ASTER

Uniform and Versatile excitation for smart, quantitative and large content Microscopies

Use of lasers in standard Wide-Field and Single Molecule Localization Microscopies (SMLM) causes inhomogeneities along the field of view, provides fixed illumination shapes and prevents quantitative sample analysis. Moreover, when focalized in the back focal plane (BFP) of the objective, a laser can be shifted to provide oblique and Total Internal Reflection Fluorescence (TIRF) optical sectioning but this scheme is incompatible with the most state of the art uniform illumination setups and will produce severe speckle artefacts.

We present a novel and highly versatile illumination scheme named Adaptative Scanning for Tuneable Excitation Regions (ASTER). ASTER provides uniform excitation, optical sectioning and resulting image quality on any field of view (FOV) sizes at high temporal resolution. Because of its high vertsatility it can adapt to numerous samples and balance the blinking or emission properties of fluorophores.

At custom laser powers, ASTER directly provides 150*150µm² super resolution images, it can also perform fast acquisitions (<2min ) on small fields, paving the way to a confident, high-throughput and quantitative data analysis

Inhomogeneities in Wide-field setups

Classical Fluorescence and SMLM are Wide-field setups, where the excitation beam is collimated at the sample plane and focused in the Back Focal Plane of the objective. Lasers are efficient excitation sources due to their high coherence, wavelength selectivity, and power concentration but they are gaussian-shaped and cause filed-dependant fluorophores responses. They also make poor use of their available power and their shape is ill-matched to image stitching schemes and square detectors cameras.

ASTER PRINCIPLE

ASTER makes use of two galvanometer mirrors to control illumination at the sample plane. While the excitation beam keeps its position in the BFP, an angular rotation of a galvanometer induces a similar angle in the BFP, corresponding to a different position in the sample plane.

By applying specific patterns such as a raster-scanning we can provide uniform excitations on tuneable FOV sizes and acquire images at up to 500fps.

In SMLM, ASTER can adapt to any sample size and blinking properties: magnifying the illumination size will provide wide uniform blinking areas at low fluorophore density while reducting the illumination area will provide faster blinking and allow imaging of high-density area by preliminary sending most fluorophores in the dark state. In classical, fluorescence, photobleaching can be controled by adjusting local irradiance.

2D to 3D imaging on 150×150µm²!

ASTER and optical sectioning

Classical uniform excitations setups fails to provide appreciable TIRF because they modify the beam in its core, hampering its focusing in the Back Focal Plane. As ASTER is scanning a beam that is still, at any instant, gaussian, it can perform great TIRF optical sectioning, with less interference pattern than a gaussian excitation thanks to the scanning effect.Implementation of TIRF and oblique illumination in classical and ASTER excitation schemes.

Wide uniform imaging applications and analysis

The large FOV provided by ASTER illumination coupled with large-chip sCMOS cameras also have interesting applications for imaging neuronal cells, which grow axons over hundreds of microns in culture. Traditionally, SMLM imaging of axons has been limited to <50 µm segments of axons, impeding the visualization of rare structures and the definition of their large-scale organization. A 150 µm x 150 µm FOV allowed visualizing the dendrites and cell body of two neurons, and a large number of long axonal segments. The zoomed views confirm the quality and resolution of the resulting image: the periodic 190 nm organization of axonal spectrin is clearly visible, as confirmed by the corresponding Fourier transform of the images. The Fourier transform of the whole image exhibits a sharp ring at the corresponding frequency, because the banded pattern of β2-spectrins appears in axons running in all directions. On the zoomed images, the β2-spectrin along axons in one direction results in a direction-dependent frequency band on the Fourier transform, corresponding to the 190 nm spacing.

ASTER’s complete uniformity allow confident analysis on uprecedented scales. Here with the example of clathrin in SMLM, image of a 140*140µm² FOV containing multiple cells.

We then detect and characterize individual clathrins to distinguish different existing populations. these can depend on numerous clathrin parameters(radius, size, holowness, number of localization …). 20 000 clathrins were imaged at once while a custom 30*30µm² field of view would have imaged 800, with approximative uniformity.

Comparison

Qualitative comparison of ASTER and other uniformization technologies. ASTER synthetizes an excitation over time, thus it performs at the same frame rate (~1kHz) that technologies using slow diffusers. Otherwise ASTER has remarkable abilities in all other categories.

Webinar

Versatile wide field uniform illumination scheme, from classical fluorescence microscopy to single molecule localization microscopy

Adrien Mau, PhD

Paper

Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

Nature Communication: Adrien Mau, Karoline Friedl, Christophe Leterrier, Nicolas Bourg & Sandrine Lévêque-Fort