Adaptive Optics enables deep imaging in 2-photon super-resolution structured illumination microscopy

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However, accessing such visualization at large depths is impaired by optical aberrations arising from tissue inhomogeneity, which limits both achievable image contrast and depth.

 

Adaptive Optics (AO) for improved image contrast, resolution and depth in 2P-MSIM

In their paper recently published in Photonix, scientists from research groups Shenzhen University implemented AO at both excitation and detection paths. To enable wavefront (WF) sensing, a non-linear guide star was generated using a Spatial Light Modulator (SLM), the corresponding fluorescence signal from a tissue structure such as e.g. a neuron body was diverted to a Shack-Hartmann wavefront sensor.
Based on the WF measurement, both the multifocal excitation pattern generated by the SLM and the shape of a Deformable Mirror (DM) in the fluorescence imaging path were adjusted to compensate for measured aberrations. When applied e.g. to deep imaging of dendritic structures in fixed mouse brain slices, the approach enabled a x5 increase in fluorescence signal a significant depths >300 µm, with a maintained 150nm resolution.

 

Key components for AO implementation

AO was implemented based on selected components from our AOKit BIO, namely Mirao 52e as the DM and as a Shack-Hartmann WF sensor. This combination, together with our software solutions, provide an efficient combination of WF sensing accuracy and WF modulation linearity and stroke. Alternatives providing even better linearity, more actuators, or an integrated solution such as an AO module can be found in our product range.

Studying brain function with Super-Resolution Microscopy

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The lab of Thomas Blanpied at the University of Maryland School of Medicine is at the forefront of this research. They employ a variety of cutting-edge microscopy methods to explore the intricacies of synaptic active zones and the functions of the postsynaptic density.
In particular, they are using PALM (Photoactivated Localization Microscopy) and STORM (Stochastic Optical Reconstruction Microscopy) single-molecule super-resolution microscopy techniques to overcome the diffraction limit of conventional light microscopy and visualize neuronal structures at the nanometer scale.
Both techniques rely on the precise localization of single molecules that are activated and deactivated stochastically. By compiling the positions of these molecules, researchers can construct a super-resolved image.

 

Improving image resolution: the role of Adaptive Optics in synaptic imaging

The localization precision of molecule positions in super-resolution microscopy is highly dependent on the number of detected photons and the quality of single-molecule spots, also known as the point spread function (PSF). In neuronal samples, differences in refractive indices between different cellular components can cause optical aberrations. These aberrations distort the PSF, leading to reduced localization precision and image resolution.
To overcome aberrations and restore image resolution, scientists in the Blanpied lab use an adaptive optics device called MicAO 3DSR. This device features a deformable mirror capable of compensating for aberrations, correcting the PSF, and thereby improving the resolution of super-resolved images.

 

How to use advanced imaging techniques, an example:

An example of such imaging was recently published in the Journal of Neuroscience: “Distinct SAP102 and PSD-95 Nano-Organization Defines Multiple Types of Synaptic Scaffold Protein Domains at Single Synapses”. This study investigates the nano-organization of two key synaptic scaffold proteins, SAP102 and PSD-95, within individual synapses.
By employing super-resolution microscopy enhanced with adaptive optics, the researchers were able to:

• Visualize spatial distribution: Precisely map the localization of SAP102 and PSD-95 at single synapses.
• Identify multiple domains: Discover multiple types of scaffold protein domains, indicating a complex and heterogeneous organization within synapses.
• Provide insights into synaptic function: Offer new understanding of how different scaffold protein organizations may contribute to synaptic function and plasticity.

 

Significance and Impact of Blanpied Lab’s work

Integration of adaptive optics with super-resolution microscopy in the Blanpied lab enhances the ability to study the fine details of neuronal synapses. The use of devices like MicAO 3DSR to correct aberrations and improve PSF quality is crucial for advancing our understanding of brain function and dysfunction. These technological advancements enable scientists to:

• Achieve high precision: Overcome challenges posed by refractive index mismatches, resulting in highly precise localization of molecules within synapses.
• Visualize dynamic processes: Capture real-time changes in protein organization, allowing for a deeper understanding of synaptic dynamics and plasticity.
• Inform neurological research: Provide crucial insights into the molecular mechanisms underlying synaptic function and their alterations in disease states.

