DRBIO Cover Image
April 2002 to September 2002
Multiphoton
Imaging the Molecular Dynamics of Living
The intrinsic fluorescence
of NADH (nicotinamide adenine dinucleotide in its reduced form) in mitochondria
is imaged by multiphoton microscopy to measure metabolic state of living
systems. Here, a few living cells in a primary culture from the hippocampus
of mouse brain are shown imaged by two-photon excitation at 737 nm with
100 fs pulse trains at 80 MHz. The NADH fluorescence (blue) primarily
in the cellular mitochondria are presented in a schematic background illustrating
the 0.5 µm diameter focal volume (white) of the focused (red) laser.
The dynamics of the changes of metabolic state throughout living brain
preparations during anoxia and recovery on re-oxygenation can be imaged
by a time series of images of the organ to show the rapid oxygen depletion
in active layers of the hippocampus and faster recovery in quiescent regions
on re-oxygenation.
(Karl Kasischke, Sam Hess, Harsh Vishwasrao and Kevin Hodgson, DRBIO,
Cornell University)
DRBIO Cover Image
December 2002-February 2003
"Non-linear
microscopy in living
brain tissue"
Selected
for the 2003 American Physical Society calendar. (Image: Karl
Kasischke)
The image shows
two-photon excited fluorescence from neurons (green) and from zinc-containing
vesicles (red) and second-harmonic generation from axonal microtubules
(blue). Anatomical region: dentate gyrus (rat hippocampus).
DRBIO Cover Image February 2003-May 2003
Zero-mode
waveguides for single molecule analysis at high fluorophore concentrations
Image relates to cover article in Science
on 31 January 2003. [274. Levene, M. J., J. Korlach, S. W. Turner, M.
Foquet, H. G. Craighead and W. W. Webb, Science 299,
682-686, 2003]
The image shows a pattern of light generated by a "zero-mode"
waveguide, a 50-nm hole in a thin aluminum film. Such a waveguide enables
observations of single molecules within zeptoliter optical volumes. Single
molecules of a DNA polymerase enzyme could be seen to incorporate fluorescent
nucleotides, as shown by the cartoon. [Illustration: K. Hodgson] .
DRBIO Cover Image,
May-June 2003
"Water-Soluble
Quantum Dots for Multiphoton
Fluorescence Imaging in Vivo"
Image
relates to article in Science
magazine 30 May 2003,
D.R. Larson et al.
Extremely bright, water-soluble quantum dots can be used to image capillaries
within the dermis with cellular resolution. In this image, collagen is
imaged by second harmonic generation (blue) and the quantum dots are imaged
by two photon fluorescence excitation (yellow). Red blood cells exclude
the quantum dots, forming shadows within the capillaries, which can be
monitored over time (yellow trace). Illustration:
Rebecca M. Williams
DRBIO Cover Image,
July-September 2003
Image relates to paper in the journal PNAS 10 June 2003,
D.A. Dombeck et al.
The active microtubule ensembles in the intrinsically polar cilia on the internal surface of the aqueductus cerebri in the brain stem are here imaged in a living slice by second-harmonic generation (green). This signal originates from polar structures when illuminated by infrared femtosecond laser pulses. Two-photon intrinsic fluorescence (red) is obtained simultaneously. llustration: Dan Dombeck, Harsh Vishwasrao, and Karl Kasischke.
DRBIO Cover Image,
October-December 2003
Image relates to cover article in the journal Nature, 23 Oct. 2003,
T. Baumgart, S.T. Hess, and W.W. Webb
DRBIO Cover Image,
December 2003 - February 2004
Image relates to article in the journal Nature Biotechnology,
Zipfel, W. R., R. M. Williams and W. W. Webb,
Nature Biotechnology 21(11), 1369-1377, 2003
The two-photon excitation volume. Axial and lateral views of Illumination Point Spread Function (IPSF) and IPSF2. Squaring the IPSF results in minimal wings relative to the center.
DRBIO Cover Image,
February 2004-March 2004
Image is the cover of the 28 Jan 2004 issue of the Journal
of Neuroscience. Cover article
is by Dombeck et al.
Second-harmonic generation microscopy image of a primary cultured Aplysia
neuron stained with the membrane dye DHPESBP.
March to May 2004
Image is of multiphoton microscopy GRadient INdex (GRIN) lens imaging. J. Neurophysiology 91 (4), 1908-12, 2004 , Levene et al. Copyright © 2004 The American Physiological Society.
Image of layer V neurons ~750 µm below the surface of cortex. Scale bar 10 µm.
