Expansion Microscopy Technique Enables 20-nm Resolution

A new expansion microscopy (ExM) technique from MIT makes it possible to use a conventional light microscope to generate high-resolution images at the nanoscale, by expanding specimens 20-fold before imaging them.

Historically, nanoscale structures in cells and tissues have been imaged with high-powered, expensive, superresolution microscopes. The new ExM protocol, which achieves 20-fold expansion in just one step, provides a simple, inexpensive method that can be used by most biology labs to perform imaging at a resolution of about 20 nm.

“What this new technique allows you to do is see things that you couldn’t normally see with standard microscopes,” professor Laura Kiessling said. “It drives down the cost of imaging because you can see nanoscale things without the need for a specialized facility.”

The original version of the ExM technique, developed by professor Edward Boyden and his team in 2015, expanded tissue about 4-fold and provided images with a resolution of around 70 nm. In 2017, Boyden’s lab modified the process to include a second expansion step, achieving an overall 20-fold expansion.

“We’ve developed several 20-fold expansion technologies in the past, but they require multiple expansion steps,” Boyden said. “If you could do that amount of expansion in a single step, that could simplify things quite a bit.”

The new method reaches the same level of performance possible with iterative expansion methods, but with the simplicity of a single-shot protocol.

To implement ExM, the researchers embed the tissue specimen in an absorbent polymer and added water, creating a hydrogel that expands the polymer and pulls the biomolecules in the specimen apart. For one-step, 20-fold expansion, the researchers use gel that is extremely absorbent and mechanically stable to ensure that the gel does not fall apart when the specimen is expanded by 20x.

To further stabilize the gel and enhance its reproducibility, the researchers remove oxygen from the polymer solution prior to gelation, preventing side reactions that could interfere with crosslinking. Unlike previous expansion gels that require another molecule to be added to form crosslinks between the polymer strands, the gel used for the single-shot, 20-fold ExM technique forms crosslinks spontaneously.

Once the gel is formed, select bonds in the proteins that hold the tissue together are broken and water is added to make the gel expand. After the gel expands, target proteins in the tissue can be labeled and imaged. The new technique supports post-expansion staining for brain tissue to facilitate biomolecular labeling.

“This approach may require more sample preparation compared to other superresolution techniques, but it’s much simpler when it comes to the actual imaging process, especially for 3D imaging,” researcher Tay Won Shin said.

In one round of expansion, the new ExM technique, which the team calls 20ExM, enabled the researchers to image hollow microtubule structures in cultured cells and synaptic nanocolumns in the mouse somatosensory cortex on a conventional confocal microscope. The team could also visualize mitochondria and the organization of individual nuclear pore complexes in the cells.

The new ExM technique could be used for a variety of experiments where high resolution and single-step simplicity are desired. The researchers are currently using the technique to image glycans — carbohydrates, found on the surface of a cell that help control how the cell interacts with its environment.

20ExM could also be used to image tumor cells, providing insight into how proteins are organized within these cells. In principle, the new ExM technique could be used to simplify or enhance the resolution of other expansion-based technologies, such as in situ RNA detection and sequencing and genome imaging.

The single-shot, 20-fold expansion microscopy method provides a robust, simple, affordable solution to nanoscale-resolution imaging of preserved cells and tissues using conventional microscopes. The researchers believe that any biology lab could use the technique at a low cost, because it relies on standard, off-the-shelf chemicals and equipment that most labs already have or can easily access.

“Our hope is that with this new technology, any conventional biology lab can use this protocol with their existing microscopes, allowing them to approach resolution that can only be achieved with very specialized and costly state-of-the-art microscopes,” researcher Shiwei Wang said.

Bio Photonics Research Award

Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee

#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,

Optogenetic Light Source Transmits Information Between Neurons

Researchers at The Institute of Photonic Sciences (ICFO) demonstrated that photons, acting as neurotransmitters, can enable communication between neurons. The researchers developed an all-optogenetic, synaptic transmission system that enabled synthetic signaling between unconnected neurons and the generation of synaptic circuits.

The team’s findings could lead to therapies that use light instead of chemicals or drugs to restore communication between nerve cells in the treatment of diseases such as Alzheimer’s and Parkinson’s. In addition to treating neurological disorders, the team’s approach could potentially be used to rewire damaged neural circuits and improve learning.

