Friday, December 10, 2010

Protein localization using electron and fluorescence nanoscopy

Molecular topography of a cell can be successfully monitored by combining powerful imaging techniques such as electron microscopy and fluorescence nanoscopy ( STED or PALM). Recently, Prof. Erik M Jorgensen and his colleagues described a correlative fluorescence electron microscopy technique to localize protein on specific organelles. Here organelles first are revealed by electron microscopy and proteins are monitored by fluorescence imaging. As a result of image correlation between these two imaging modalities, proteins can be localized with nanometer accuracy. The paper also demonstrates localization of histone proteins on mitochondria.

Here is the paper that was published in Nature Methods: "Protein localization in electron micrographs using fluorescence nanoscopy"

X-ray microscopy resolves three-dimensional cellular ultrastructures

Partially coherent object illumination allowed researchers to reconstruct the three-dimensional ultrastructures of cells such as the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions. These results demonstrated visualization of structures at ~36-nm (Rayleigh) and ~70-nm (Fourier ring correlation) resolution.

Here is the paper that was reported in Nature Methods Journal: "Three-dimensional cellular ultrastructure resolved by X-ray microscopy"

Sunday, October 17, 2010

Zero-cost diagnostics on papers

George Whitesides is a Chemistry professor at Harvard University, and his recent work seems to have the potential to change the way diagnostic medicine works. Dr. Whitesides and his team have recently developed a prototype “paper chip” that is capable of diagnosing multiple disease simply with the application of a blood drop.

Here is the talk given by Prof. Whitesides on paper diagnostics:

Here are the papers on paper diagnostics from the same research group:

Patterned Paper as a Platform for Inexpensive, Low-Volume, Portable Bioassays†, Angew Chem. 2007

Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis, Anal Chem, 2008

Three-dimensional microfluidic devices fabricated in layered paper and tape, PNAS, 2008

FLASH: A rapid method for prototyping paper-based microfluidic devices, Lab Chip, 2008

Understanding Wax Printing: A Simple Micropatterning Process for Paper-Based Microfluidics, Anal Chem, 2009

Paper-supported 3D cell culture for tissue-based bioassays, PNAS, 2009

Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices, Anal Chem, 2009

Electrochemical sensing in paper-based microfluidic devices, Lab Chip, 2010

Programmable diagnostic devices made from paper and tape, Lab Chip, 2010

Paper‐Based ELISA, Angew Chem, 2010

And more and more publications over the last 3 years have been published by Prof. Whitesides' research group. Known with his contibutions to microfludics, Prof. Whitesides is opening up a field on paper based diagnositics...

Tuesday, October 12, 2010

High speed Atomic Force Microscopy unveils the steps of Myosin V

In Nature's October issue, the direct visualization of Myosin V motor proteins has been reported by using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative ‘visual evidence’ for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism.

Here is the recent report in Nature:
Video imaging of walking myosin V by high-speed atomic force microscopy
Noriyuki Kodera,Daisuke Yamamoto,Ryoki Ishikawa,Toshio Ando, Nature, 2010

Friday, October 1, 2010

Super-resolution Microscopy collection

Nature methods highlights the recent developments in the super-resolution imaging field. This collection of articles from several leaders in the field highlights the diversity of super-resolution microscopy techniques being developed and the principles that allow them to overcome this long-standing limitation.

Click here to see the collection, which is also sponsored by Nikon.

Here are the articles in the collection:

1- Primer: fluorescence imaging under the diffraction limit. D. Evanko. Nat. Methods 6, 19–20 (2009)

2- Microscopy and its focal switch. S.W. Hell. Nat. Methods 6, 24–32 (2009)

3- Putting super-resolution fluorescence microscopy to work. J. Lippincott-Schwartz & S. Manley. Nat. Methods 6, 21– 23 (2009)

4- Subdiffraction resolution in continuous samples. R. Heintzmann & M.G.L. Gustafsson. Nat. Photonics 3, 362–364 (2009)

5- Single-molecule mountains yield nanoscale cell images. W.E. Moerner. Nat. Methods 3, 781–782 (2006)

6- Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. H. Shroff et al. Nat. Methods 5, 417– 423 (2008)

7- Spherical nanosized focal spot unravels the interior of cells. R. Schmidt et al. Nat. Methods 5, 539–544 (2008)

8- Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. B. Huang et al. Nat. Methods 5, 1047–1052 (2008)

9- Super-resolution video microscopy of live cells by structured illumination. P. Kner et al. Nat. Methods 6, 339– 342 (2009)

Friday, August 27, 2010

Label-Free Nonlinear Microscopy reveals Zebrafish Cell Cycling

Together with more explorations of intrinsic nonlinear properties of the biological samples, the nonlinear microscopy has become an extensively used tool to demonstrate morphological visualization of biological structures.

