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Light Microscopy Techniques


Figure 4 : Maximum intensity projection of live zebrafi sh transgenically expressing yellow fl uorescent protein (YFP), showing the optic nerves. Zebrafi sh head is 350 μ m wide. Image acquired in grayscale with 4 mm 25× objective. Image courtesy of Phillip Williams and Rachel Wong, University of Washington, Seattle, WA.


if they were a single excitation photon of high energy (short wavelength). T is combined-energy light excites the fl uorophore and stimulates fluorescence, just as if the fluorophore had been stimulated by illumination of higher energy. Because the long-wavelength illumination photons coincide only at the location of interest, multiphoton fl uorescence excitation occurs only at the plane of focus at a single point. This differs from confocal microscopy, where fluorescence is generated throughout the entire volume of the specimen. As a result, there is less phototoxicity and photobleaching, and the specimen can be imaged for a longer period of time. Multiphoton microscopy allows the imaging of brain slices, eye tissue, developing embryos, and other living tissue that is exceptionally thick or dense ( Figure 4 ).


Cell biologists, neuroscientists, physiologists, and other researchers who wish to study dynamic processes over time in living cells and tissues use multiphoton systems to keep specimens alive for as long as possible while imaging life processes. Oſt en, such studies last days, weeks, or even longer. Because of this, in-vivo imaging with multiphoton excitation is becoming one of the technique’s main applications.


Clearing Technologies


Until recently, it was impossible to image deeper than one millimeter into biological tissue using a light microscope, but the clearing methodologies developed over the past fi ve years have allowed researchers to image up to 8,000 nm (8 mm) deep using fixed specimens. These new systems use multiphoton microscopes, ultra-long-working-distance optics, and propri- etary clearing reagents to facilitate the imaging of contiguous fi xed tissue deep within dense organs.


In August 2011, a team at the RIKEN Brain Science Institute in Japan led by Dr. Atsushi Miyawaki published data in Nature Neuroscience [ 6 ] showing that they had created a clearing agent called SCALE, which, when used with a newly developed


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Figure 5 : Fast high-resolution imaging of clarifi ed brain using CLARITY- optimized light-sheet microscopy (COLM). The 1 mm × 2 mm × 5 mm volume was acquired from an intact clarified Thy1-EYFP mouse brain using COLM with 25× magnification. Camera exposure time of 20 ms was used; RI liquid 1.454 was used as the immersion medium. Image courtesy of Raju Tomer, Stanford University School of Medicine, Stanford, CA.


www.microscopy-today.com • 2016 May


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