As a new step towards better imaging solutions incorporating adaptive optics (AO), we recently demonstrated, in collaboration with several research groups from CNRS, a fast aberration correction method, designed as an AO module compatible with light-sheet fluorescence microscopy (LSFM) for enhanced neuroimaging of live samples.

Focusing on the Neuroscientific Challenge

In the dynamic realm of neuroscience, understanding the brain’s functional organization and connectivity poses a great challenge.
Indeed, to be able to unveil mechanisms behind neurodegenerative diseases like Alzheimer’s and Parkinson’s, demands advanced imaging techniques allowing to map the brain activity with high spatio-temporal resolution at large depths.
Such requirements led to the development of new 3D imaging strategies such as light-sheet fluorescence microscopy (LSFM).
Even though the technique is perfectly suited for live imaging (thanks to great spatio-temporal capabilities and minimal phototoxicity), LSFM still encounters limited imaging depth capability due to strong optical aberrations compromising signal-to-background ratio (SBR).

Overcoming Challenges and Pushing Boundaries thanks to AO-LSFM combination

In response to this limitation, adaptive optics (AO) emerges as a game-changing technology when integrated into LSFM setups to counteract optical aberrations.
Recently, our research consortium highlighted the transformative impact of AO on LSFM, employing direct wavefront sensing with an extended-scene Shack-Hartmann (ESSH) wavefront sensor (link here). As a proof of concept, we were able to show significant improvement of image quality in-depth with structural imaging of ex-vivo Drosophila brain.
In this new study, an enhanced AO-LSFM set-up which does not require any guide star and is compatible with calcium neuroimaging is demonstrated.
Using dual labelling in the Drosophila brain, a strongly scattering sample :
– Structural and calcium signal images down to 80 and 40 μm were reported respectively, with an increased contrast of at least a factor of 2.
– Fast AO correction at a speed up to 10 Hz was demonstrated, positioning the approach as a game-changer in 3D imaging and dynamic process observation in depth in scattering samples.

Next steps and Future Frontiers

The instrumental approach designed by the team offers a compact and versatile prototype for integration into various LSFM setups. Ongoing testing on different configurations reveals the potential for a commercial AO add-on.
Researchers now delve into new challenges such as light-sheet thickness and excitation optimization, showcasing the continuous evolution of AO-LSFM technology for even greater image quality and sensitivity.
In conclusion, with its potential to unlock the mysteries of the brain, AO-LSFM is one of the technologies standing at the forefront of neuroimaging innovation. To address current & future challenges in this promising field, we are dedicated to developing tailored adaptive optics solutions to propel research forward.

You have questions regarding adaptive optics in microscopy? Do not hesitate to contact us!

In their quest to better understand the disassembly of NPCs during mitosis, the team of Prof. Ulrike Kutay at Zurich ETH decided to dig deeper into Y-complex behaviour.

Why observe Y-complexes?

During mitotic entry, the dismantling of NPCs seems to be a two-tier process and if disassembly of most components occurs rapidly, Y-complex Nups and cytoplasmic rings display slower dissociation kinetics, indicating a mechanistic difference in their splitting. To understand this phenomenon better, researchers needed to image and compare the structure of these Y-complexes before (images b, c and d) and during mitosis (images e, f and g).

The challenge here? Observing such small cellular elements requires sufficient optical resolution, going below 20nm laterally and 50nm axially. This is where single molecule localization microscopy (SMLM) in a combination with adaptive optics (AO) comes into play.

Single Molecule Localization Microscopy for a better lateral resolution

First, let’s see what SMLM can bring. This group of imaging techniques enable scientists to breach the conventional diffraction limit, allowing them to visualize structures at the nanoscale. The diverse methods that are regrouped under the umbrella term SMLM are using the properties of some fluorophores to switch from dark to emissive state and vice-versa. In this case, Prof. Kutay team used dSTORM, a method allowing to capture details on the order of tens of nanometres – a sufficient resolution to observe Y-complexes in both interphasic (image b) and mitotic (image g) states in lateral dimension. But what about 3D?

Playing with astigmatism to get an axial dimension

While SMLM offers remarkable lateral resolution, understanding the complete three-dimensional architecture of Y-complexes requires tackling the axial dimension. One of ways to address this question is the shaping of point spread function (PSF) with astigmatism using adaptive optics. With this method, researchers were able to reach localization precision around 5-10nm and obtained 3D images of Y-complexes in their ring shape (during interphase, images c and d) and in their ER-bound shape (during mitosis, images e and f).

How can research benefit from adaptive optics?

In conclusion, boosting your SMLM system with adaptive optics will help you to see new details in cellular structures, complexes and molecules, just as Prof. Kutay group did (read their full article here).

But how to implement adaptive optics? Well, in their paper, researchers were using MicAO 3DSR device. This adaptive optics module, which is simply inserted between the microscope and imaging camera, can indeed enable you to perform 3D SMLM using astigmatism as mentioned above. If adaptive optics are a real game changer for 3D super-resolution microscopy, it is also a very powerful tool for 2D. Indeed, MicAO 3DSR can correct sample-induced aberrations and help increasing photon collection, resulting in increase of localization precision that, in the end, will allow you to obtain the highest quality super resolved 3D structures.

You have questions regarding adaptive optics in microscopy? Do not hesitate to contact us!

One of the interests of Siegfried Musser group from Texas AM University is to study molecular transport via Nuclear Pore Complexes (NPCs) across the nuclear membrane inside the cell. As a key tool in their research, scientists are using Single Molecule Localization Microscopy (SMLM) boosted by Adaptive Optics (AO), which provides increased resolution both in time and space (localization precision).

Recently, a second paper was published on this topic, providing a systematic analysis of the influence of some key imaging parameters – including AO – on the quality of SMLM images. Since the Point Spread Function (PSF) is symmetrical along the Z axis, SMLM techniques intrinsically are two-dimensional and provide information only in lateral dimension.

To break the axial symmetry and to retrieve the 3D information, several PSF shaping methods are currently available. To name a few, one can use astigmatism, double-helix, or tetrapod approaches.

Astigmatism is probably the simplest and most efficient PSF shaping method, allowing to systematically reach localization precisions around 5-10nm @4000 arriving photons (a typical amount in real-life situation). Astigmatic PSF can be obtained by simply adding a cylindrical lens to the emission pathway of the microscope. But the amplitude of astigmatism in this case is fixed. Another way to induce astigmatism is by using a deformable mirror, which enables to correct aberrations and to introduce pure astigmatism with an amplitude that can be instantaneously varied over a large range.

In this paper, scientists carefully evaluated the following factors influencing both lateral and axial localization precision: the number of incident photons, the amplitude of induced astigmatism and even the type of acquisition camera (EMCCD and sCMOS). As for AO, it was shown that by changing the amount of induced astigmatism it is possible to fine tune the ratio of lateral and axial localization precision. A homogeneous localization precision is obtained when using 90nm RMS of astigmatism on the deformable mirror: it allows reaching localization precision of about 10nm in all three dimensions at 3000 incident photons.

In this study scientists used AOKit Bio product bundle, which is composed of Mirao 52E deformable mirror, HASO4 First wavefront sensor for mirror calibration, and an adaptive optics software including a 3N image-based sensorless aberration correction process. The implementation design of AOKit Bio is described in this paper to a great detail. For easier, faster integration of the same AO approach, our add-on device MicAO 3DSR can be used, enabling easy implementation on any inverted frame microscope.