This post is copied from my English scientific blog on wordpress, Adelboden winterschool 2010: bridging physics with biology

On 26th to 29th of January, I attended a winter school together with other 4 group members in Adelboden in Switzerland. The winter school was organized by LCPPM (which stands for Laboratory of Physical Chemistry of Polymers and Materials) lead by Prof. Horst Vogel in EPFL. So far as I know, H.V. initiated this tradition almost 20 years ago, while this time the purpose of setting it up is mainly for seeking collaborations among attending groups to apply newly developed tools to investigate and answer biomedical problems.

This year’s winter school started with the evening session on 26th from the introduction of NK cells by Prof. Petter Höglund in Sweden, followed up by Prof. Werner Held in Lausanne and Prof. Daniel M Davis in London who are also working in immunology related fields. Being in Karolinska Institute where NK cell is first discovered, Petter pioneered in a vast range of studies in understanding of NK cell functionality (sorry for not mentioning any details of his studies, but trust me that he is recognized as a very good scientist if not the best in the field). Werner, also considered as a immunologist, is the first one to verify the existence and the functional role of cis interaction of Ly49 A inhibitory receptor to the MHC class I molecule on the NK cell itself [%Doucey, M., Scarpellino, L., Zimmer, J., Guillaume, P., Luescher, I., Bron, C., & Held, W. (2004). Cis association of Ly49A with MHC class I restricts natural killer cell inhibition Nature Immunology, 5% (3), 328-336 DOI: 10.1038/ni1043]. The direct consequence of this is that less inhibitory receptors would be left to allow binding of MHC molecules on the target cells, thus less inhibitory signals can be generated and therefore killing can be initiated, though the expression level of MHC molecules on the target may be normal. Dan, in my view, is a biophysicist who applies various of microscopic tools to investigate immune synapse (when I wrote this article, I started to look for some of his information and his educational background actually support my opinion formed at the first sight). He is probably the first one to take seriously the nanotubes developed by NK cells to their targets, though he might not be the first one to have seen the phenomenon. The interesting thing is that NK cells extend some nanotubes (one may think of these nanotubes as extension of cell membrane or cytoskeleton) to reach the target cells which are probably a bit far away from the NK cells. Funny enough is that the NK cells try to drag the targets towards themselves while or course the targets always want to run away. The biological roles of developing these nanotubes is still vaguely understood. It is nevertheless eye-catching.

27th, the second day of the winter school, there were some people talking about odorant receptors. Not super interesting to me. It is however intriguing to know how fast some people respond to the most state-of-the-art experimental tools. There are already quite some people applying super resolution microscopic tools to tackle biological problems, namely PALM, STORM and structured illumination developed in the US and STED invented by Stefan W Hell in Göttingen. For instance, Dan’s group is applying all these techniques to study immunosynapses. Horst is also establishing PLAM/STORM in his lab. Since most of the research activities from Horst’s group are centered around odorant receptors, I assume the use of such super-resolution-microscopy is no exception. I may not skip mentioning that I am very deeply impressed by the vast knowledge of Prof. Horst Vogel. It is hard for anyone to overlook how many advanced techniques have been applied to biological studies in his lab, and to not be amazed by the level of his lab can maintain in each aspect. His lab activities, as a perfect example, reflect the trend of grater impact that physical and chemical methodologies can offer to biological investigations. And of course, it is by no means a singularity (I am not talking about gravitational singularity, so don’t make me wrong) in geographic distribution of science or in history . Go back to the winter school. During the evening session, my supervisor" Prof. Jerker Widengren":http://www.biomolphysics.kth.se/ gave an overview of the activities in our lab. He started from the basic introduction of fluorescence, went to FCS, and newly established modulation excitation method to monitor the populations of different energy states of the fluorophores built up from ground state upon laser excitation, and how to use so-obtained information to gain information about how molecules interact with the local environment [%Sandén, T., Persson, G., & Widengren, J. (2008). Transient State Imaging for Microenvironmental Monitoring by Laser Scanning Microscopy Analytical Chemistry, 80% (24), 9589-9596 DOI: 10.1021/ac8018735], and he also mentioned about other activities, like applying STED imaging along with single molecule multi-parameter spectroscopy and other methods to do early diagnosis of cancer, applying FCCS to study biomolecular interactions to gain quantitative information about how NK cells regulate their killing, etc.