The Photons as Synaptic Transmitters (PhAST) system connects two neurons by using light-emitting enzymes and light-sensitive ion channels. The researchers tested the PhAST system on the roundworm model C. elegans and showed that photon-based synaptic transmission can facilitate the modification of animal behavior.

After genetically modifying the roundworms to have faulty neurotransmitters, which made the worms insensitive to mechanical stimuli, the researchers engineered a luciferase enzyme to generate light inside the worms and a specially designed microscope for viewing the light being emitted by the worms. They also selected ion channels for the postsynaptic cells. The channels are highly sensitive, so that very little light is needed to open them.

The genetically engineered enzyme introduced into the worms as a light source requires calcium and the binding of a co-factor to emit light. The calcium increase occurs when the presynaptic cell activates, so the light is only on when the presynaptic neuron is on as well. When the light source and the presynaptic cell are activated, blue light is emitted from the presynaptic cell. The ion channel in the postsynaptic cell senses the blue light and activates the postsynaptic cell, which then transmits the information to the downstream pathway.

To follow the information flow, the researchers developed a device that delivered mechanical stresses to the animals’ noses, while at the same time measuring the calcium activity in the sensory neurons. To acquire the dim light signals coming from the neurons, they built a microscope with a sensor that is strong enough to detect a faint signal from just a few photons. The researchers then took a default model of an existing microscope and removed the optical elements that were not needed for bioluminescence and that could interfere with imaging. The researchers also used artificial intelligence to enhance the bioluminescence imaging capabilities of the microscope.

In experiments, the researchers established a new transmission between two unconnected cells, restoring neuronal communication in a defective circuit. They also suppressed the worms’ response to a painful stimulus, and they switched the worms’ response to an olfactory stimulus from attractive to aversive behavior. The researchers also used PhAST to study the calcium dynamics of the temporal pattern generator in a motor circuit for ovipositioning. The experimental results showed that the PhAST system can facilitate the modification of animal behavior.

Further, the PhAST system could help researchers better understand the underlying mechanisms of brain function and complex behaviors, and how different brain regions communicate with each other. It could lead to new ways to image and map brain activity with higher spatiotemporal resolution.

Limitations to the widespread use of the technology remain. Further improvements in the engineering of the bioluminescent enzymes and the ion channels and in the targeting of molecules would allow greater optical control of the neuronal function, with higher specificity and precision. However, the ICFO study demonstrated that chemical neurotransmitters can be replaced with light to overcome malfunctioning in neural circuits and help neurons communicate again. Now that the technology has been shown to work in vivo in worms, a potential next step could be use of the PhAST system for the study of more complex neural circuits.


Bio Photonics Research Award

Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee

#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,

Light-Activated Tool Controls Protein Bonds and Tracks Cell Adhesion

Optical tools can be used to activate biological functions, but with current methods the effects are slow to appear, and sustained effects require continuous light activation. As a result, these light-activation tools provide limited control of fast biological processes and can lead to toxicity in cells and organisms.

Although light is a well-established tool for control of bond breakage, it is less firmly established for the control of specific bond formation in complex environments.

A team at Tampere University worked with researchers at the University of Cambridge and the University of Pittsburgh to develop a way to use visible light to control irreversible protein binding. The new optical technique for fast, irreversible protein conjugation could be especially valuable in processes where a short initial signal leads to long-term changes in cell or tissue function. Examples include the regulation of gene expression during stem cell differentiation and the activation of immune cells in viral infections.

The researchers built on their previous work with proteins to develop a system for the rapid, light-activated control of protein bond formation. Their “protein superglue” is a peptide/protein pair called SpyTag003/SpyCatcher003 that exhibits fast, irreversible binding. Based on an engineered protein, the SpyTag003/SpyCatcher003 peptide/protein pair allows the modular assembly of complex protein structures.

To achieve optical control of the protein superglue, the researchers looked beyond the 20 amino acids constituting human proteins. Using modified protein synthesis machinery from archaebacteria, they incorporated a light-reactive, unnatural amino acid into the SpyCatcher003 protein to make the protein photoreactive. The amino acid was strategically placed to block the peptide/protein pairing until it was activated by light.