A recent report in Science August(20) issue achieves 3 dimensional reconstruction of early Zebrafish Embryos. In this study, researchers designed a framework for imaging and reconstruction unstained whole zebrafish embryos for their 10 cell division cycles and also they reported the measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy.

Click here to read more about this report:
Cell Lineage Reconstruction of Early Zebrafish Embryos Using Label-Free Nonlinear Microscopy
Nicolas Olivier, Miguel A. Luengo-Oroz, Louise Duloquin, Emmanuel Faure, Thierry Savy, Israël Veilleux, Xavier Solinas, Delphine Débarre, Paul Bourgine, Andrés Santos, Nadine Peyriéras, and Emmanuel Beaurepaire (20 August 2010)
Science 329 (5994), 967.

Sunday, August 22, 2010

Second harmonic generating (SGH) nanoprobes

A new type of nanoprobe is introduced for in vivo imaging, circumventing many of the limitations of classical fluorescence probes. These second harmonic generating (SGH) probes are nanocrystals that converts two photons into one photon of half of the wavelength under intense illumination. Unlike fluorescent probes, they don't photobleach or saturate with increasing illumination intensity.

The report onf SGH nanoprobes is reported in July issue of PNAS. Click here to read more..

Getting around the diffraction limit

Optical microscopy has been extensively used to observe biological processes, where counting and identifying of molecular structures are achieved for accurate measurements. To date, several promising technologies have been introduced to break the resolution limits of conventional microscopes (i.e diffraction limit ~200nm), including PALM,STORM and STED. These methods are called super-resolution techniques,where resolution is defined as the minimum distance or volume that can be measured between two identical particles in a given period of time. Since biological molecules are <5-10nm,getting molecular details requires imaging at this scale, which can be achieved by super-resolution methods. Another important method to break the diffraction limit is localization accuracy, where it's defined as the minimum distance or volume that one can locate a particle's position within a certain time period.Localization have paved the way to understand how some biological molecules move or change its position, including the motor protein analysis.

Simply, one should not confuse localization super-accuracy with super-resolution as aforementioned. Recently, Toprak et al. reviewed some of the methods that were used for both localization and super-resolution in fluorescence microscopy. Here is the article for further details:

Erdal Toprak, Comert Kural, Paul R. Selvin, "Super-accuracy and super-resolution getting around the diffraction limit," Methods in Enzymology 475:1-26 (2010).

Wednesday, July 14, 2010

Plasmonic Structured Illumination Microscopy

Another super resolution imaging method is reported in Nanoletters. Click here to read the paper.

We propose a super resolution imaging technique called plasmonic structured illumination microscopy (PSIM), which combines the structured illumination microscopy technique with the tunable surface plasmon interference. Because of the high-resolution enabled by using surface plasmon interference as an illumination source, PSIM possesses higher image resolving power compared with conventional structured illumination microscopy. To demonstrate the technique, we present two specific types of plasmonic structure designs for PSIM. The final images from the simulations show 3-fold and 4-fold resolution improvement compared with conventional epi-fluorescence microscopy.

Saturday, July 10, 2010

Combining digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy

A high-contrast imaging method is introduced to visualize nontransparent objects by combining two powerful techniques(digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy).

Here is the summary of the novel imaging method that is reported in Nature Methods July 2010 issue:
Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.

Friday, July 9, 2010

Subnanometer Single-molecule Analysis

Over the last decade, Sub-diffraction measurement techniques have achieved nanometer resolution using stochastic processes of switchable molecules. Recently, Steven Chu and his research group demonstrated subnanometer localization, registration and distance measurements using closed-loop feedback control systems. Click here to see the recent report published in Nature.

