Amazing Science

The growing class of fluorescent proteins useful for detecting events in living cells and animals has almost single-handedly launched and fueled a new era in biology and medicine.

It took over thirty years, and the advent of recombinant DNA as well as vastly improved molecular biological approaches to see the pioneering work of Osamu Shimomura developed into a useful tool for live-cell imaging by Doug Prasher and Martin Chalfie. Just in the past decade, however, we have witnessed a truly remarkable expansion in the fluorescent protein palette, largely driven by the innovative studies from Roger Tsien's laboratory. Most of the fluorescent proteins that are commonly used today have been modified through mutagenesis to optimize their expression in biological systems. Continued efforts using directed evolution approaches will no doubt improve the spectral characteristics, photostability, maturation time, brightness, acid resistance, and utility of the fluorescent protein tags for cellular imaging.

The current thrust of fluorescent protein development strategies is centered on fine-tuning the current palette of blue to yellow variants from jellyfish, while simultaneously developing monomeric fluorescent proteins emitting in the orange to far-red regions of the visible light spectrum. We now have jellyfish proteins that span an 80-nanometer portion visible spectrum from deep blue to yellow-green, providing a wide choice of genetically encoded markers for studies in cell biology. Fluorescent proteins derived from Anthozoa species (corals and anemones), as well as other sources, span the entire visible spectrum and feature a wide range of useful properties. The unique optical highlighter properties of fluorescent proteins can allow the investigator to change the color or the emission state, providing unique opportunities to track the dynamic behavior of proteins in living cells and animals.

Still the "gold standard" in fluorescent protein technology, the enhanced version of GFP features a chromophore based on a para-hydroxybenzylidene substituted imidazolinone. The chromophore of the first reported red fluorescent protein extends conjugation into the polypeptide backbone to generate fluorescence in the longer wavelength regions. The ZsYellow fluorescent protein chromophore features a novel three-ring system and peptide backbone cleavage due to the substitution of lysine for serine as the first amino acid residue in the chromophore tripeptide. The final step in mKO chromophore maturations involves the formation of a novel five-member thiazole ring system when the Cys65 hydroxyl moiety attacks the carbonyl of Phe64 and cyclizes. In a manner similar to mKusabira Orange, mOrange chromophore maturation involves the formation of a novel five-member oxazole (rather than a thiazole) ring system.

http://nic.ucsf.edu/FPvisualization/

Methodology designed to circumnavigate the classical Abbe diffraction barrier in optical microscopy is rapidly advancing using both ensemble and single-molecule techniques.

Over the past several decades, fluorescence microscopy has become an essential tool for examining a wide variety of biological molecules, pathways, and dynamics in living cells, tissues, and whole animals. In contrast to other techniques (such as electron microscopy), fluorescence imaging is compatible with cells that are being maintained in culture, which enables minimally invasive optical-based observation of events occurring on a large span of timescales. In terms of spatial resolution, several techniques including positron-emission tomography, magnetic resonance imaging, and optical coherence tomography can generate images of animal and human subjects at resolutions between 10 centimeters and 10 micrometers, whereas electron microscopy and scanning probe techniques feature the highest spatial resolution, often approaching the molecular and atomic levels (see Figure ). Between these two extremes in resolving power lies optical microscopy. Aside from the benefits derived from being able to image living cells, the most significant drawback to all forms of fluorescence microscopy (including widefield, laser scanning, spinning disk, multiphoton, and total internal reflection) are the limits to spatial resolution that were first elucidated and described by Ernst Abbe in the late 1800s.

The Abbe diffraction limit (or at least the recognition of this limit) stood for almost a century before inventive microscopists began to examine how their instruments could be improved to circumvent the physical barriers in order to achieve higher resolution. Due to the fact that axial resolution is far lower than lateral resolution (by at least a factor of two), much of the work conducted in the latter part of the twentieth century addressed improvements to performance in the axial dimension. Researchers discovered that laser scanning confocal instruments produced very modest increases in resolution at the cost of signal-to-noise, and that other associated technologies (including multiphoton, structured illumination, and spinning disk) could be used for optical sectioning, but without significant improvement in axial resolution. An important concept to note, and one of the most underappreciated facts associated with optical imaging in biology, is that the achieved microscope resolution often does not reach the physical limit imposed by diffraction. This is due to the fact that optical inhomogeneities in the specimen can distort the phase of the excitation beam, leading to a focal volume that is significantly larger than the diffraction-limited ideal. Furthermore, resolution can also be compromised by improper alignment of the microscope, the use of incompatible immersion oil, coverslips having a thickness outside the optimum range, and improperly adjusted correction collars.

