Spatio-temporal dynamics challenges from fluorescence data.

July 13-16^th July 2010. Warwick University, UK.

Titles and Abstracts:

Dan Axelrod, University of Michigan - Visualizing submicroscopic dynamics in cells: polarized TIRF and membrane deformation during secretion.

By utilizing combinations of the many rich properties of photons, new forms of optical microscopy can now visualize subtle dynamic features of samples, beyond classical thickness and density variations. These features include lateral motions, orientations and tumbling, binding kinetics and specific transient associations of previously ‘submicroscopic’ cellular structures and single molecules As a very particular example, we highlight the combination of total internal reflection fluorescence (TIRF) microscopy with polarization microscopy to study the dynamics of secretion from a cell. In the process releasing the contents of a secretory granule, the plasma membrane must become deformed before, during, and/or after the fusion event. The extent and timing and possible biochemical regulation of this membrane deformation is a crucial part of both exocytosis and endocytosis. To correctly interpret the results of such polarized TIRF microscopy experiments, a detailed understanding of the ability of a high aperture objective to capture near-field emission is required.

 

Paul Barber, University of Oxford - Automated high-throughput FLIM for the analysis of protein-protein interactions

The most highly resolved imaging technique for the study of live cells is fluorescence microscopy but the study of protein interactions over a few nanometres requires a higher resolution. Förster (or Fluorescence) Resonance Energy Transfer (FRET) acts over a few nanometres and can be detected optically with fluorescence microscopy. Fluorescence Lifetime Imaging (FLIM) of the FRET donor fluorophore is a robust way to detect FRET and give insight into protein interactions. We are building microscopes to collect “high-content” information, including FLIM, about a large numbers of cells in a “high-throughput” manner.

Results from live cells and tissue micro arrays will be presented from automated microscopes incorporating time-domain TCSPC FLIM. Novel hardware and software with a modular approach and scripting abilities allow us to work towards speed-optimized acquisition and ease of use to bring FLIM into the high-throughput regime.

FLIM can be a time consuming process as both overloading the detection electronics and photo-bleaching the sample are to be avoided; getting the most from the photons detected is an important consideration. We have worked on algorithms for the analysis of FLIM data for several years¹ and our techniques for analysis will be presented. These range from pixel by pixel analysis using a Levenburg-Marquardt algorithm to global analysis. Most exciting is the use of Bayesian techniques that offer improved fitting performance when photon counts are very low.

1 P R Barber, S M Ameer-Beg, J Gilbey, L M Carlin, M Keppler, T C Ng, B Vojnovic (2009) , “Multiphoton time-domain FLIM: Practical application to protein-protein interactions using global analysis”, J R Soc Interface 6 p. S93-S105.

Ernst Stelzer, EMBL Heidelberg - Fluorescence microscopy based on spatially modulated light sheets reduces phototoxic effects and estimates scattering properties

 

Most optical technologies are applied to flat, basically two-dimensional cellular systems. However,

physiological meaningful information relies on the morphology, the mechanical properties and thebiochemistry of a cell’s context (Pampaloni 2007). Cells require the complex three-dimensional

relationship to other cells (Pampaloni 2010). However, the observation of multi-cellular biological

specimens remains a challenge: Specimens scatter and absorb light, thus, the delivery of the probing

light and the collection of the signal light become inefficient; many endogenous biochemical

compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces

malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a

single plane is observed, the entire specimen is illuminated (Verveer 2007). Recording stacks of

images along the optical z-axis thus illuminates the entire specimen once for each plane. Hence,

cells are illuminated 10-20 and fish embryos 100-300 times more often than they are observed

(Keller 2008). This can be avoided by changing the optical arrangement. The basic idea is to use

light sheets, which are fed into the specimen from the side and overlap with the focal plane of a

wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an

azimuthal fluorescence arrangement uses two independently operated lenses for illumination and

detection (Stelzer 1994; Huisken 2004). Optical sectioning and no photo-toxic damage or photobleaching

outside a small volume close to the focal plane are intrinsic properties. Light sheet-based

fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be

used together with laser cutters (e.g. Colombelli 2009) and for fluorescence correlation

spectroscopy (FCS, Wohland 2010). During the last few years, LSFM was used to record zebrafish

development from the early 32-cell stage until late neurulation with sub-cellular resolution and

short sampling periods (60-90 sec/stack). The recording speed was five four Megapixel large

frames/sec with a dynamic range of 12-14 bit. We followed cell movements during gastrulation,

revealed the development during cell migration processes and showed that an LSFM exposes an

embryo to 200 times less energy than a conventional and 5,000 times less than a confocal

fluorescence microscope (Keller 2008). Most recently, we implemented incoherent structured

illumination in our DSLM (Keller 2010). The intensity modulated light sheets can be generated

with dynamic frequencies and allow us to estimate the effect of the specimen on the image

formation process at various depths in objects of different age.

