Archives des Super resolution - mu-Imagine https://mu-imagine.com/category/news/super-resolution/ Adaptive optics, adapted to microscopy Mon, 02 Sep 2024 15:18:22 +0000 en-US hourly 1 https://wordpress.org/?v=6.7.1 https://mu-imagine.com/wp-content/uploads/2023/03/cropped-Logo-muImagine-32x32.png Archives des Super resolution - mu-Imagine https://mu-imagine.com/category/news/super-resolution/ 32 32 Studying brain function with Super-Resolution Microscopy https://mu-imagine.com/studying-brain-function-with-super-resolution-microscopy/ Thu, 18 Jul 2024 14:20:44 +0000 https://mu-imagine.com/?p=1056 Understanding brain function, particularly at the level of neuronal synapses, is a complex and crucial endeavor in neuroscience. Neuronal synapses are where many neurological processes occur, including signal transmission and plasticity, which underlie learning and memory. Imaging these tiny structures...

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Studying brain function with Super-Resolution Microscopy
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Advancing synaptic imaging: the Blanpied Lab’s approach

Understanding brain function, particularly at the level of neuronal synapses, is a complex and crucial endeavor in neuroscience.
Neuronal synapses are where many neurological processes occur, including signal transmission and plasticity, which underlie learning and memory. Imaging these tiny structures presents significant challenges due to their minuscule size, often requiring advanced microscopy techniques for adequate resolution.

Example of Mirao 52e implementation on a super-resolution microscope

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.

 

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Adaptive optics enhances image quality in white-light confocal microscopy https://mu-imagine.com/adaptive-optics-enhances-image-quality-in-white-light-confocal-microscopy/ Wed, 22 May 2024 14:51:46 +0000 https://mu-imagine.com/?p=931 In their paper published in Journal of Microscopy, researchers from Oxford's adaptive optics group, led by Prof. Martin Booth, describe an implementation of Adaptive Optics (AO) into Aurox Clarity optical setup. This original, laser-free, spinning disk microscope enables the use of incoherent sources for illumination...

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Adaptive optics enhances image quality in white-light confocal microscopy
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Addressing aberrations in high-resolution microscopy

In the paper published in Journal of Microscopy, researchers from Oxford’s adaptive optics group, led by Prof. Martin Booth, describe an implementation of Adaptive Optics (AO) into Aurox Clarity optical setup.
Clarity system is an original, laser-free, spinning disk microscope, which enables the use of incoherent sources for illumination. In this method, optical sectioning is achieved using a patterned disk which is spinning in both illumination and imaging paths.

Example of Mirao 52e implementation on a super-resolution microscope

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.

 

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Case study: revealing secrets of Nuclear Pore Complex (NPC) disassembly during cellular mitosis thanks to SMLM & AO combination https://mu-imagine.com/case-study-revealing-secrets-of-nuclear-pore-complex-npc-disassembly-during-cellular-mitosis-thanks-to-smlm-ao-combination/ Tue, 10 Oct 2023 09:21:36 +0000 https://mu-imagine.com/?p=679 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...

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Case study: revealing secrets of Nuclear Pore Complex (NPC) disassembly during cellular mitosis thanks to SMLM & AO combination
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Nuclear pore complexes scheme plus images (obtained with dSTORM technique)

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!

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Variable astigmatism allows fine-tuning the 3D localization precision in PALM/STORM https://mu-imagine.com/variable-astigmatism-allows-fine-tuning-the-3d-localization-precision-in-palm-storm/ Tue, 09 May 2023 13:13:23 +0000 https://mu-imagine.com/?p=581 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)...

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Variable astigmatism allows fine-tuning the 3D localization precision in PALM/STORM
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Astigmatism on beads at different depth

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.

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