The most significant advances in superresolution imaging have been achieved in what is termed far-field microscopy and involve either spatially or temporally modulating the transition between two molecular states of a fluorophore (such as switching between a dark and bright state) or by physically reducing the size of the point-spread function used in the excitation illumination. Among the methods that improve resolution by PSF modification, the most important techniques are referred to by the acronyms STED (stimulated emission depletion; from the Stefan Hell laboratory) and SSIM (saturated structured illumination microscopy; pioneered by Mats Gustafsson). Techniques that rely on the detection and precise localization of single molecules include PALM (photoactivated localization microscopy; introduced by Eric Betzig and Harald Hess) and STORM (stochastic optical reconstruction microscopy; first reported by Xiaowei Zhang). As will be discussed, there are many variations on these techniques, as well as advanced methods that can combine or even improve the performance of existing imaging schemes. Even more importantly, new superresolution techniques are being introduced with almost breathtaking speed (relative to traditional advances in microscopy) and it is not unreasonable to suggest that at some point in the near future, resolution of a single nanometer may well be attainable in commercial instruments.

This might just look like a microscope image of some strange, small life-form. But actually its a view of a massive 281-gigapixel image of a zebrafish embryo, which can be zoomed in on to show sub-cellular levels of detail. The image is the product of a new technique called virtual nanoscopy. The process involves stitching together nanometer resolution photographs of what's placed under the microscope, and the result is an image which can be explored a little like a Google Map.

Traditional microscopy is a powerful tool for imaging small objects, such as cells. However, anybody who has used one will know its limitations: a tiny field of view and a shallow depth of field. That makes it hard to get a sense of the three dimensional shape of objects like cells. One clever way round this is to make a hologram of the sample. The idea here is to split a laser beam in two, use one as a reference beam and bounce the other off the sample to record the pattern of phase shifts that this produces using a digital camera.

Recombining the beams produces an interference pattern that can be analysed to retrieve three-dimensional information about the sample in high resolution.

This method is clearly powerful. A traditional microscope records only variations in the intensity of light bouncing off a sample. The holographic technique records this and phase information and so obviously stores more information about the subject. With the appropriate image-processing software. it’s possible to change the depth of focus, correct optical aberrations and reconstruct the 3D shape of the sample. There's another advantage too. Digital holographic microscopy has the potential to be incredibly cheap. Various research groups have created devices for as little as $1000 or so.

Atsushi Shiraki at the Kisarazu National College of Technology in Japan, and a few pals, show how to cut the cost even further. These guys have built a digital holographic microscope using a web camera, a small solid state laser, an optical pinhole and free open source software. The total cost: just $250 (with the optical pinhole accounting for about half of this!). The entire machine is light and portable, fitting in the palm of the hand (details in the paper below).

That's a handy machine that could play a useful role both in field studies for professional researchers and in schools where value for money is a key factor in equipment purchases. It might also be a fun to put together at home.

Ref: arxiv.org/abs/1211.0336: Handheld and Low-Cost Digital Holographic Microscopy

Most plankton organisms are too small to be seen with the naked eye. But despite their size, they are vital in marine and freshwater ecosystems, serving as food for larger animals and as oxygen producers in the ocean’s carbon cycle. Microscopy has made it possible to photograph these tiny creatures in the lab, but capturing their behavior in their natural environment has been impossible—until now. Researchers in San Diego are developing the world’s first underwater video microscope, capable of imaging these miniscule organisms in 3D. When plankton are swept into the submersible microscope’s collection chamber, multiple cameras reveal the minute life-forms floating inside. The microscope has been engineered to capture detailed 3D images of plankton moving freely in the salt water chamber. These never-before-seen views of living plankton are a missing piece of the puzzle for scientists who study life in water. Observing activity on a microscopic scale will inform the bigger picture of interactions among creatures of all sizes in oceans, lakes, and rivers around the world.

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