One method that allows "see" beyond the diffraction of light is the super resolution microscope, which provides Unrivaled views through the cells and their intrinsic structures and organelles. Recently, the method has gained increasing attention, specifically because the researchers who designed it won, in 2014, the Nobel Prize in Chemistry.
However, there is a great disadvantage that restricts the application of super resolution microscopy: it provides only spatial resolution. Although this is suitable for static samples such as fixed cells or solid materials, in the case of biology, the issues are very complicated. Living cells are highly dynamic and are based on a complex set of biological processes that are carried out on scales of time inferior to the second, and are constantly modified. Therefore, to visualize and understand the behavior of healthy and diseased living cells, a temporary or "high" resolution is also needed.
A research group headed by Professor Theo Lasser, Head of the Laboratory of Biomedical Optics (LOB)) at EPFL, has now taken longer steps to overcome the challenge by creating a method with the potential to carry out Fast 3D phase images and 3D superresolution microscopy in a single instrument. The phase image is a method in which alterations in the light phase caused by the cells and their organelles are converted into refractive index maps of the same cells.
The distinctive platform, termed as "4D microscope" integrates the high time resolution and sensitivity of the phase image with the high spatial resolution and specificity of fluorescence microscopy. The scientists created an innovative algorithm with the ability to retrieve phase information from a bunch of bright field images captured by a classical microscope.
With this algorithm, we present a new way to achieve 3D quantitative phase microscopy using a conventional brightness -field microscope. This allows direct visualization and analysis of subcellular structures in living cells without labeling .
Adrien Descloux, lead author
To achieve fast 3D images, the researchers developed a custom image – divisible prism that allows concurrent recording of a stack of eight displaced z images. This indicates that the microscope can perform high-speed 3D phase imaging on a volume with dimensions of 2.5 μm & # 39; 50 μm & # 39; 50 μm. The speed of the microscope is fundamentally restricted by the speed of your camera. For this illustration, the researchers were able to obtain images of the intracellular dynamics at almost 200 Hz.
" With the prism as a complement, you can turn a classical microscope into an ultra-fast 3D imager ," Kristin said. Grussmayer, other main authors of the study.
The prism can also be used for 3D fluorescence imaging, which was researched by researchers by adopting super-resolution optical fluctuation (SOFI) images. This technique takes advantage of the flicker of the fluorescent dyes to improve the 3D resolution by analyzing the correlation of the signal. By adopting this, the team carried out 3D images of superresolution of stained structures in the cells, and integrated them with 3D phase images without labels. The two methods complemented each other quite well, exhibiting intriguing images of the intrinsic structure, the cytoskeleton and the organelles in living cells at distinctive temporal points.  " We are delighted with these results and the possibilities offered by this technique " declared Professor Hilal Lashuel, whose laboratory at EPFL collaborated with Professor Lasser's laboratory by adopting the innovative method to investigate the mechanisms by which the aggregation of proteins plays a vital role in the development and progress of neurodegenerative diseases, for example, Alzheimer's and Parkinson's. " Technical advances allowed high-resolution visualization of the formation of pathological aggregates of alpha-synuclein in hippocampal neurons ."
Researchers have called the innovative microscopy platform PRISM, which means Phase Recovery Instrument with super resolution microscopy.
We offer PRISM as a new microscopy tool and anticipate that it will be used rapidly in the life sciences community to expand the reach of high-speed 3D imaging for biological investigations. We hope it becomes a regular workhorse for neuroscience and biology .
This study was funded by the European Union (Horizon 2020, Marie Skłodowska-Curie Grant Agreement and AD-gut European Consortium) and the Swiss National Science Foundation (SNSF).
A. Descloux, K. S. Grußmayer, E. Bostan, T. Lukes, A. Bouwens, A. Sharipov, S. Geissbuehler, A-L. Mahul-Mellier, H. A. Lashuel, M. Leutenegger, T. Lasser. Combined phase recovery of multiple planes and generation of superresolution optical fluctuation images for 4D cell microscopy. Nature Photonics 12, 165-172 (February 26, 2018). DOI: 10.1038 / s41566-018-0109-4