In experiments, the researchers showed a uniform, specific reaction in cell lysate upon light activation.

“A short pulse of light was enough to trigger the rapid and efficient formation of the irreversible peptide/protein complex, both in the test tube and in living cells,” said Mark Howarth, a professor at the University of Cambridge. “Importantly, the activation only took place with specific wavelengths of light, making it possible to combine protein control with live-cell fluorescence microscopy.”

After validating their approach to optically controlling irreversible protein coupling, the researchers applied the technique to the covalent reconstitution of a talin protein that was split in half. The researchers used light to activate the talin — a central adhesion protein — inside living cells.

Optical control of talin reconstitution allowed the researchers to probe the timescale of the initial adhesion complex formation. By tracking the timing of protein recruitment into the adhesion complex, the team could determine a timeline of the events leading to the formation of the adhesion complex, and the hierarchy of the recruitment of key components for cell adhesion.

Cell-matrix adhesions — large protein complexes consisting of hundreds of different proteins — are highly dynamic. “Their dynamic structure and vast complexity make cell adhesions difficult to study,” Tampere University professor Vesa Hytönen said. “The details of how cell-matrix adhesions initially form and how they react to different stimuli have remained largely unknown.”

Researcher Rolle Rahikainen said that the team observed an immediate cell response after activating the talin protein with a short pulse of light. “We got very excited when we first realized how well the system worked in controlling complex cellular processes, such as the formation of adhesion and cell spreading.”

The findings demonstrate the potential of the light-activated protein superglue for investigating complex cellular processes. The results could also lead scientists to a more comprehensive understanding of the complex structure and function of adhesion.

The modular, Lego brick-like structure of the system makes it applicable to the study and control of diverse cellular functions. The precise, irreversible assembly of biological building blocks has many applications, from biomaterials to vaccines.

Beyond adhesion, SpyCatcher003 could be used for the photocontrol of biomolecules. The robust cellular response, initiated in seconds, opens possibilities for spatiotemporal control of highly dynamic intracellular and extracellular processes.

Bio Photonics Research Award

Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee

#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,

Simple Approach to Laser Color Conversion Uses SRS in Ionic Liquids

S

cientists from Brookhaven National Laboratory showed that ionic liquids provide an efficient means to convert one color of laser light into another. The discovery could lead to a way to create lasers with desired colors for a range of medical, scientific, and technological applications.

The method is based on the interaction between the laser and different types of ionic liquids (also known as liquid salts). The vibrational energy in the chemical bonds in the ionic liquid cause the laser’s energy to shift and change color.

“By adding a certain ion that has a particular vibrational frequency, we can design a liquid that shifts the laser light by that vibrational frequency,” chemist James Wishart said. “And if we want a different color, then we can switch out one ion and put in another that has a different vibrational frequency. The component ions can be mixed and matched to shift laser colors by different degrees as needed.”

The new approach to changing laser wavelengths has its roots in a project to boost the capabilities of a CO2 laser at Brookhaven’s Accelerator Test Facility. To improve the laser’s beam quality and repetition rate, the researchers wanted to pump the laser using optical excitation instead of electric discharge.

To create a laser with the appropriate wavelength for optical pumping, the researchers used stimulated Raman scattering (SRS) to shift the wavelength of an existing laser. SRS can be used to harness the vibrational frequencies of molecules in a solid, liquid, or gas form.

“Basically, the laser deposits energy into the molecular vibrations — the squishing and stretching of the chemical bonds that make up the material,” researcher Rotem Kupfer said. “Then the photons (particles of light) that come out have the original energy, minus the energy of those vibrations.” The lower-energy photons have a longer wavelength and, thus, a different color.

The researchers demonstrated that 1-ethyl-3-methylimidazolium dicyanamide (EMIM DCA), an ionic liquid, was an effective medium for converting 532-nm pulses from a Q-switched Nd:YAG laser to 603 nm. This corresponded to an approximate 2200 cm−1 shift, which could be used to generate mid-infrared radiation for optical pumping of CO2 lasers.

While choosing the best ionic liquid for pumping the CO2 laser, the researchers considered that their color-shifting approach could have a broader use.

