentration as low as 0.2% PFA already induces very strong cellular changes at the molecular level, which are translated in an observation that does not meet the characteristics of living cells. These modifications are much more accentuated amongst membranes (mitochondrial, nuclear and plasma).

These conclusions correlate with previous research on the subject[1], [2]. PFA fixation has been shown to induce strong perturbations of cellular nanostructures[2], and inefficiently fix glycosylphosphatidylinositol-anchored, phospholipid-anchored or cholesterol-anchored membrane proteins, leading to observation of aberrant clustering of proteins observed by subsequent immune-staining[1].

[1]         K. A. K. Tanaka et al., “Membrane molecules mobile even after chemical fixation,” Nature Methods, vol. 7, no. 11. pp. 865–866, Nov-2010.

[2]         Y. Li et al., “The effects of chemical fixation on the cellular nanostructure,” Exp. Cell Res., vol. 358, no. 2, pp. 253–259, Sep. 2017.

Please scroll the table right to see the full comparison

Example of Embryonic Stem Cells (ESC) during mitosis

Comparison between ESCs (4 & 5) as observed with the two technologies