Sunday, April 17, 2011
Further developments in the application of multi-photon microscopy have lead to a relatively new technology that promises to be a a new means for enabling better human diagnostic procedures and a better means for visualizing structures within the body, such as the brain, during surgical procedures. In the 1990s I collaborated with Scott Fraser's lab at Cal Tech using multi-photon microscopy to visualize the dynamics of synaptic movement in the living retina, which is a part of the brain (CNS) in the back of the eye. At the time, Fraser's multi-photon microscope was one of the few in the world. Now, not much more than a decade later, the technology is widespread and new variants are promising to change the way we visualize our world in many different applications, including human surgery.
Third Harmonics Generated (THG) microscopy is based on third harmonic light generated at the focal point of a tightly focused short-pulse laser beam. When the medium at the focal point is homogenous, the third
harmonic waves generated before and after the focal point interfere destructively, resulting in zero net THG. However, when there are inhomogeneities near the focal point, such asan interface between two media, the symmetry along the optical axis breaks and measurable amount of third harmonic is generated. Due to its nonlinear nature, the third harmonic light is generated only in a close proximity to the focal point. Therefore, high spatial resolution can be obtained, enabling THG microscopy to section the tissue and to construct three dimensional images of transparent samples. Because biological tissues have non-vanishing third order susceptibilities, THG microscopy can be utilized as a general-purpose microscopy technique, and is now being used as a surgical microscope in laboratory experiments. This means that different tissues, because of their varying optical properties, can be visualized with the THG microscope without the use of any staining procedures and can be performed in natural, live tissue. Below are two examples of relevant papers:
Opt Express. 2010 Mar 1;18(5):5028-40. doi: 10.1364/OE.18.005028. Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea. Olivier N, Aptel F, Plamann K, Schanne-Klein MC, Beaurepaire E. Laboratory for optics and biosciences, Ecole Polytechnique ParisTech, CNRS, and INSERM U696, 91128 Palaiseau, France
Proc Natl Acad Sci U S A. 2011 Apr 12;108(15):5970-5. Epub 2011 Mar 28. Label-free live brain imaging and targeted patching with third-harmonic generation microscopy. Witte S, Negrean A, Lodder JC, de Kock CP, Testa Silva G, Mansvelder HD, Louise Groot M.
Biophysics Group, Institute for Lasers, Life, and Biophotonics Amsterdam, VU University, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.
Monday, April 11, 2011
Modifying a protein from a plant much favored by science, researchers at the University of California, San Diego School of Medicine and colleagues have created a new type of genetic tag visible under an electron microscope, illuminating life in never-before-seen detail.
Led by Nobel laureate Dr. Roger Tsien, PhD, Howard Hughes Medical Institute investigator and UCSD professor of pharmacology, chemistry and biochemistry, a team of scientists radically re-engineered a light-absorbing protein from the cress plant Arabidopsis thaliana. When exposed to blue light, the altered protein produces abundant singlet oxygen, a form of molecular oxygen that can be made visible by electron microscopy (EM).
The work is published in the April 5 issue of the journal Public Library of Science(PLoS) Biology. Tsien was co-winner of the 2008 Nobel Prize in chemistry for his role in helping develop and expand the use of green fluorescent proteins (GFP), a tool widely employed in light microscopy to peer inside living cells or whole animals and observe molecules interacting in real-time.
Tsien said the development of the small, highly engineered Arabidopsis protein, dubbed “miniSOG,” may elevate the abilities of electron microscopy in the same way GFPs have made modern light microscopy in biological research much more powerful and useful.
The big advantage of EM is that it has always had much higher spatial resolution, that is you can see more detail, than light microscopy. You can achieve up to a hundred-fold higher useful magnification from EM than from light microscopy. The result has been extraordinarily detailed, three-dimensional images of microscopic objects at resolutions measuring in the tens of nanometers, tiny enough to meticulously render the internal anatomy of individual cells.
But current EM technologies do not distinguish or highlight individual proteins in these images. These can be tagged with GFP or other fluorescent proteins, but they are visible only with the limited resolution of light microscopy.
Existing EM images of cell structures are analogous to satellite or aerial photographs showing major landmarks and populations centers. Therefore with EM, difficulties arise when visualizing individual protein types and their location, just as most geographical maps do not show the location of individual classes of people, such as everyone with the surname Smith, or all of the orthodontists in an area. Tsien's new technique enables scientists to put beacons on just about any protein and visulaize a snapshot of the protein's location at the much higher resolution of EM.
To create this ability, the scientists began with a protein from Arabidopsis, a small flowering plant that has long used as a research model. The original protein absorbs incoming blue light, triggering biochemical signals that inform the plant how much sunlight it is receiving. Tsien rationally engineered the protein based on its atomic model so that it changes incoming blue light into a little bit of green fluorescence and a lot of singlet oxygen.
Established methods were then used to convert singlet oxygen production into a tissue stain that the electron microscope can see. The scientists tested the modified protein’s utility as an EM marker by first using it to confirm the locations of several already well-understood proteins in mammalian cells, nematodes and rodents, then used miniSOG to successfully tag two neuronal proteins in mice whose locations had not been known.
The UCSD scientists are optimistic that miniSOG will grant new powers to electron microscopy, permitting scientists to pursue answers to questions previously impossible to ask.
MiniSOG would be much appreciated by scientists who investigate cellular and subcellular structures including neuronal circuits at nanometer resolution in multicellular organisms, because previous methods have great difficulty in achieving both efficient labeling and good preservation of the structures under study.
However, EM will not replace light microscopy for a number of reasons.
When using miniSOG, the tagged proteins plus the landmarks are used to navigating. On the other hand, EM has the disadvantage that it gives a snapshot of cells before we sacrificed them (to make the image), whereas light microscopy can show the dynamics in live cells. Each technique has different complementary strengths and weaknesses.
The new electron microscopy technique reveals the previously unknown locations of two neuronal proteins called SynCAM1 and SynCAM2. The first is an adhesion protein found at the synapse – or communications link – of neurons sending information. Its close relative, SynCAM2, is used by neurons receiving information. Neurons that send information are distinguishable because they contain synaptic vesicles, which are used to store neurotransmitters for communications use. In these images, the vesicles resemble small hollow circles.