In a proof-of-principle, single-pass conversion setup, the researchers obtained a threefold-higher Raman conversion efficiency in the ionic liquid compared with water under identical conditions, resulting in an efficient generation of high-quality orange laser pulses in a wavelength region that is difficult to access at high energies.

“There are a lot of hard ways to do Raman shifting. But for this one, we just filled a tube with a properly selected ionic liquid, shot a laser in from one end, and we got the color we wanted out — without any fine tuning,” Wishart said.

Kupfer said that other methods for achieving a shift in laser color require complex optical setups or the use of toxic materials. “Plus, those other processes ‘break’ the molecules; they wear out and have to be replaced,” he said. “In our case, it is a balance sheet. The molecules stay unharmed.”

The researchers determined that ionic liquids provide a framework to engineer liquids suitable for wavelength conversion over a broad spectral range. Careful selection of the molecular structures of the ionic liquid anions and cations lead to specific characteristics, such as a desirable Raman shift, low Brillouin scattering, and good optical transmission in the pump and Stokes wavelengths. An ionic liquid can interact with photons while offering a high density of energy-swapping sites.

Gas molecules have limited vibrational frequencies, and diffuse gaseous molecules mean scattering efficiency is low. Solids have more tightly packed molecules, making them more efficient, but their complex vibrational frequencies make them costly to produce.

“Liquids are somewhere in between,” Wishart said. “You’re still dealing with single molecules, but denser, meaning higher efficiency than gases. And with ionic liquids, you can engineer the molecules to give you the frequency you need.”

The large number of ionic liquids available makes it possible to precisely tune the energy loss caused by the ionic liquid-photon interaction, providing greater selective control over color. Optically transparent ionic liquids prevent background absorption of light. In addition, their viscosity prevents laser scattering from acoustic waves, which can diminish the color-shifting effect in low-viscosity liquids.

Although further improvements could optimize the process, the researchers said that overall, the made-to-order ionic liquids provide a suitable platform for efficient, simple, adjustment-free laser color shifting using SRS.

Bio Photonics Research Award

Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee

#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,

Computational Technique Harnesses the Benefits of Spectral, Photographic Insights

Using an approach that combines computer vision, color science, and optical spectroscopy, researchers at Purdue University devised an approach that enables conventional photography to be used for optical spectroscopy and hyperspectral imaging. Using the mechanism, the researchers realized spectral resolution comparable to the resolution of scientific spectrometers with photos from a smartphone camera.

A range of industries, running the gamut from agriculture, environmental monitoring, and food quality analysis, to industrial quality control, defense and security, and medical diagnostics, could benefit from the technique, according to its developers.

The researchers hypothesized that the RGB values of reference colors, as captured by a traditional camera, could be used to design a spectral color chart that could then be used to decode spectral information. The fidelity of spectral recovery would be determined mainly by the spectral incoherence among the reference colors in the chart.

They developed a general computational framework, co-designed with spectrally incoherent color reference charts, to recover spectral information from a single-shot photograph. They optimized reference color selection and the computational algorithm to eliminate the need for training data or pretrained models.

The spectral color chart, together with the device-informed computation, can be used to recover spectral information from RGB values acquired using conventional cameras, such as smartphone cameras.

In transmission mode, data is acquired by photographing the spectral color chart through the sample of interest. Altered RGB values of reference colors are used to recover the spectral intensity of the sample. In reflection mode, the sample of interest is placed alongside the spectral color chart to recover the sample’s spectral hypercube without needing a hyperspectral imaging system. A spectral hypercube of the sample can be constructed from a single-shot photo, analogous to hyperspectral imaging.

The technique, which the researchers called computational photography spectrometry (CPS), has the potential to make optical spectroscopy and hyperspectral imaging accessible with off-the-shelf smartphones. Instead of limiting the user to multispectral data with only a few bands, the technique enables a high spectral resolution of 1-2 nm.

“Importantly, the spectral resolution — around 1.5 nm — is highly comparable to that of scientific spectrometers and hyperspectral imagers,” researcher Semin Kwon said. “Scientific-grade spectrometers have fine spectral resolution to distinguish narrow spectral features. This is critical in applications like biomedical optics, material analysis, and color science, where even small wavelength shifts can lead to different interpretations.”

Although methods exist to estimate and reconstruct spectral information from RGB values acquired using conventional cameras, they are limited in their ability to achieve a high degree of spectral resolution.