Keller PJ, Schmidt AD, Santella A, Khairy K, Bao Z, Wittbrodt J, Stelzer EHK (2010) Fast high-contrast imaging of animal development with

scanned light sheet-based structured illumination microscopy, in press. Pampaloni F, Stelzer EHK., Leicht S, Marcello M (2010) MDCK cells are

increased in aerobic glycolysis when cultured on flat and stiff collagen-coated surfaces rather than in physiological three-dimensional cultures,

Proteomics, in press. Wohland T, Shi X, Sankaran J, Stelzer EHK (2010) Single Plane Illumination Fluorescence Correlation Spectroscopy (SPIMFCS)

probes inhomogeneous three-dimensional environments, Optics Express, 18(10):10627-10641. Colombelli J, Besser A, Kress H, Reynaud EG,

Girard P, Caussinus E, Haselmann U, Small JV, Schwarz US, Stelzer EHK (2009) Mechanosensing in actin stress fibers revealed by a close

correlation between force and protein localization, Journal of Cell Science, 122:1665-1679, Epub 30 April 2009. Keller PJ, Schmidt AD, Wittbrodt

J, Stelzer EHK (2008b) Reconstruction of zebrafish early embryonic development by Scanned Light Sheet Microscopy, Science, 322(5904):1065-

1069. Pampaloni F, Reynaud EG, Stelzer EHK (2007) The third dimension bridges the gap between cell culture and live tissue, Nat Rev MCB,

8(10):839-845. Keller PJ, Pampaloni F, Stelzer EHK (2007) Three-dimensional preparation and imaging reveal intrinsic microtubule properties, Nat

Methods, 4(10):843-846. Verveer PJ, Swoger J, Pampaloni F, Greger K, Marcello M, Stelzer EHK (2007) High-resolution three-dimensional imaging

of large specimens with light-sheet based microscopy, Nat Methods, 4:311-313. Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EHK (2004)

Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science, 305: 1007-1009. Stelzer EHK, Lindek S (1994)

Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta

microscopy, Optics Commununication, 111:536-547

George Patterson, NIH Bethesda - Development of fluorescent proteins for single molecule localization techniques.

In conventional biological imaging, diffraction places a limit on the minimal xy distance that two marked objects can be discerned. Consequently, resolution of target proteins is often two orders of magnitude greater than the spatial distribution of the molecules. Localization techniques, such as photoactivated localization microscopy (PALM), fluorescence-PALM (F-PALM), and stochastic optical reconstruction microscopy (STORM), are capable of optically resolving photoactivated subsets of proteins at mean separations of <50 nanometers. In PALM, subsets of individual photoactivatable fluorescent proteins (PA-FPs) are photoactivated and imaged until photobleaching. They are then localized by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. This position information is assembled into a higher-resolution image containing fluorescent molecules at molecular densities up to 10⁵ molecules/µm². Thus, PALM imaging offers structural and molecular density information within fixed cells beyond that of conventional diffraction-limited fluorescence imaging. Molecular localization techniques, such as PALM, F-PALM, and STORM, alone can provide more information than conventional diffraction-limited fluorescence microscopy, but combination with existing techniques extends their capabilities and has led to new approaches in the study of cell biology. Some of these techniques require photoactivatable, photoconvertible, or photoswitchable probes which can be turned on or turned off to maintain a density of fluorescing single molecules low enough to distinguish individual molecules. Our recent advances in developing these molecules for single molecule localization techniques will be discussed.

Christian Eggeling - Observing the Nanoscale Far-Field STED Microscopy

Fluorescence far-field microscopy is a very sensitive analysis tool. However its resolution is limited by the diffraction of light, meaning that similar objects closer than about the wavelength of light, i.e., about 200 nm cannot be discerned or adressed. We present concepts that break this barrier. The key of these nanoscale microscopy approaches is the exploitation of the fluorophore properties, in particular of their states. Specifically, by utilizing at least two distinguishable molecular states, such as a ‘bright’ and a ‘dark’ state, it is possible to ensure that the measured signal stems from a region of the sample that is much smaller than these 200 nm [1]. Examples are based on Stimulated Emission Depletion (STED), on the use of photoswitchable fluorescent markers [2], or on optical shelving into the marker’s dark triplet state [3]. The presented results range from imaging with macromolecular resolution [4] to fluorescence correlation spectroscopy (FCS) in dynamically reduced sub-diffraction foci [5] and are helpful in solving fundamental biological problems.

 

[1] S.W. Hell Science 2007, 316, 1153-1158.

[2] M. Hofmann, C. Eggeling, S. Jakobs, S.W. Hell PNAS 2005, 102, 17565-17569.

[3] S. Bretschneider, C. Eggeling, S.W. Hell Phys. Rev. Lett. 2007, 98, 218103.

[4] G. Donnert et al. PNAS 2006, 103, 11440-11445.

[5] L. Kastrup, H. Blom, C. Eggeling, S.W. Hell Phys. Rev. Lett. 2005, 94, 178104.

 

Gaudenz Danuser, Havard Medical School – Forces ans signals at the leading edge

Michael Unser, EPFL

Xavier Darzacq, IBENS, Paris

Gerhard Schuetz, Biophysics Institute, Linz - Addressing plasma membrane nanostructures by single molecule techniques

 

Current scientific research throughout the natural sciences aims at the exploration of the

Nanocosm, the collectivity of structures with dimensions between 1 and 100nm. In the life

sciences, the diversity of this Nanocosm attracts more and more researchers to the emerging

field of Nanobiotechnology. In my lecture, I will show examples how to obtain insights into

the organization of the cellular Nanocosm by single molecule experiments.