DRBIO
Cover Image,
May to July 2004
Image: Schematic kinetic model demonstrates light- and pH-dependent interconversion
between different electronic states of GFP mutants. Cover for the Journal
of Physical Chemistry B, July 15, 2004. Article
by S. Hess et al.
Copyright © 2004 American Chemical Society.
DRBIO Cover Image,
August to September 2004
Image relates to Report in Science
[Kasischke, Vishwasrao, Fisher, Zipfel and Webb, Science 305(5680),
99-103, 2004].
It has long been suspected that activity dependent metabolism in the brain
(yellow plot) is partitioned between astrocytes (red cells) and neurons
(blue cells). Multiphoton fluorescence imaging of NADH now reveals that
early oxidative metabolism in neurons (blue plot) is eventually sustained
by late activation of the astrocyte-neuron lactate shuttle (red plot). Image
by K.A. Kasischke,
H.D. Vishwasrao,
and P.J.
Fisher.
DRBIO Cover Image
October 2004 to January 2005
By D.W. Piston and E. Elson. Copyright © 2004, SPIE.
DRBIO
Cover Image
January to April 2005
In the left panel, an image from a 19-day tendon collected in the forward direction shows mostly mature fibrils. In the right, colored panel, as indicated by their polarization, immature fibril segments are aligned to the fibril directionality. The backward-directed Second Harmonic Generation (SHG) is separated into two channels depending on the emission polarization (purple = perpendicular and yellow = parallel to the tendon axis . In the last case, the fundamental was polarized perpendicular to the tendon axis.
Citation: Williams, R. M., W. R. Zipfel and W. W. Webb, "Interpreting Second Harmonic Generation Images of Collagen I Fibrils," Biophysical Journal 88, 1377-1386, 2005.
Copyright © 2005, by the Biophysical Society.
April to June 2005
Excerpt: "I changed fields many times, not to avoid trouble, but to move on to new challenges. Each time I entered a new field, I have built on what I learned from earlier experiences. For example, my research in the 1960s on continuous phase transitions or critical phenomena, then the hot topic in chemical physics, guided our understanding of phase separations in membrane structure and function, which we study today. It is fun to see this kind of connection evolve."
BioTechniques Vol. 38, No. 4: pp 515 (Apr 2005)
July to September 2005
|
Caption: "Metabolic dynamics in the brain are imaged using the fluorescence of endogenous reduced beta-nicotinamide adenine dinucleotide (NADH). Fluorescence measurements, however, are complicated by the dependence of the quantum efficiency of NADH on its free/bound state. Time-resolved fluorescence anisotropy discriminates free/bound NADH and shows a preferential increase in free NADH during the normoxic (blue curve) to hypoxic (red curve) metabolic transition."
Illustration by H.D. Vishwarao, K.A. Kasischke, M.A. Williams and W.W. Webb.
Copyright © 2005, by the American Society for Biochemistry and Molecular Biology.
October 2005-January 2006
Caption: "Individual bright and dark water-soluble quantum dots. (Upper) 3D intensity plot of individual green-colored quantum dots labeled with red-colored organic dye molecules. The green-plus-red peaks (appearing yellow) indicate bright quantum dots and the red peaks indicate dark quantum dots. (Lower) Array of widefield images of individual bright and dark quantum dots trapped in agarose gel."
Illustration by J. Yao.
February-September 2006
Rebecca M. Williams, Andrea Flesken-Nikitin, Alexander Nikitin, Watt W. Webb and Warren R. Zipfel
October 2006-March 2007
Image at left relates to Yao, J., Munson, K., Webb, W. W. & Lis, J. T. (2006) Nature 442, 1050-1053.
The central question of molecular biology is to understand when, where and how every gene is expressed. However people have not been able to “see” directly the activation of a native gene in living cells. Multiphoton microscopy imaging of the polytene nucleus has successfully resolved individual gene loci and enables “watching” gene activation in real time. In the optical section series of a cell nucleus from Drosophila (fruit fly) salivary gland tissue which is under heat shock (36.5°C), heat shock factor (green) is bound to chromosomes (red) and is activating the transcription of heat shock genes. The direct visualization of transcription activation of native genes will have a profound impact on the fundamental understanding.
March 2007 to January 2008
Giant plasma membrane vesicles (GPMVs) derived from chemically-treated mammalian cells release from cytoskeletal constraints and undergo macroscopic phase separation below 25°C. Membrane proteins and lipids partition differentially into liquid-ordered and liquid-disordered phases, identified by fluorescent probes. Contrasting with ternary lipid model membranes, GPMVs reveal influence of their complex lipid plus protein mixtures, providing effective approaches to studying plasma membrane heterogeneity. Image by Tobias Baumgart, Mark A. Williams and Watt W. Webb.
