Overview
Biological systems are both three-dimensional (3D) and dynamic. Visualizing the 3D structure and dynamics at the molecular scale is a current and critical need in biomedical research. In our group, we are developing and applying new fluorescence (far-field) microscopy techniques with spatial and/or temporal resolutions far exceeding current technology. Our laboratory houses the only 4Pi Microscope in the United States, which provides the highest 3D resolution currently commercially available in fluorescence microscopy. We are currently working on several biological efforts, including the characterization of the dendritic spines of neurons in unprecedented detail. We are also researching histone distribution and signaling response to double-strand breaks of DNA in the cell nucleus. Looking ahead, we are in the process of developing several novel microscopy techniques that will go beyond the 3D imaging capabilities of our 4Pi Microscope, creating the microscopes for tomorrow's biological research.
Scientific report
Towards Molecular Resolution in Light Microscopy
Biological systems are both three-dimensional (3D) and dynamic. Visualizing the 3D structure and dynamics at the molecular scale is a current and critical need in biomedical research. Unfortunately, current microscopic techniques cannot resolve 3D cellular sub-structures, such as chromatin in interphase nuclei, at the nanometer level on a millisecond time scale. Thus, radically enhancing the 3D spatial and temporal resolution is essential for new breakthroughs in biomedical research and a seminal challenge in modern light microscopy.
In our group, we are developing and applying new fluorescence (far-field) microscopy techniques with spatial and/or temporal resolutions far exceeding current technology. We focus especially on the 3D aspect of imaging and its application to biological questions.
Our laboratory houses the only 4Pi Microscope in the United States. 4Pi Microscopy provides the highest 3D resolution currently available in fluorescence microscopy. By utilizing two opposing high-numerical aperture objective lenses, 4Pi Microscopy increases the axial resolution of laser scanning microscopy ~6-fold. With 100-nm resolution along the optic axis (z axis), it allows far more defined images of cellular structures than conventional (confocal) microscopy. Recently, we have expanded the field of 4Pi Microscopy applications to cell nuclei and even tissue sections several tens of microns thick (see images).
The embedding of our group in The Jackson Laboratory and the Institute for Molecular Biophysics provides the ideal environment for interdisciplinary research. Close collaborations with biologists at The Jackson Laboratory and external institutions give 
rise to many cutting-edge biological projects. For example, together with Ken Knight (U Mass Med School) and Brian Bennett (U Mass Med School, Lake Placid Biologicals), we have characterized the spatial distribution of the histone H2AX and its phosporylated variant gamma-H2AX in the cell nucleus using 4Pi Microscopy. This phosphorylation event is an important early step in the repair of double-strand breaks of DNA. The 4Pi data allowed for identification and quantification of separate clusters of H2AX and gamma-H2AX at an unprecedented level of detail and led to the suggestion that H2AX clusters provide a platform from which signaling and repair events can be coordinated.
We are currently working on several biological applications of 4Pi Microscopy ranging from the extension of the H2AX-project to other histones and DNA-repair related proteins to the characterization of fine dendritic spines of neurons in 50-70 micron thick mouse brain tissue sections. For that purpose, we are optimizing preparation protocols, especially refractive index matching of cell nuclei and tissue.
Furthermore, we are in the process of developing several novel microscopy techniques that will go beyond the 3D imaging capabilities of our 4Pi Microscope, creating the microscopes for tomorrow's biological research.
Collaborators (list incomplete):
- Lindsay Shopland, The Jackson Laboratory
- Kevin Mills, The Jackson Laboratory
- Sam Hess, University of Maine
- Jeffrey Marchant, Tufts University
- Mark Ellisman, National Center for Microscopy and Imaging Research (NCMIR), University of California, San Diego
- Gabriel Popescu, Massachusetts Institute of Technology / U Illinois Urbana-Champaign
- Kendall Knight, University of Massachusetts Medical School
- Brian Bennett, University of Massachusetts Medical School / Lake Placid Biologicals
- Joachim Spatz, Max Planck Institute for Metals Research / University of Heidelberg, Germany
Lab staff
Principal Investigator: Joerg Bewersdorf, Dr. rer. nat., Research Scientist (The Jackson Laboratory), Adjunct Assistant Prof. (Dept. of Physics and Astronomy, U. Maine)
Research Assistant II: Mark Lessard, B.S., Michael Mlodzianoski, M.S.
Undergraduate Students: Manuel Juette (U. Heidelberg, Germany), Stefanie Kirschbaum (U. Heidelberg, Germany)
Publication listings
J. Bewersdorf, R. Pick and S.W. Hell. 1998. “Multifocal Multiphoton Microscopy”, Opt. Lett. 23(9): 655-657
J. Bewersdorf and S.W. Hell. 1998. “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz”, J. Microsc. 191: 28-38
C. Blanca, J. Bewersdorf and S.W. Hell. 2001. “Single sharp spot in fluorescence microscopy of two opposing lenses”, Appl. Phys. Lett. 79(15): 2321-2323
N. Martini, J. Bewersdorf and S.W. Hell. 2002. “A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy”, J. Microsc. 206(2): 146-151
S.W. Hell, C.M. Blanca and J. Bewersdorf. 2002. “Phase determination in interference-based superresolving microscopes through critical frequency analysis”, Opt. Lett. 27(11): 888-890
C.M. Blanca, J. Bewersdorf and S.W. Hell. 2002. “Determination of the unknown phase difference in 4Pi-confocal microscopy through the image intensity”, Opt. Commun. 206: 281-285
H. Gugel*, J. Bewersdorf*, S. Jakobs, J. Engelhardt, R. Storz and S.W. Hell. 2004. “Cooperative 4Pi excitation and detection yields 7-fold sharper optical sections in live cell microscopy”, Biophys. J. 87: 4146-4152 * contributed equally
J. Bewersdorf, R. Schmidt and S.W. Hell. 2006. “Comparison of I5M and 4Pi microscopy”, J. Microsc. 222: 105-117
A. Schauss, J. Bewersdorf and S. Jakobs. 2006. “Fis1p and Caf4p, but not Mdv1p, are required for a polar localization of Dnm1p clusters on the mitochondrial surface”, J. Cell Sci. 119: 3098-3106
R. Medda, S. Jakobs, S.W. Hell, J. Bewersdorf. 2006. “Enhanced high-resolution imaging with quantum dots in 4Pi microscopy”, J. Struct. Biol. 156: 517–523.
J. Bewersdorf, A. Egner, S.W. Hell. 2006. “Multifocal multiphoton microscopy” in J. Pawley, Handbook of Biological Confocal Microscopy, Springer, New York
J. Bewersdorf, A. Egner, S.W. Hell. 2006. “4Pi-microscopy” in J. Pawley, Handbook of Biological Confocal Microscopy, Springer, New York
J. Bewersdorf*, B.T. Bennett*, K.L. Knight. 2006. “H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy”, Proc. Nat. Acad. Sci. 103: 18137-18142. * contributed equally
M. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, S.W. Hell. 2007. “Wide-field sub-diffraction RESOLFT microscopy using fluorescent protein photoswitching”, Microsc. Res. Techn. 70(3): 269-80.
R. Khanna, Q. Li, J. Bewersdorf, E.F. Stanley. 2007. “The Presynaptic CaV2.2 Channel-Transmitter Release Site Core Complex”, Eur. J. Neurosci. 26: 547-559.