Thursday, July 8, 2010

Single-molecule ELISA

More sensitive biomarker detection in blood requires single protein molecule screening for diagnostic purposes. Microscopic beads with specific antibodies provide flexibility to detect low-abundance proteins in blood. Conventional ELISA can work with an ensemble of proteins linked with antibodies, however, analyzing single proteins is needed for low concentration targets in blood. Towards this end, Quanterix Corporation introduced a technology that can isolate single beads with 50-fl reaction chambers, which also are monitored by fluorescent imaging. They demonstrated the detection of as few as ~10–20 enzyme-labeled complexes in 100 μl of sample (~10−19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA

Please check out the recent report in Nature Biotechnology on Single-molecule ELISA.

4D Ultrafast Electron Microscopy

Electron microscopy has been extensively used for cell biology to explain biological processes. But the problem is that the technique is quite invasive (i.e sample requires metal coating) and it requires long exposure times to average fast fluctuations. Recently, Ahmet Zewail and his research group introduced a microscopy technique that can monitor nanometer structures with femtosecond resolution. More importantly, this method mitigates the problems of cell labeling and challenging sample preparation steps.

Researcher called this technology as photon-induced, near-field electron microscopy(PINEM), where they demonstrate the performance of this system initially to image carbon nanotubes and silver nanowires. Click here to read Nature PINEM paper.

Recently, they also used this method to image unstained e-coli with an enhanced contrast and protein vehicles. Click here to read the PNAS Report on Biological Imaging.

This microscope is also commercialized by the company FEI.

Monday, June 21, 2010

Imaging through Turbid Media

A major limitation of conventional microscopes is the penetration depth in turbid media (e.g tissue, thick cells). It is known that turbid media scatter the incoming light, resulting in a messy image after the sample. But there is clever way to undo the effect of scattering medium by simply modulating the incoming light using a Spatial Light Modulator(SLM) or an equivalent device to adjust the wavefront.

Some recent articles on Imaging through turbid media:

Scattered light fluorescence Microscopy: imaging through turbid layers, Optics Letters

Focusing coherent light through opaque strongly scattering media, Optics Letters

Exploiting disorder for perfect focusing, Nature Photonics

Maximum Likelihood Fitting for Localization and Microscopy

In may 2010 issue of Nature methods, there are several papers on using maximum likelihood estimators(MLE) to achieve fast and single molecule localization. It turns out this approach is very powerful to fit photon distributions, assuming that Poisson noise is dominant in real measurements (i.e. microscopic images).

Here are the articles on MLE:

Efficient maximum likelihood estimator fitting of histograms

Fast, single-molecule localization that achieves theoretically minimum uncertainty

Optimized localization analysis for single-molecule tracking and super-resolution microscopy

Sunday, June 20, 2010

Merging Confocal with Wide Field Imaging: Image Scanning Microscopy ( ISM)

A new microscopy technique is introduced, image scanning microscopy (ISM), which combines conventional confocal-laser scanning microscopy with fast wide-field CCD detection. The technique allows for doubling the lateral optical resolution in fluorescence imaging. The physical principle behind ISM is similar to structured illumination microscopy, by combining the resolving power of confocal-laser scanning microscopy with that of a wide-field imaging microscopy. This Letter describes the theoretical foundation and experimental realization of ISM.

The article was published in PRL, click here to read the paper

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

A new super- resolution method was developed by Prof. Shimon Weiss and his research group. They demonstrated 5-fold improvement in spatial resolution based on the statistical analysis of temporal fluctuations in the images. The article was published in PNAS, click here to read the paper.

Learn Super-Resolution technologies from their inventors

Nature Method's Method of the Year 2008 was given to super-resolution fluorescence microscopy..

Here is the link that you can hear super-resolution imaging methods from their inventors:

Super-Resolution Microscopy

Pushing the Envelope in Biological Imaging By Eric Betzig

Eric Betzig of HHMI gave an interesting talk on the advancements in Light Microscopy at CNSI (Los Angeles) this week. Known with his contributions to near field microscopy, he recently reviewed his new ideas on resolution improvements, labeling technology, deep tissue imaging, and Non-invasive imaging.

He used to reject invitations for such talks, but then he decided to explain his ideas thinking about the fact that life is short.

