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Super-resolution imaging in vivo

Super-resolution imaging in vivo


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Are there any microscopy modalities or techniques for in vivo imaging at higher resolutions than the diffraction limit?

I was looking at this list: Super resolution microscopy, but they don't talk about in vivo imaging.


There have been experiments with Live-cell dSTORM with SNAP-tag fusion proteins in Markus Sauer's lab in Würzburg. If you are using the physiology definition of in-vivo, I'm pretty sure some of my co-workers have measured on live organisms. However, this is work in progress and not fully published; if you are interested, you should ask Markus Sauer directly.

You can use the dSTORM-like techniques (STORM, dSTORM, GSDIM, RPM, etc.) in live cells because the chemical environment in the cell is favourable and the redox system of the cell works for you. The key problem is to get the fluorophores into the cell, but SNAP-tags can help you to do it.

As one of the co-authors, I can tell you that live cell was some pain, but considerably less than getting 3D localization microscopy to work.

PALM is a different story because you generally have longer acquisition times. STED is difficult: If the cell wasn't dead before, the STED beam will ensure it is, either through the intensity or by unbalancing the chemical environment sufficiently. You might have your image before the cell dies, or maybe not.


Since the question was asked, the field has seen tremendous growth and even a Nobel prize has been awarded for Super-resolution microscopy. Since other answers have already touched upon how STED, PALM, STORM could help, I would talk about Expansion Microscopy (ExM) and how it has been recently used to image intact zebrafish brain tissue and gastrulating zebrafish embryos [Freifeld et al]. Since the optical resolution is limited by the wavelength of light which gets diffracted if the object being imaged is smaller than the wavelength of light (~300 nm). In order to image small object, what if we could enlarge the object itself to cross the diffraction barrier? This has been done by a team of researchers at MIT who imaged mouse brain tissue in 2015 [Chen et al].

Chen et al. have solved this problem by digesting the mouse brain away after transforming it into a polymer gel but before inducing matrix expansion. But how can one study a brain in which all the neurons are gone? Here, a second idea came into play: For some questions, it would be enough to study a “shadow” of the brain in the polymer matrix. This was realized by first marking molecules of interest in neurons with antibodies. The antibodies then were labeled with fluorescent markers that bind both to the antibodies and the matrix. The brain was then digested away and the matrix expanded. The fluorescent neuronal shadows also expanded and could be studied in “superresolution.” Chen et al. could thus increase specimen size by about a factor of 5 while preserving general morphology, and could visualize cultured neurons and brain slices at 70-nm resolution. This allowed them to observe proteins localized to synapses.

The best part is that conventional confocal microscopes can be used to image these enlarged samples, so the imaging speed is also good.

  • Freifeld, L., Odstrcil, I., Förster, D., Ramirez, A., Gagnon, J. A., Randlett, O.,… & Martin-Alarcon, D. A. (2017). Expansion microscopy of zebrafish for neuroscience and developmental biology studies. Proceedings of the National Academy of Sciences, 114(50), E10799-E10808.
  • Chen, F., Tillberg, P. W., & Boyden, E. S. (2015). Expansion microscopy. Science, 347(6221), 543-548.

Any superresolution techniques relying on stochastic sampling (e.g. PALM, STORM) is extremely difficult to accomplish in vivo, as the tissue would be moving (e.g. due to respiration) and this would hinder a lot the reconstruction of the image.

Apparently it may be possible to do it with STED but I could not find the related paper*, although the same authors recently published some interesting work on living brain slices.

* Note that the images in that article come from this paper, which is not done in vivo.

Thanks to @vkehayas for pointing out that in vivo STED has been done for instance here Nanoscopy in a living mouse brain


Yes. A decade ago I've been working on a software for microscope that does what you want.

Here's how the product looks after 12 years of development.

It's optical, however it produces Z map instead if image, and has resolution is order of magnitudes below the diffraction limit: http://www.amphoralabs.com/production/10404/10708


Super-resolution imaging breakthrough in living cells

Edinburgh scientists have developed a new imaging technique that reveals the inner workings of living cells in stunning detail and could pave the way to a better understanding of many diseases.

The new super-resolution imaging technique - LIVE-PAINT- provides a flexible and powerful way of tracking individual proteins inside living cells, without disrupting their activity.

