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Optics beyond the Abbe Limit
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Optics beyond the Abbe Limit

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Present State of Super Resolution Far Field Light Microscopy realized in the Christoph Cremer Lab (December 2011) …

Present State of Super Resolution Far Field Light Microscopy realized in the Christoph Cremer Lab (December 2011)
Optical Resolution OR (resolvable distance): OR ~ 5 nm (~ 1/100 λexc, from localization precision)
Structural Resolution (imaging capability): ● Mean (2D) distance between individual molecules actually detected: ~ 6 nm ( ~ 1/80 λexc) ● Density of individually detected Molecules : ~ 2,8 • 104/μm2 –
Multicolor- Localization Microscopy: ● 2 – 3 different molecules types ● 3D single Molecule Resolution inside cells (best values): 3D Observation Volume about 1*104 times smaller (i.e. 3D resolution 104 times better) than in conventional Light Microscopy (“Abbe-limit”)
Super Resolution Microscopy with viruses (single molecule imaging)

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  • 1. January 1, 2012 Letizia Mancino Heidelberg Jackson Lab Mainz Optics beyond the Abbe Limit: “Molecular Constellations“ C. Cremer Institute of Molecular Biology (IMB), Mainz/DEInstitut for Pharmacy & Molecular Biotechnology; Kirchhoff-Institute of Physics, University Heidelberg; Institute for Molecular Biophysics, The Jackson Laboratory,ME/USA For more information see www.imb-mainz.de
  • 2. Conventional Resolution Localization Microscopy From: C. Cremer et al., Biotechnology J. 6 (2011) 200 nm___ From: All scale bars 500 nm Y. Markaki et al., CSH Symposia 75 (2011) 100 nmAbove: Viruses; Below: 2 Protein types within a human cell nucleus. The crosses/circlesdenote positions of single molecules (exciting wavelength 488 nm)
  • 3. Principle of Spectrally Assigned Localization Microscopy(SPDM): Optical Isolation of Diffraction Images of individualMolecules: Determination of Gravity Centers 3 point-like objects in x,y plane with next neigbour distances 50 nm a) Labelling with same spectral signature b) Labeling with different unique spectral signatures B,G,R Computation (scalar Theory): NA = 1.4, λexc = 488 nm Optical Isolation by the “Supernova“ Mode: Induction of stochastically distributed short flashes of light emission byCremer et al. 1996, 1999, 2010, 2011 individual molecules
  • 4. Present State of Superresolution Far Field Light Microscopyrealized @ C. Cremer Lab (December 2011)- Optical Resolution OR (resolvable distance): OR ~ 5 nm (~ 1/100 λexc, from localization precision)- Structural Resolution (imaging capability):● Mean (2D) distance between individual molecules actuallydetected: ~ 6 nm ( ~ 1/80 λexc)● Density of individually detected Molecules : ~ 2,8 · 104/µm2- Multicolor- Localization Microscopy:● 2 – 3 different molecules types● 3D single Molecule Resolution inside cells (best values): 3DObservation Volume about 1*104 times smaller (i.e. 3Dresolution 104 times better) than in conventional LightMicroscopy (“Abbe-limit”)
  • 5. Some recent references:C. Cremer, Optics far Beyond the Diffraction Limit: From Focused Nanoscopy toSpectrally Assigned Localization Microscopy. Springer Handbook of Lasers and Optics,2nd edition (F. Träger, Edit.), in press.Y. Markaki et al. (2011) Functional nuclear organization of transcription and DNAreplication: a topographical marriage between chromatin domains and the interchromatincompartment. Cold Spring Harbor Symposia on Quantitative Biology 75: 1–18.doi:10.1101/sqb.2010.75.042.R. Kaufmann et al. (2011) Analysis of Her2/neu membrane protein clusters in breastcancer cells using localisation microscopy. J. of Microscopy 242: 46–54.G. Best et al. (2011) Structured illumination microscopy of autofluorescent aggregationsin human tissue. Micron 42: 330–335.C. Cremer et al. (2011) Superresolution imaging of biological nanostructures by spectralprecision distance microscopy, Biotechnology 6: 1037–1051.C. Cremer (2011) Lichtmikroskopie unterhalb des Abbe-Limits, Physik in Unserer Zeit 42:21 – 29.