Here is his amazing life story:

Before he moved to the Janelia Farm Research Campus, Eric Betzig—physicist, inventor, and engineer—didn't have a lab. There was an office in his Michigan cottage where he did most of his work, but some days he packed it all up and took his boat out on Hiland Lake, finding a secluded spot to serve as his workspace. The tools of his trade, which he says amounted to "a laptop and a couple of really good ideas," packed easily, after all.

But now Betzig's ideas have outgrown the laptop and the cottage. After two and a half years of theoretical research, he has taken his theories into the lab, where he can apply them to one of the major technological challenges of biological research. As a JFRC group leader, Betzig will work to develop a microscope that will allow biologists to peer inside living cells with unprecedented resolution.

Betzig is trained as an experimental physicist, and he made waves in that field early on by helping to develop a technique known as near-field microscopy, which brought into focus structures that scientists had long considered too small to see with a light microscope. As a graduate student at Cornell University, and then during six years at Bell Labs, he advanced the technology to make it more practical for biologists, allowing powerful imaging of dead cells.

The size of a typical protein is about one or two nanometers—some 200 times smaller than what can be seen with an ordinary light microscope. Near-field microscopes, on the other hand, can discriminate structures as small as 30 nanometers. That's much larger than a protein, but according to Betzig, "there's still a lot you can learn." He was frustrated, however, when he realized the limitations inherent in the overall approach meant it would probably never be useful for imaging living cells. Sensing he'd taken the technology as far as it could go, Betzig decided it was time to move on.

Betzig turned his back on Bell Labs, and the world of science altogether, to join his father Robert's machine tool company in Chelsea, Michigan. He spent seven years at the Ann Arbor Machine Company, tackling the automated high-volume production of machine parts. The problem, he explains, is that a multi-ton machine and its tools must be moved to many points in order to cut a single part. "So more time is spent moving the machine," he says, "than actually cutting metal." Betzig used his engineering savvy to create a method to move machines with extraordinary speed without sacrificing the necessary precision, greatly reducing the time devoted to that aspect of manufacturing.

Once he'd seen his latest invention through development and marketing, Betzig says, he became restless, and started to think about returning to science. But with no scientific publications for the past ten years, "there was this big gap on my résumé. So I knew I had to come up with some intellectual capital to get people to listen to me again."

"So I holed up in my cottage, and just started thinking. Eventually those thoughts brought me back to microscopy," he recalls. Progress in the imaging field, such as the development of fluorescent proteins, makes the need for advanced microscopy even more critical today than when he worked in science a decade ago, Betzig says. "We can at least dream now about being able to see within the cell on the molecular level, which is where all the action is. If we can do that, and study dynamics at that level, our understanding of cell biology and molecular biology should skyrocket."

However, his experience with the near-field microscope made him keenly aware of the trade off between spatial resolution and signal strength, which normally would doom any possibility of studying macromolecular dynamics in real time. "That led to a theoretical development of mine," he says—an approach to microscopy that relies on a massive three-dimensional array light foci. By collecting light from all these foci at once, he believes he can compensate for the rapid loss of signal that made the earlier technique unsuitable for imaging living cells. He expects the new technique, which he calls optical lattice microscopy, will have further advantages over conventional methods, such as improved sensitivity to single fluorescent molecules and less damage to cells.

Betzig has filed a patent for his design, and has already begun preliminary experiments to demonstrate that the approach is feasible. At Janelia Farm, he will continue testing his theories and work toward translating them into a functional instrument. His initial approach should enable rapid imaging of the dynamic changes within living cells, but the spatial resolution will still be limited, Betzig says. "So from there you need to do various types of tricks to try to get beyond the diffraction limit to super-high resolution. And that's another thrust of what I'll be doing at Janelia."

Janelia is the ideal environment for this work, he says, largely because of the opportunity to interact with people who will ultimately use the tool he creates. "I learned from my business experience that there is nothing more important than constant contact with the customer as you're developing new products," he says. "And that's exactly what we have at Janelia. The people who will use the microscope will be right there; they'll guide the design."

Equally important, Betzig says, are the mechanisms Janelia has in place to "take this rubber band-and-bubblegum thing that a physicist can get to work, and take it through the development phase to turn it into something that biologists are really going to be able to use."

"Ultimately," he says, "it comes down to impact. You want to create an instrument that's going to have an impact." And, by bringing his ideas to Janelia Farm, he expects to do just that.