The game-changing advance could lead to new insights into diseases by revealing the behaviour of proteins involved in disease processes and those essential to health.


Abstract

We have developed a method for super-resolution ultrasound imaging, which relies on a new class of blinking nanometer-size contrast agents: laser-activated nanodroplets (LANDs). The LANDs can be repeatedly optically triggered to undergo vaporization the resulting spatially stationary, temporally transient microbubbles provide high ultrasound contrast for several to hundreds of milliseconds before recondensing to their native liquid nanodroplet state. By capturing high frame rate ultrasound images of blinking LANDs, we demonstrate the ability to detect individual recondensation events. Then we apply a newly developed super-resolution image processing algorithm to localize the LAND positions in vivo almost an order of magnitude better than conventional ultrasound imaging. These results pave the way for high resolution molecular imaging deep in tissue.


In Vivo Imaging

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The IVIS Lumina S5 and X5 in vivo imaging systems: It’s never been easier or faster to get robust data—and answers—on anatomical and molecular aspects of disease.

For research use only. Not for use in diagnostic procedures.

The More You Image, The More You Understand With The Quantum GX2 MicroCT System

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Quantum GX2 microCT imaging - flexibility and performance you need to further understand your disease models.

Whether you're trying to better understand biology pathways, monitor disease progression, or evaluate potential drug candidates earlier in the development process, our portfolio of preclinical in vivo imaging solutions from standalone optical and microCT imaging to integrated systems are designed to meet your needs.

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For research use only. Not for use in diagnostic procedures.

See Disease In All It’s Dimensions with Multimodality Imaging

It’s simple – for today’s researchers there’s an increasing need for in vivo imaging that enables you to visualize multiple events simultaneously and to extract the maximum amount of information from each subject – leading to greater biological understanding.

At PerkinElmer, we offer systems with integrated x-ray or microCT facilitating the quickest workflow for data acquisition and analysis. Furthermore, you can co-register data from your single-mode optical or microCT platform using our Living Image ® Software to provide you with more insight into molecular and anatomical features of disease.


In Vivo Preclinical Imaging Solutions

Tracking, monitoring, and visualizing biological processes and disease progression is key to not only helping you better understand biology, but also crucial in evaluating the effectiveness of your potential drug candidates earlier in the development process.

In vivo imaging has a profound role in advancing researchers understanding of molecular and physiological research across a broad range of disease models as well as accelerating preclinical development of therapeutics in a non-invasive manner and in real-time.

Let us help you achieve your research and discovery goals with our best-in-class small animal imaging solutions. From basic research models to clinically translatable applications, our in vivo imaging community publishes on a daily basis with thousands of papers published in peer reviewed journals, covering major disease areas including cancer, infectious disease, cardiopulmonary, metabolic disease and more.

Whatever answer you’re seeking, in vivo preclinical imaging is the most direct path in helping you gain deeper insight into your basic research and drug discovery & development projects.

Explore our in vivo imaging solutions.

The more you see – the more you know

Your cutting edge research commands high-sensitivity and reliable imaging data. As a leader in optical imaging we have the tools you need, from 2D optical, to 3D tomography and integrated optical imaging systems, to optical reagents to help you reach your research goals.

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Stunning First 3D Super-Resolution Images Captured Inside Living Mice

Researchers developed a 3D-2PE-STED system that can image dendritic spines deep inside the brain of a living mouse. Their system showed subtle changes that occurred between day 1 and 3 (left images). These changes are hard to distinguish using two-photon microscopy alone (right). Credit: Joerg Bewersdorf, Yale School of Medicine

New STED technique enables deep-tissue imaging, reveals subcellular dynamics of neurons.

Researchers have developed a new microscopy technique that can acquire 3D super-resolution images of subcellular structures from about 100 microns deep inside biological tissue, including the brain. By giving scientists a deeper view into the brain, the method could help reveal subtle changes that occur in neurons over time, during learning, or as result of disease.

The new approach is an extension of stimulated emission depletion (STED) microscopy, a breakthrough technique that achieves nanoscale resolution by overcoming the traditional diffraction limit of optical microscopes. Stefan Hell won the 2014 Nobel Prize in Chemistry for developing this super-resolution imaging technique.

