Ultra-High Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy (FPALM)

Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Final images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, in order to significantly advance the understanding of biological systems.

[1]  Samuel T. Hess,et al.  Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories , 2007, Proceedings of the National Academy of Sciences.

[2]  Mark Bates,et al.  Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes , 2007, Science.

[3]  S W Hell,et al.  Photochromic rhodamines provide nanoscopy with optical sectioning. , 2007, Angewandte Chemie.

[4]  Christian Eggeling,et al.  Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters , 2007, Biophysical journal.

[5]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[6]  J. Bewersdorf,et al.  H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy , 2006, Proceedings of the National Academy of Sciences.

[7]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[8]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[9]  Stephan J. Sigrist,et al.  Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release , 2006, Science.

[10]  S. Hell,et al.  Comparison of I5M and 4Pi‐microscopy , 2006, Journal of microscopy.

[11]  R. Tsien,et al.  The Fluorescent Toolbox for Assessing Protein Location and Function , 2006, Science.

[12]  V. Verkhusha,et al.  Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light , 2006, Nature Biotechnology.

[13]  Shimon Weiss,et al.  Using photon statistics to boost microscopy resolution. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[15]  V. Verkhusha,et al.  Photoactivatable fluorescent proteins , 2005, Nature Reviews Molecular Cell Biology.

[16]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[17]  R. Heintzmann,et al.  Superresolution by localization of quantum dots using blinking statistics. , 2005, Optics express.

[18]  Paul R. Selvin,et al.  Kinesin and Dynein Move a Peroxisome in Vivo: A Tug-of-War or Coordinated Movement? , 2005, Science.

[19]  Volker Westphal,et al.  Nanoscale resolution in the focal plane of an optical microscope. , 2005, Physical review letters.

[20]  Hilmar Gugel,et al.  Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy. , 2004, Biophysical journal.

[21]  A. Miyawaki,et al.  Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting , 2004, Science.

[22]  J. Wiedenmann,et al.  EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Konstantin A Lukyanov,et al.  Photoswitchable cyan fluorescent protein for protein tracking , 2004, Nature Biotechnology.

[24]  L. Mets,et al.  Nanometer-localized multiple single-molecule fluorescence microscopy. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[25]  T. Ha,et al.  Single-molecule high-resolution imaging with photobleaching. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[26]  W. Webb,et al.  Fluorescence Photoconversion Kinetics in Novel Green Fluorescent Protein pH Sensors (pHluorins) , 2004 .

[27]  Samuel T Hess,et al.  Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells. , 2003, Biophysical journal.

[28]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

[29]  D. Axelrod.  Total Internal Reflection Fluorescence Microscopy in Cell Biology , 2001, Traffic.

[30]  A. Miyawaki,et al.  An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[31]  George H. Patterson,et al.  A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells , 2002, Science.

[32]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[33]  Alexander Egner,et al.  Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Ahmed A. Heikal,et al.  Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid–base specificity ☆ , 2001 .

[35]  Irving L. Weissman,et al.  "Fluorescent timer": protein that changes color with time. , 2000, Science.

[36]  S T Hess,et al.  Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  C Cremer,et al.  Three‐dimensional spectral precision distance microscopy of chromatin nanostructures after triple‐colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome , 2000, Journal of microscopy.

[38]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[39]  W. E. Moerner,et al.  Single-Molecule Fluorescence Resonant Energy Transfer in Calcium Concentration Dependent Cameleon , 2000 .

[40]  W E Moerner,et al.  Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[41]  F. Pinaud,et al.  Ultrahigh-resolution multicolor colocalization of single fluorescent probes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[42]  M. Gu,et al.  Advanced Optical Imaging Theory , 1999 .

[43]  Agard,et al.  I5M: 3D widefield light microscopy with better than 100 nm axial resolution , 1999, Journal of microscopy.

[44]  W. Webb,et al.  Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  M. E. Cox.  Handbook of Optics , 1980 .

[46]  Tirunelveli S. Ramalingam,et al.  Nanoscale Complexity of Phospholipid Monolayers Investigated by Near-Field Scanning Optical Microscopy , 1995, Science.

[47]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[48]  W. Webb,et al.  Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes. , 1995, Applied optics.

[49]  S. Hell,et al.  Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit , 1995 .

[50]  David A. Agard,et al.  Sevenfold improvement of axial resolution in 3D wide-field microscopy using two objective lenses , 1995, Electronic Imaging.

[51]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[52]  W. Webb,et al.  Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes. , 1994, Applied optics.

[53]  S. Hell,et al.  Properties of a 4Pi confocal fluorescence microscope , 1992 .

[54]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[55]  D H Burns,et al.  Strategies for attaining superresolution using spectroscopic data as constraints. , 1985, Applied optics.

[56]  N. Thompson,et al.  Total internal inflection fluorescent microscopy. , 1983, Journal of microscopy.

[57]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[58]  Stanley R. Sternberg,et al.  Biomedical Image Processing , 1983, Computer.

[59]  W. Webb,et al.  Diffusion of low density lipoprotein-receptor complex on human fibroblasts , 1982, The Journal of cell biology.