 

Adaptive optics enhances image quality in white-light confocal microscopy

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Like for other high-resolution microscopy methods, here the imaging performance is also compromised by aberrations due to refractive index mismatch and refractive index variations within the biological sample.
In this paper, researchers demonstrated that correction of such aberrations with AO brings significant improvement of the fluorescence signal, contrast, and image resolution. They studied different samples at various depths and significant enhancement of image quality was observed at every depth inside the sample. In some cases, the fine details that were not visible in the noncorrected version became visible in the corrected one.

 

Implementing AO for improved image quality

To implement AO, researchers detached the Clarity module from the microscope base and created an intermediate pupil plane in which they placed Mirao52e deformable mirror.
For aberration detection, they used one of the sensorless adaptive optics methods which estimates the best correction by detecting sample aberrations from a sequence of images taken when a sequence of predetermined aberrations is applied using deformable mirror.
For each image, an image quality metric was determined and, in this case, researchers used a weighted sum of the power of all spatial frequencies obtained from the image spectrum. This specific metric has been chosen because of the specific image formation process in this microscopy technique. The weighting curve applied on the image spectrum enhances mid-range spatial frequencies, which are the most affected by aberrations (low frequencies vary slightly with aberrations, and high frequencies are largely affected by noise).

 

Exploring AO options

As a key element of the proposed AO method, the wavefront modulator used was a Mirao 52e deformable mirror, based on a set of 52 electromagnetic actuators, and providing large stroke and high linearity, the latter being a key driver of the accuracy of sensorless AO operation.
Alternatives providing even better linearity, more actuators, or an integrated solution such as an AO module can be found in our product range.

 

Boosting Neuroimaging: The Synergy of Adaptive Optics and Light-Sheet Fluorescence Microscopy

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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!

mu-Imagine at Focus On Microscopy 2023

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In the beginning of April we participated at the 2023 edition of Focus On Microscopy. This conference always was and still is a great opportunity to discover latest developments in microscopy & its applications in fields such as biology, medicine, or material sciences. At the exhibitor’s area this year we showcased mu-DM, a new deformable mirror dedicated for microscopy and ophthalmology applications. We also presented a poster describing this product : Modulating phase for adaptive optics and PSF shaping in microscopy : state of the art, requirements & development of a new phase modulator tailored to microscopy needs. F. Harms¹, C. Veilly¹, A. Jasaitis¹, P. Treimany¹, X. Levecq¹ (¹Imagine Optic, Orsay)

We would like to thank our collaborators for their presentations during conference : – A closed-loop adaptive optics two-photon microscope more resilient to scattering for in-vivo neuroimaging. A. Guillaume-Manca¹²³, S. Imperato¹, M. Mercier¹³, R. Lobo¹, F. Harms⁴, L. Bourdieu², A. Fragola³ (¹LPEM, ESPCI, CNRS, Sorbonne Université; ²IBENS, Univ. PSL, INSERM, CNRS; ³ISMO, Université Paris Sud, CNRS; ⁴Imagine Optic, Orsay) – Adaptive Optics Light Sheet Fluorescence Microscopy for High Resolution In Vivo Imaging in Zebrafish. M. Mercier¹², S. Imperato¹, A. Hubert¹, A. Guillaume-Manca¹²³, C. Veilly³, F. Harms³, W. Supatto⁴, A. Fragola² (¹LPEM, ESPCI, CNRS, Sorbonne Université; ²ISMO, CNRS, Université Paris-Saclay ; ³Imagine Optic, Orsay ; ⁴LOB Ecole Polytechnique, Palaiseau) – 3D time-lapse high-resolution imaging in acute brain slices with lattice light-sheet microscopy. A. Getz¹, M. Malivert¹², D. Choquet¹, M. Ducros¹ (¹Univ. Bordeaux, CNRS, INSERM, BIC; ²Imagine Optic, Orsay) We would also like to thank all our visitors for coming to our booth ! For those who missed the mu-DM demo, do not worry, we will organize a webinar soon. Until then, we will gladly answer all your questions or discuss about your microscopy project requiring adaptive optics or wavefront sensing via email. Please don’t hesitate to contact us.