The researchers developed a generalizable method for extracting high-resolution spectral information from a single-shot photo of a sample without having to rely on task-specific training datasets or predetermined models. This provides an advantage over existing machine learning models for spectral reconstruction, which depend on task-specific training data or fixed models.

“From an algorithmic standpoint, to the best of our knowledge, our paper presents the first computational spectrometry method with 1.5-nm spectral resolution using a photograph of an arbitrary sample without relying on specific training data or predetermined algorithms,” professor Young Kim said.

The spectral color chart and device-informed computation eliminate the need for complex hardware, simplifying the hardware requirements for the CPS technique. And, the CPS approach could potentially offer a simple, affordable, portable way to use smartphones for optical spectroscopy and hyperspectral imaging in day-to-day applications.

“Many mobile spectrometers require additional accessories and bulky components as mandatory attachments to smartphones,” Kwon said. “In contrast, our method leverages the built-in camera of the smartphone.”

The team is currently using the algorithm for digital and mobile health applications in both domestic and resource-limited settings.

“Photography is central to these applications, but color distortion has posed a persistent challenge, which is why we are focusing on these settings,” Kim said. “This algorithm provides a basis for quantifying and correcting colors, enhancing the reliability of medical diagnostics.”

The researchers believe that the generalized computational photography spectrometry technique could change how industry uses smartphones.

“A photograph is more than just an image. It contains abundant hyperspectral information,” Kim said. “We are one of the pioneering research groups to integrate computational spectrometry and spectroscopic analyses for biomedical and other applications.”

A patent for the algorithm is pending. Industry partners interested in developing or commercializing the algorithm should contact Patrick Finnerty, assistant director of business development and licensing-life sciences, at Purdue University.

Bio Photonics Research Award

Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee

#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,

Spatial Light Modulation Gauges How Lenses Slow Progress of Myopia

Myopia, or nearsightedness, is one of the most common ocular disorders worldwide and a leading cause of visual impairment in children. Although specialized eyeglass lenses have been clinically tested to treat myopia progression, an in-depth optical characterization of the lenses has not yet been performed.

Researchers from the ZEISS Vision Science Lab at the University of Tübingen and the University of Murcia undertook a comprehensive characterization to investigate the properties of spectacle lenses designed to slow the progress of myopia. The results of their study could help increase the efficacy of future lens designs.

Myopia is typically caused when a person’s eyes become elongated, which affects how the eyes focus on faraway objects. The condition can progress in children and teens as their bodies grow.

To reproduce pupil shape and myopic ocular aberrations, researchers developed an instrument that reproduced the aberrations in myopic eyes and enabled physical simulation of the pupil. They based their instrument on spatial light modulation (SLM) technology.

“After exploring the state of the art, we didn’t find a method that could be used to characterize the optical properties of these eyeglass lenses under real viewing conditions,” said researcher Augusto Arias-Gallego. As a result, Arias-Gallego said, the researchers endeavored to build an instrument that can measure the lens’ optical response to different angles of illumination, while also reproducing the myopic eye’s pupil and refractive errors.

The team’s instrument uses an illumination source mounted on an arm that rotates around the lens. After the light passes through the lens, it is guided to an SLM by a rotating mirror. The SLM is composed of tiny liquid crystal cells that modify the propagating light, boosting its spatial resolution.

The SLM reproduces the refractive errors and pupil shape of myopic eyes, allowing the researchers to re-create myopic aberrations and to produce different aberrations depending on the angle of illumination. Using the SLM, the researchers programmed the aberrations as phase maps and induced programmed amounts of defocus to perform through-focus testing.

Tests helped the researchers determine the image quality within the proximity of a simulated retinal position, shedding light on how the special lens interacts with eye elongation signaled at the retina. “By combining the through-focus results with light-scattering measurements, we were able to accurately characterize several types of eyeglass lenses,” Arias-Gallego said. The researchers then compared measurements for each lens with their reported clinical efficacy for slowing myopia progression, he said.

The researchers quantified and compared the focusing and scattering properties of a single vision lens with two types of spectacle lenses for myopia progression management: defocus incorporated multiple segments (DIMS) and diffusion-optical technology (DOT). They calculated four optical metrics potentially related to myopia progress and quantified the scattered light from the peripheral lens zones. Scattering was quantified by implementing the optical integration method.