In Optica, The Optical Society’s (OSA) journal for high impact research, the researchers describe how they used their new STED microscope to image, in super-resolution, the 3D structure of dendritic spines deep inside the brain of a living mouse. Dendric spines are tiny protrusions on the dendritic branches of neurons, which receive synaptic inputs from neighboring neurons. They play a crucial role in neuronal activity.

“Our microscope is the first instrument in the world to achieve 3D STED super-resolution deep inside a living animal,” said leader of the research team Joerg Bewersdorf from Yale School of Medicine. “Such advances in deep-tissue imaging technology will allow researchers to directly visualize subcellular structures and dynamics in their native tissue environment,” said Bewersdorf. “The ability to study cellular behavior in this way is critical to gaining a comprehensive understanding of biological phenomena for biomedical research as well as for pharmaceutical development.”


Researchers used their 3D-2PE-STED microscope to image the brain of a living mouse. Zooming in on part of a dendrite reveals the 3D structure of an individual spine. Credit: Joerg Bewersdorf, Yale School of Medicine

Going deeper

Conventional STED microscopy is most often used to image cultured cell specimens. Using the technique to image thick tissue or living animals is a lot more challenging, especially when the super-resolution benefits of STED are extended to the third dimension for 3D-STED. This limitation occurs because thick and optically dense tissue prevents light from penetrating deeply and from focusing properly, thus impairing the super-resolution capabilities of the STED microscope.

To overcome this challenge, the researchers combined STED microscopy with two-photon excitation (2PE) and adaptive optics. 𔄚PE enables imaging deeper in tissue by using near-infrared wavelengths rather than visible light,” said Mary Grace M. Velasco, first author of the paper. “Infrared light is less susceptible to scattering and, therefore, is better able to penetrate deep into the tissue.”

The researchers also added adaptive optics to their system. “The use of adaptive optics corrects distortions to the shape of light, i.e., the optical aberrations, that arise when imaging in and through tissue,” said Velasco. “During imaging, the adaptive element modifies the light wavefront in the exact opposite way that the tissue in the specimen does. The aberrations from the adaptive element, therefore, cancel out the aberrations from the tissue, creating ideal imaging conditions that allow the STED super-resolution capabilities to be recovered in all three dimensions.”

Seeing changes in the brain

The researchers tested their 3D-2PE-STED technique by first imaging well-characterized structures in cultured cells on a cover slip. Compared to using 2PE alone, 3D-2PE-STED resolved volumes more than 10 times smaller. They also showed that their microscope could resolve the distribution of DNA in the nucleus of mouse skin cells much better than a conventional two-photon microscope.

After these tests, the researchers used their 3D-2PE-STED microscope to image the brain of a living mouse. They zoomed-in on part of a dendrite and resolved the 3D structure of individual spines. They then imaged the same area two days later and showed that the spine structure had indeed changed during this time. The researchers did not observe any changes in the structure of the neurons in their images or in the mice’s behavior that would indicate damage from the imaging. However, they do plan to study this further.

“Dendritic spines are so small that without super-resolution it is difficult to visualize their exact 3D shape, let alone any changes to this shape over time,” said Velasco. 𔄛D-2PE-STED now provides the means to observe these changes and to do so not only in the superficial layers of the brain, but also deeper inside, where more of the interesting connections happen.”

Reference: 𔄛D super-resolution deep-tissue imaging in living mice” by Mary Grace M. Velasco, Mengyang Zhang, Jacopo Antonello, Peng Yuan, Edward S. Allgeyer, Dennis May, Ons M’Saad, Phylicia Kidd, Andrew E. S. Barentine, Valentina Greco, Jaime Grutzendler, Martin J. Booth and Joerg Bewersdorf, 25 March 2021, Optica.
DOI: 10.1364/OPTICA.416841


Recent advances in super-resolution fluorescence imaging and its applications in biology

Fluorescence microscopy has become an essential tool for biological research because it can be minimally invasive, acquire data rapidly, and target molecules of interest with specific labeling strategies. However, the diffraction-limited spatial resolution, which is classically limited to about 200 nm in the lateral direction and about 500 nm in the axial direction, hampers its application to identify delicate details of subcellular structure. Extensive efforts have been made to break diffraction limit for obtaining high-resolution imaging of a biological specimen. Various methods capable of obtaining super-resolution images with a resolution of tens of nanometers are currently available. These super-resolution techniques can be generally divided into three primary classes: (1) patterned illumination-based super-resolution imaging, which employs spatially and temporally modulated illumination light to reconstruct sub-diffraction structures (2) single-molecule localization-based super-resolution imaging, which localizes the profile center of each individual fluorophore at subdiffraction precision (3) bleaching/blinking-based super-resolution imaging. These super-resolution techniques have been utilized in different biological fields and provide novel insights into several new aspects of life science. Given unique technical merits and commercial availability of super-resolution fluorescence microscope, increasing applications of this powerful technique in life science can be expected.

