- Researchers develop a new technique (PRISM) that can observe beyond the diffraction of light.
- It can capture exceptional views inside living cells through super-resolution spatial and temporal imaging.
For the last couple of decades, we’ve been using wide-field fluorescence imaging methods, like PALM and STORM, to observe subcellular structures. These methods require hundreds and thousands of raw images in long sequences. Thus, increasing spatial resolution decreases time resolution.
Now, Researchers at EPFL (research institution in Lausanne, Switzerland) have designed a system that can capture exceptional views inside living cells through super-resolution spatial and temporal imaging (both in space and time).
The new microscopy platform, named PRISM (Phase Retrieval Instrument with Super-resolution Microscopy), can observe beyond the diffraction of light. It integrates 3-dimensional microscopy and a novel technique for white light 3-dimensional phase retrieval.
It combines the spatial resolution of fluorescence super-resolution and molecular specificity with the high-speed and sensitive quantitative phase imaging, enabling multimodal 4-dimensional imaging.
How 4D Cell Microscopy Works?
Researchers used Fourier filtering to extract 3D quantitative phase from an array of white-light images. Then they explained this bright-field depth-resolved phase imaging through 3D partially coherent image formation.
They demonstrated their concept by retrieving the high-resolution 3D phase data of stable cells from a big stack of z-displaced intensities. They developed PRISM for concurrent acquisition of 8 planes – it can perform high speed 3D phase imaging across 2.5*50*50-micrometer of volume. Finally, they sequentially imaged samples of cells with 3D with phase microscopy and super-resolution optical fluctuation imaging (SOFI).
SOFI supports 3D imaging of live cell with nearly one second per reconstructed picture of time resolution in a multi-plane microscope. Furthermore, it offers a quantitative assessment of molecular parameters, and tolerates high labelling densities.
In simple language, PRISM is an add-on to current wide-field microscopy platforms, which enables simple and fast implementation of 3D fluorescence super resolution imaging and 3D quantitative phase. Bottom line, the new system presents a better opportunity to observe the temporal physiology and complex spatial of live cells.
A cell observed with the PRISM technique | Credit: T. Lasser / EPFL
Based on the Helmholtz wave equation, the technique is embedded in the Wiener–Khintchine theorem’s framework. The process of decoding phase data along the z-axis is done by calculating the forward weak scattering interference.
They built an efficient algorithm to recover the 3D phase data from an acquired volumetric intensity stack. The high detection numerical aperture and white light Koehler illumination provide speckle-free high-resolution and stable quantitative phase imaging of live cells. Moreover, the simulations verified an axial resolution of 350*560 nanometers.
Overall, the procedure enables to upgrade a conventional bright-field microscope into a simple, fast and reliable 3D phase microscope, which is supposed to fulfill the expectations for numerous investigations and applications in life sciences and biology.