Despite many achievements in the field of super-resolution microscopy in the past few years with spatial resolution advances, live-cell imaging has remained a challenge because of the need for high temporal resolution.
Now, Lei Zhu, assistant professor in Georgia Tech’s George W. Woodruff School of Mechanical Engineering, and Bo Huang, assistant professor in UCSF’s Department of Pharmaceutical Chemistry and Department of Biochemistry and Biophysics, have developed an advanced approach using super-resolution microscopy to resolve cellular features an order of magnitude smaller than what could be seen before. This allows the researchers to tap previously inaccessible information and answer new biological questions.
The research was published April 22, 2012 in the journal Nature Methods. The research is funded by the National Institutes of Health, UCSF Program for Breakthrough Biomedical Research, Searle Scholarship and Packard Fellowship for Science and Engineering.
The previous technology using the single-molecule-switching approach for super-resolution microscopy depends on spreading single molecule images sparsely into many, often thousands of, camera frames. It is extremely limited in its temporal resolution and does not provide the ability to follow dynamic processes in live cells.
“We can now use our discovery using super-resolution microscopy with seconds or even sub-second temporal resolution for a large field of view to follow many more dynamic cellular processes,” said Zhu. “Much of our knowledge of the life of a cell comes from our ability to see the small structures within it.”
Huang noted, “One application, for example, is to investigate how mitochondria, the power house of the cell, interact with other organelles and the cytoskeleton to reshape the structure during the life cycle of the cell.”
Currently, light microscopy, especially in the modern form of fluorescence microscopy, is still used frequently by many biologists. However, the authors say, conventional light microscopy has one major limitation: the inability to resolve two objects closer than half the wavelength of the light because of the phenomenon called diffraction. With diffraction, the images look blurry and overlapped no matter how high the magnification that is used.
“The diffraction limit has long been regarded as one of the fundamental constraints for light microscopy until the recent inventions of super-resolution fluorescence microscopy techniques,” said Zhu. Super-resolution microscopy methods, such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM), rely on the ability to record light emission from a single molecule in the sample.
Using probe molecules that can be switched between a visible and an invisible state, STORM/PALM determines the position of each molecule of interest. These positions ultimately define a structure.
The new finding is significant, said Zhu and Huang, because they have shown that the technology allows for following the dynamics of a microtubule cytoskeleton with a three-second time resolution, which would allow researchers to study the active transports of vesicles and other cargos inside the cell.
Using the same optical system and detector as in conventional light microscopy, super-resolution microscopy naturally requires longer acquisition time to obtain more spatial information, leading to a trade-off between its spatial and temporal resolution. In super-resolution microscopy methods based on STORM/PALM, each camera image samples a very sparse subset of probe molecules in the sample.
An alternative approach is to increase the density of activated fluorophores so that each camera frame samples more molecules. However, this high density of fluorescent spots causes them to overlap, invalidating the widely used single-molecule localization method.
The authors said that a number of methods have been reported recently that can efficiently retrieve single-molecule positions even when the single fluorophore signals overlap. These methods are based on fitting clusters of overlapped spots with a variable number of point-spread functions (PSFs) with either maximum likelihood estimation or Bayesian statistics. The Bayesian method has also been applied to the whole image set.
As a result of new research, Zhu and Huang present a new approach based on global optimization using compressed sensing, which does not involve estimating or assuming the number of molecules in the image. They show that compressed sensing can work with much higher molecule densities compared to other technologies and demonstrate live cell imaging of fluorescent protein-labeled microtubules with three-second temporal resolution.
The STORM experiment used by the authors, with immunostained microtubules in Drosophila melanogaster S2 cells, demonstrated that nearby microtubules can be resolved by compressed sensing using as few as 100 camera frames, whereas they were not discernible by the single-molecule fitting method. They have also performed live STORM on S2 cells stably expressing tubulin fused to mEos2.
At the commonly used camera frame rate of 56.4 Hertz, a super-resolution movie was constructed with a time resolution of three seconds (169 frames) and a Nyquist resolution of 60 nanometers, much faster than previously reported, said Zhu and Huang. These results have proven that compressed sensing can enable STORM to monitor live cellular processes with second-scale time resolution, or even sub-second-scale resolution if a faster camera can be used.
John Toon | Newswise Science News
Nerves control the body’s bacterial community
26.09.2017 | Christian-Albrechts-Universität zu Kiel
Ageless ears? Elderly barn owls do not become hard of hearing
26.09.2017 | Carl von Ossietzky-Universität Oldenburg
Controlling electronic current is essential to modern electronics, as data and signals are transferred by streams of electrons which are controlled at high speed. Demands on transmission speeds are also increasing as technology develops. Scientists from the Chair of Laser Physics and the Chair of Applied Physics at Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) have succeeded in switching on a current with a desired direction in graphene using a single laser pulse within a femtosecond ¬¬ – a femtosecond corresponds to the millionth part of a billionth of a second. This is more than a thousand times faster compared to the most efficient transistors today.
Graphene is up to the job
At the productronica trade fair in Munich this November, the Fraunhofer Institute for Laser Technology ILT will be presenting Laser-Based Tape-Automated Bonding, LaserTAB for short. The experts from Aachen will be demonstrating how new battery cells and power electronics can be micro-welded more efficiently and precisely than ever before thanks to new optics and robot support.
Fraunhofer ILT from Aachen relies on a clever combination of robotics and a laser scanner with new optics as well as process monitoring, which it has developed...
Plants and algae use the enzyme Rubisco to fix carbon dioxide, removing it from the atmosphere and converting it into biomass. Algae have figured out a way to increase the efficiency of carbon fixation. They gather most of their Rubisco into a ball-shaped microcompartment called the pyrenoid, which they flood with a high local concentration of carbon dioxide. A team of scientists at Princeton University, the Carnegie Institution for Science, Stanford University and the Max Plank Institute of Biochemistry have unravelled the mysteries of how the pyrenoid is assembled. These insights can help to engineer crops that remove more carbon dioxide from the atmosphere while producing more food.
A warming planet
Our brains house extremely complex neuronal circuits, whose detailed structures are still largely unknown. This is especially true for the so-called cerebral cortex of mammals, where among other things vision, thoughts or spatial orientation are being computed. Here the rules by which nerve cells are connected to each other are only partly understood. A team of scientists around Moritz Helmstaedter at the Frankfiurt Max Planck Institute for Brain Research and Helene Schmidt (Humboldt University in Berlin) have now discovered a surprisingly precise nerve cell connectivity pattern in the part of the cerebral cortex that is responsible for orienting the individual animal or human in space.
The researchers report online in Nature (Schmidt et al., 2017. Axonal synapse sorting in medial entorhinal cortex, DOI: 10.1038/nature24005) that synapses in...
Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...
19.09.2017 | Event News
12.09.2017 | Event News
06.09.2017 | Event News
26.09.2017 | Life Sciences
26.09.2017 | Physics and Astronomy
26.09.2017 | Information Technology