Our primary goal is an understanding of the role of such structures for immune recognition.

For this, we apply single molecule tracking to resolve the plasma membrane structure at subdiffraction-

limited length-scales by employing the high precision for localizing biomolecules

of ~15nm (1-5). Brightness and single molecule colocalization analysis allows us to study

stable or transient molecular associations in vivo (6). In particular, I will present results on the

interaction between antigen-loaded MHC and the T cell receptor directly in the interface

region of a T cell with a mimicry of an antigen-presenting cell (7).

1. Wieser, S., Axmann, M., and Schütz, G. J. (2008) Biophys J 95, 5988-6001

2. Wieser, S., Moertelmaier, M., Fuertbauer, E., Stockinger, H., and Schütz, G. J. (2007) Biophys

J 92, 3719-3728

3. Wieser, S., and Schütz, G. J. (2008) Methods 46, 131-140

4. Wieser, S., Schütz, G. J., Cooper, M. E., and Stockinger, H. (2007) Appl Phys Lett 91, 233901

5. Wieser, S., Weghuber, J., Sams, M., Stockinger, H., and Schütz, G. J. (2009) Soft Matter 5,

3287-3294

6. Moertelmaier, M., Brameshuber, M., Linimeier, M., Schütz, G. J., and Stockinger, H. (2005)

Appl Phys Lett 87, 263903

7. Huppa, J. B., Axmann, M., Mortelmaier, M. A., Lillemeier, B. F., Newell, E. W.,

Brameshuber, M., Klein, L. O., Schütz, G. J., and Davis, M. M. (2010) Nature 463, 963-967

Sandy Simon, Rockefeller – Dynamics of proteins in macromolecular machines

We have been using fluorescence to probe the dynamics of proteins in macromolecular machines. The work will be illustrated using examples from the assembly of the retrovirus HIV-1 at the plasma membrane and the dynamics of proteins in the nuclear pore complex. We will demonstrate how the dynamics can be used to generate quantative simulations to test models for macromolecular function.

Justin Molloy, NIMR - TIRF microscopy of single molecules inside live cells

Over the past decade, there have been remarkable advances in live cell imaging. In our work, we have developed methods to visualise individual protein molecules within living cells. The motivation is to use multi-parameter, single molecule, imaging to help understand molecular mechanisms and biochemical pathways in situ. Direct observation of single fluorophores enables the temporal and spatial trajectories of individual molecules to be recorded so that their distribution, diffusion coefficients and binding kinetics can be calculated. By recording the spatial trajectories of differentially labelled molecules, formation and disruption of molecular complexes can be studied. We have initially focused our efforts on making measurements of single molecule mobility and residency times at the plasma membrane; analysis of diffusional paths of individual molecules has allowed measurement of protein mobility, membrane properties and rates of binding and dissociation. The behaviour of M₁ muscarinic receptors, KCNQ1 potassium channels, and a molecular motor, called myosin 10, will be described in the talk.

Karsten Rippe, Heidelberg - Dissecting chromatin dynamics and epigenetic networks in living cells by fluorescence fluctuation microscopy

Deutsches Krebsforschungszentrum and BioQuant, Research Group Genome Organization & Function, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

The genome of eukaryotes is organized into a dynamic nucleoprotein complex termed chromatin that directs the establishment of different functional cell states. Both the DNA and the protein component of chromatin are subject to various post-translational modifications like DNA/histone methylation, as well as acetylation and phosphorylation of histones. These epigenetic marks define the cell’s gene expression program and can be transmitted through cell division. The interplay of highly dynamic binding of chromatin-interacting proteins and the establishment of certain patterns of epigenetic modifications can be dissected by nalyzing the protein mobility in living cells with fluorescence microscopy based approaches. In particular the combination of fluorescence bleaching and correlation methods in conjunction with an integrative multi-scale analysis of the corresponding data sets is ideally suited to determine parameters like intracellular concentration, association state, diffusion coefficient, kinetic binding and dissociation rate constants. However, the complex environment of the nucleus makes it difficult to separate contributions from chromatin binding, protein complex formation and the confinement of the accessible nuclear space from the mobility data. I will present our ongoing work on the acquisition and analysis of protein mobility and interaction maps in the nucleus, and will discuss the application of these approaches to dissect chromatin dynamics and epigenetic networks.

 

Erdel, F., K.P. Müller, M. Baum, A. Matveeva, T. Höfer, W. Wachsmuth, and K. Rippe (2010) Acquisition of protein mobility maps to dissect chromatin dynamics and epigenetic networks in living cells. Chrom. Res., submitted.

Heuvelman, G., F. Erdel, M. Wachsmuth, and K. Rippe (2009) Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy. Eur. Biophys. J. 38, 813-828.

Müller, K.P., F. Erdel, M. Caudron-Herger, C. Marth, B.D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe (2009) Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy. Biophys. J. 97. 2876-2885.

 

Zvi Kam