Keywords: Bio-imaging FPALM Fluorescence microscopy NSOM Optical diffraction limit PALM RESOLFT SSIM STED STORM Super-resolution TIRF fluorescence photoactivation localization microscopy near-field scanning optical microscopy photoactivated localization microscopy reversible saturable optically linear fluorescence transitions saturated structured-illumination microscopy stimulated emission depletion stochastic optical reconstruction microscopy total internal reflection fluorescence microscopy.


Imaging (super-resolution)

John Klingenstein Professor of Neuroscience and Professor of Cell Biology Investigator, Howard Hughes Medical Institute Chair, Department of Neuroscience Director, Kavli Institute for Neuroscience and Program in Cellular Neuroscience, Neurodegeneration and Repair (CNNR)

Lucille P. Markey Professor of Microbial Pathogenesis and Professor of Cell Biology Chair, Department of Microbial Pathogenesis

Fergus F. Wallace Professor of Genetics Chair, Genetics

Carolyn Walch Slayman Professor of Genetics

Associate Professor of Genetics and Neuroscience Director of Graduate Studies, Genetics

Sterling Professor of Genetics and Professor of Pediatrics Investigator HHMI

Professor of Molecular, Cellular, and Developmental Biology Co-Director, Yale Interdepartmental Neuroscience Program

Professor with Tenure of Pediatrics (Critical Care Medicine)

Associate Professor Tenure Associate Professor of Microbial Pathogenesis

Associate Professor of Cell Biology

Associate Professor of Microbial Pathogenesis and of Immunobiology Member, Yale Systems Biology Institute Investigator, Howard Hughes Medical Institute


Researchers capture first 3D super-resolution images in living mice

Researchers developed a 3D-2PE-STED system that can image dendritic spines deep inside the brain of a living mouse. Their system showed subtle changes that occurred between day 1 and 3 (left images). These changes are hard to distinguish using two-photon microscopy alone (right). Credit: Joerg Bewersdorf, Yale School of Medicine

Researchers have developed a new microscopy technique that can acquire 3-D super-resolution images of subcellular structures from about 100 microns deep inside biological tissue, including the brain. By giving scientists a deeper view into the brain, the method could help reveal subtle changes that occur in neurons over time, during learning, or as result of disease.

The new approach is an extension of stimulated emission depletion (STED) microscopy, a breakthrough technique that achieves nanoscale resolution by overcoming the traditional diffraction limit of optical microscopes. Stefan Hell won the 2014 Nobel Prize in Chemistry for developing this super-resolution imaging technique.

In Optica, the researchers describe how they used their new STED microscope to image, in super-resolution, the 3-D structure of dendritic spines deep inside the brain of a living mouse. Dendric spines are tiny protrusions on the dendritic branches of neurons, which receive synaptic inputs from neighboring neurons. They play a crucial role in neuronal activity.

"Our microscope is the first instrument in the world to achieve 3-D STED super-resolution deep inside a living animal," said leader of the research team Joerg Bewersdorf from Yale School of Medicine. "Such advances in deep-tissue imaging technology will allow researchers to directly visualize subcellular structures and dynamics in their native tissue environment," said Bewersdorf. "The ability to study cellular behavior in this way is critical to gaining a comprehensive understanding of biological phenomena for biomedical research as well as for pharmaceutical development."

Conventional STED microscopy is most often used to image cultured cell specimens. Using the technique to image thick tissue or living animals is a lot more challenging, especially when the super-resolution benefits of STED are extended to the third dimension for 3-D-STED. This limitation occurs because thick and optically dense tissue prevents light from penetrating deeply and from focusing properly, thus impairing the super-resolution capabilities of the STED microscope.