The characterization showed an increased contrast and sharpness of images through the DIMS lens at the peripheral retina when inducing myopic defocus, with respect to the single vision and DOT lenses. It further showed that contrast reduction by the DOT lens was dependent on the luminance at the pupil.

According to Arias-Gallego, the results both raised new questions and pointed to potential strategies that could increase the efficacy of future designs.

“Insights into the link between the optical properties of myopia progression management lenses and effectiveness in real-world scenarios will pave the way to more effective treatments,” Arias-Gallego said. “This could help millions of children and is fundamental in understanding the mechanisms by which these lenses work.”

The researchers are working to adapt the SLM instrument to include sources with varying wavelengths.

Bio Photonics Research Award

Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee

#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,

DNA-Barcoded Fluorescence Imaging Illuminates Core Cell Components

Since many core components of cells — like DNA, RNA, proteins, and lipids — are just a few nanometers in size and substantially smaller than the resolution limit of traditional light microscopy, the exact composition and arrangement of these molecules and structures is thus often unknown. This results in a lack of mechanistic understanding of fundamental aspects of biology.

Drawing on recent improvements to superresolution imaging, including single-molecule localization microscopy, or SMLM, researchers from the Max Planck Institute of Biochemistry and Ludwig-Maximilians-Universität Munich have developed a technique that enhances the resolution of fluorescence microscopy down to the angstrom scale. The researchers’ technique enables the study of whole and intact cells over individual proteins — all the way down to the distance between two adjacent bases in DNA.

The researchers, from the group of Ralf Jungmann, called the technique resolution enhancement by sequential imaging, or RESI.

Current SMLM resolve structures on the order of 10 nm by temporally separating the structures’ individual fluorescence emission. As individual targets stochastically light up in an otherwise dark field of view, their location can be determined with subdiffraction precision. The SMLM technique of DNA points accumulation for imaging in nanoscale topography, or DNA-PAINT, uses the transient hybridization of dye-labeled DNA “imager” strands to their target-bound complements to achieve the light-up necessary to achieve superresolution.

To date, however, neither DNA-PAINT nor other superresolution methods have been able to resolve the smallest cellular structures.

RESI builds on DNA-PAINT and capitalizes on its ability to encode target identity via DNA sequences. By labeling adjacent targets, too close to each other to be resolved even by superresolution microscopy, with different DNA strands, an additional degree of differentiation — a barcode — is introduced into the sample. By sequentially imaging first one and then the other sequence to thereby capture the full target, the strands can now be unambiguously separated.

Critically, as they are imaged sequentially, the targets can be arbitrarily close to one other, which is a dynamic that existing techniques are unable to resolve. Further, RESI does not require specialized instrumentation and can be applied using any standard fluorescence microscope.

To demonstrate RESI’s leap in resolution compared to other methods, the researchers sought to resolve the separation between individual bases along a double helix of DNA, which is separated by less than 1 nm. They designed a DNA origami nanostructure that presented single-stranded DNA sequences that protrude from a double helix at one base pair distance.

The researchers then imaged these single strands sequentially and resolved a distance of 0.85 nm, or 8.5 Å between adjacent bases. They accomplished these measurements with a precision of 1 Å, or one ten-billionth of a meter.

According to the researchers, the technique is universal, with application beyond DNA nanostructures. In separate tests, Jungmann and his team investigated the molecular mode of action of rituximab, an anti-CD20 monoclonal antibody used for treatment of CD20-positive blood cancer. Investigating the effects of such drug molecules on molecular receptor patterns has been beyond the spatial resolution capabilities of traditional microscopy techniques. Understanding whether and how such patterns change in health and disease as well as upon treatment is important for mechanistic research and the design of targeted therapies.

The researchers used RESI to reveal the natural arrangement of CD20 receptors in untreated cells as dimers and uncovered how CD20 re-arranged to chains of dimers upon drug treatment.

Because RESI is performed in whole, intact cells, the technique closes the gap between purely structural techniques such as x-ray crystallography or cryogenic electron microscopy and traditional lower-resolution whole-cell imaging approaches.


Bio Photonics Research Award

Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee

#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,