July 5th, 12:00 - 3:00 pm, Warwick Systems Biology Centre

This workshop is dedicated to advancements in high resolution imaging techniques to study the interactions and dynamics of single molecules in biological systems (Organiser: Till Bretschneider, T dot Bretschneider at warwick dot ac dot uk).

This workshop is part of a series of Challenges in Imaging workshops, FLUO, within the EPSRC funded symposium year on Complexity and Systems Biology.

There is no formal registration, but please email Anne Maynard J.A.Maynard@warwick.ac.uk if you would like to attend.
Numbers will be restricted to 25 so please notify Anne of your wish to attend as soon as possible. Lunch will be provided.

 

Programme:

• 12:00 Lunch
• 13:00 "Structured Illumination and Image Inversion Interferometry"
  Rainer Heintzmann, Randall Division of Cell & Molecular Biophysics King's College London
• 14:00 “Single-molecule based methods for sub-diffraction optical imaging and biological dynamics” 
  Thorben Cordes, Biological Physics Research Group and Department of Physics, University of Oxford
  

Abstracts:

 

Structured Illumination and
Image Inversion Interferometry

K. Wicker¹⁾, M. Walde¹⁾, E. R. Oldewurtel¹⁾, S. Boehme²⁾, L. Hirvonen¹⁾,
O. Mandula¹⁾, S. Sindbert¹⁾, G.U. Nienhaus²⁾ and R. Heintzmann^1,3) 

1) King’s College London, Randall Division, London SE1 1UL, U.K.
2) University of Karlsruhe, Institute of Applied Physics, 76128 Karlsruhe, Germany
3) Institute of Photonics Technology (IPHT), 07745 Jena, Germany
Email: heintzmann@gmail.com

We present three recent high resolution fluorescence microscopy imaging modalities. Linear and non-linear structured illumination are based on illuminating with a fine grating and imaging the emission. Image inversion interferometry is based on single spot scanning and sending the emitted light through a special type of inteferometer.

An overview of our recent advances in high resolution fluorescence microscopy will be given. In structured illumination the sample is illuminated with a number of different patterns of light. In our case this is a series of sinusoidal grids at different grid positions and orientations generated by a programmable spatial light modulator or a physical phase grating. Experimental datasets acquired under these conditions and reconstructed results from these data, demonstrating a resolution improvement of up to a factor of two over standard widefield microscopy are presented.

The non-linear approach of saturating optical transitions (for structured illumination as well as beam-scanning approaches) has a great potential especially in combination with photo-switchable dyes such as the recently described IrisFP protein (manufactured in Ulrich Nienhaus’ group) or the Cy3-Alexa647 system used in Xiaowei Zhuang’s group [1,2]. An interesting approach is to push molecules into dark states in a patterned way shortly before imaging and exploiting the saturation of this transition.

Finally a method will be presented in which the emitted fluorescence of a confocal microscope passes through two separate paths [3]. These paths are interferometrically recombined in such a way that the images undergo a mutual rotation of 180 degrees. The self-interference of the fluorescent light is only constructive, if it originated from the optical axis of the scanning laser beam, thus leading to an efficient detection of a high resolution fluorescence images.

References 

[1] R. Heintzmann, T.M. Jovin, and C. Cremer. Saturated patterned excitation microscopy (SPEM) - a novel concept for optical resolution improvement. J. Opt. Soc. Am. A, 19, 1599-1609, 2002.

[2] L. Hirvonen, O.Mandula, K. Wicker, R. Heintzmann. Structured illumination microscopy using photoswitchable fluorescent proteins. Proc. SPIE 6861, J.-A. Conchello, C. J. Cogswell, T. Wilson, T.G. Brown (eds.), doi 10.1117/12.763021, 2008

[3] K. Wicker, S. Sindbert, R. Heintzmann, Characterisation of resolution enhancing image inversion interferometers, Optics Express 17, 15491-15501, 2009

 

 

“Single-molecule based methods for sub-diffraction optical imaging and biological dynamics”

Thorben Cordes (Oxford, Munich)*, Philip Tinnefeld (Munich), Achillefs Kapandis (Oxford)
Contribution from the Biological Physics Research Group and Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU, United Kingdom and Applied Physics – Biophysics, Faculty of Physics ,Ludwig-Maximilians-Universität München, Amalienstr. 54, 80799 München, Germany.

In this talk I will discuss two different state-of-the-art applications of single-molecule based fluorescence methods: (i) localization-based sub-diffraction optical imaging and calibration approaches; (ii) single-molecule Förster-resonance energy transfer (FRET) and alternating laser excitation (ALEX) for the study of biological dynamics.
At first a combination of a recently developed AFM-based molecule-by-molecule assembly (single-molecule cut-and-paste, SMCP) with sub-diffraction resolution fluorescence imaging is presented. Using “Blink-Microscopy”, which exploits the fluctuating emission of single molecules for the reconstruction of superresolution images, SMCP assembled structures with features below the diffraction limit are resolved. Artificial line patterns then serve as calibration structures to characterize parameters, such as the labeling density, that can influence resolution besides the localization precision of a single molecule. Finally, the adjustability of blink parameters is used to demonstrate the general connection of photophysical parameters with spatial resolution and acquisition time in super-resolution microscopy.

In the second part a single-molecule based FRET-assay is introduced that is capable of monitoring different stages of initial transcription of RNA polymerase in real time. The donor- and acceptor-fluorophores are placed into that region of the promoter DNA where the transcription bubble is formed. This suppresses FRET between the two fluorophores in the double-stranded form of the promoter DNA due to proximity and resulting static quenching. Upon formation of stable open complexes between promoter and RNA polymerase the static quenching is removed, fluorophores become bright and a high FRET efficiency can be observed. The feasibility of the assay to map transcription bubble expansion and compaction during abortive initiation with RNA synthesis and abortive release is demonstrated using different fluorophore pairs and labelling positions. The results are consistent with an existing assay reporting on downstream DNA movement toward the RNAP main channel. Finally, real-time studies were performed exploiting total-internal-reflection (TIRF) microscopy of surface-immobilized transcription complexes with a time-resolution in the lower millisecond time range. Recorded single-molecule trajectories allow us to perform kinetic analysis, determining the rates of RNA-synthesis and release, and their dependence on nucleotide concentration, temperature, and promoter sequence.