Researchers used their 3D-2PE-STED microscope to image the brain of a living mouse. Zooming in on part of a dendrite reveals the 3D structure of an individual spine. Credit: Joerg Bewersdorf, Yale School of Medicine

To overcome this challenge, the researchers combined STED microscopy with two-photon excitation (2PE) and adaptive optics. "2PE enables imaging deeper in tissue by using near-infrared wavelengths rather than visible light," said Mary Grace M. Velasco, first author of the paper. "Infrared light is less susceptible to scattering and, therefore, is better able to penetrate deep into the tissue."

The researchers also added adaptive optics to their system. "The use of adaptive optics corrects distortions to the shape of light, i.e., the optical aberrations, that arise when imaging in and through tissue," said Velasco. "During imaging, the adaptive element modifies the light wavefront in the exact opposite way that the tissue in the specimen does. The aberrations from the adaptive element, therefore, cancel out the aberrations from the tissue, creating ideal imaging conditions that allow the STED super-resolution capabilities to be recovered in all three dimensions."

Seeing changes in the brain

The researchers tested their 3-D-2PE-STED technique by first imaging well-characterized structures in cultured cells on a cover slip. Compared to using 2PE alone, 3-D-2PE-STED resolved volumes more than 10 times smaller. They also showed that their microscope could resolve the distribution of DNA in the nucleus of mouse skin cells much better than a conventional two-photon microscope.

After these tests, the researchers used their 3-D-2PE-STED microscope to image the brain of a living mouse. They zoomed-in on part of a dendrite and resolved the 3-D structure of individual spines. They then imaged the same area two days later and showed that the spine structure had indeed changed during this time. The researchers did not observe any changes in the structure of the neurons in their images or in the mice's behavior that would indicate damage from the imaging. However, they do plan to study this further.

"Dendritic spines are so small that without super-resolution it is difficult to visualize their exact 3-D shape, let alone any changes to this shape over time," said Velasco. "3-D-2PE-STED now provides the means to observe these changes and to do so not only in the superficial layers of the brain, but also deeper inside, where more of the interesting connections happen."


Application of super-resolution imaging methods for cell biology and translation medicine

Prof. Vito Mennella has pioneered the application of multimodal super-resolution imaging in cell biology, in particular to study organelle architecture (Trends in Cell Biology, 2014, 2015).

By leveraging the power of 3D-SIM, dSTORM microscopy and quantitative image analysis, he has made together with his team a series of paradigm-shifting discoveries in centrosome and cilia biology: 1. The organized molecular architecture of the pericentriolar material of centrosomes (Nature Cell Biology, 2012) The architecture and function of a novel centrosomal complex in situ (Elife, 2018) and characterization of a novel type of cilium in the airway (Developmental Cell, 2020, 2020 in press).

Most recently he collaborated with clinicians to build super-resolution based tools for translational research in diseases caused by motile cilia defects and apply machine learning methods for disease diagnosis (Science Translational Medicine, 2020). In his presentation he will give an overview of how different super-resolution modalities and quantitative image analysis were applied to drive discovery in these different areas of research.


Super-resolution imaging in vivo - Biology

a State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China
E-mail: [email protected]

b Wallace H. Coulter Department of Biomedical Engineering, Emory University School of Medicine, Emory University, Atlanta, Georgia 30322, USA
E-mail: [email protected]

Abstract

RNA imaging in living animals helps decipher biology and creates new theranostics for disease treatment. Due to their low delivery efficiency and high background, however, fluorescence probes for in situ RNA imaging in living mice have not been reported. We develop a new cell-targeting fluorescent probe that enables RNA imaging in living mice via an in vivo hybridization chain reaction (HCR). The minimalistic Y-shaped design of the tripartite DNA probe improves its performance in live animal studies and serves as a modular scaffold for three DNA motifs for cell-targeting and the HCR circuit. The tripartite DNA probe allows facile synthesis with a high yield and demonstrates ultrasensitive RNA detection in vitro. The probe also exhibits selective and efficient internalization into folate (FA) receptor-overexpressed cells via a caveolar-mediated endocytosis mechanism and produces fluorescence signals dynamically correlated with intracellular target expressions. Furthermore, the probe exhibits specific delivery into tumor cells and allows high-contrast imaging of miR-21 in living mice. The tripartite DNA design may open the door for intracellular RNA imaging in living animals using DNA-minimal structures and its design strategy can help future development of DNA-based multi-functional molecular probes.