On 28th, the third day, the morning session started from my introduction of inverse fluorescence correlation spectroscopy (IFCS) [Stefan Wennmalm, etc., Inverse-Fluorescence Correlation Spectroscopy. Anal. Chem., 2009, 81 (22), pp 9209-9215.] (for which, I was a coauthor.) I paused for quite a while at the beginning leaving the room freakishly silent because I was a bit too nervous to think. It was good that it went well after I passed the phase of being absent of my mind. For people who are not familiar with the principle of IFCS, I should say in contrary to look at bursts of fluorescence intensities when fluorophore diffuse through the focal volume, we look at sudden drop of intensities when dark particles pass through a highly fluorescent background (generated by high concentration of dyes), and therefore molecules under scrutiny no longer needs to be labeled. Tor Sandén, originally from our lab, and currently a postdoc in Horst’s lab, extended this method to allow detection of non-labeled particles down to 10 nm in diameter, and he gave a presentation of his work on the last day of the winter school. On the same day of my presentation, Sofia Johansson and Johan Strömqvist from our lab talked about the NK cell project which I had participated partly. Though this research mainly addresses quantitative understanding of NK cell functionality of regulating its molecular binding pattern, they (actually solely by Johan) also patched the lack of mathematical description of non-perfectly overlapping foci which was usually the case but largely ignored by the FCCS community, which as a result introduced error in interpreting the co-diffusion of different color-labeled species. Evangelos Sisamakis also from our group introduced the concept of single molecule multi-parameter spectroscopy which allow single molecule FRET and FCS measurements.
The evening session on 28th gave me a surprise. Prof. Detlev Schild from Göttingen presented a wonderful talk. What he presented is mainly from a recent publication of his group, Junek, S., Chen, T., Alevra, M., & Schild, D. (2009). Activity Correlation Imaging: Visualizing Function and Structure of Neuronal Populations Biophysical Journal, 96 (9), 3801-3809 DOI: 10.1016/j.bpj.2008.12.3962. I am amazed by the idea of their applying online correlation calculation of temporal fluctuation of fluorescent intensities in each pixel of the recorded image to trace Ca2+ flow in individual neurons. Due to the fact that each neural cell possess different patterns of temporal fluorescence intensity fluctuation of Ca2+ sensitive dye, they were able to assign different artificial colors to different neurons. Nevertheless, the networking of different neurons is also possible to be provided by correlating the intensity fluctuations in different pixels. The correlation maps can provide the details of the fine structures of neural dendrites which were vaguely (actually not) visible in correspondent intensity images. Their line scanning microscope (in contrary to confocal microscopy which only allows point scanning ) is also very fast to acquire a 3D image of the neuronal network. It is truly a beautiful work, and deserves a more thorough introduction. I am thinking of writing another blog article about this work.

29th, it was the day to departure. But of course it does not mean there weren’t any interesting talks on that day. My colleague, Evangelos is very much fascinated by Horst’s group combining multiple optical trapping and microfluidic device to isolate cells and study their responses to different triggers carried along with the laminar flows in different islets in the same microfluidic device without interfering each other due to the unmixing property of laminar flow.

Quite recently, my former group lead by Prof. Lene Oddeshede in Niels Bohr Institute, University of Copenhagen published two findings with regards to optical trapping, which definitely will pave the road for its applications in bio-related scientific research.

I don’t know how many people in Nature network knows about optical trapping (OT). Simply allows me to say a few words about OT prior to my brief introduction of the work described in the two recent papers. OT is invented by Ashkin and now becoming the-state-of-the-art tool in investigation of biological problems, because it renders the researchers with high spatial resolution (nm) and well-calibrated force (picoNewton). It is generated by tightly focusing a laser beam to an oil immersion objective with high numeric aperture ( NA). Since light carries momentum, once the laser photons hit on the transparent microspheres, where will be momentum change of the light (i.e. due to reflection, or more precisely scattering, and refraction because the differences of the refractive index of the sphere from water environment) and this give rises to the forces. For intuitive purposes, we categorize the force to two main components, namely scattering force and gradient force. The scattering force pushes the sphere away from the focus of the trap, while the gradient force acts on the direction towards the highest light intensity, which is of course the focus. And the gradient force is main player which keeps the sphere in trap, and has to balance against the scattering component.

Normally, for small displacements the sphere in trap, the trapping potential is safely assumed to be a harmonic one. And the trapping force is approximated as a hookean spring with a linear dependence on the displacements from the equilibrium position. It is known that the lateral trapping force is much larger than the axial component, and this is because the larger gradient force in the lateral than the axial direction. And thus trapped sphere (normally a polystyrene bead is used) tends to escape the trap from the axial direction before the full lateral trapping force can be utilized.

As is known that spherical aberration (due to the mismatch of the refractive index of the immersion oil with the water environment) plays a dominant role in diminishing the trapping power other than the focusing ability of the objective, previously Lene together with Nader Reihani who was a postdoc in our group but now works as a professor in the Institute for Advanced Studies in Basic Sciences in Iran found that by correcting the spherical aberration larger forces than in ordinary case that one can obtain from a single optical tweezers (Optics Letters, vol.32, p.1998-2000). By doing this they use optical tweezers to pull stiffer tethers, or even to unfold some covalent binding in proteins or such they wanted to know what extreme can optical tweezers go to. By driving a piezoelectric stage on which the sample is held at certain velocity, the bead in trap is dragged to an extreme position before it escapes, Andrew C. Richardson (a PhD student in our group, who has done most of the experimental work) together with Nader and Lene found an increase of trapping stiffness beyond normal linear trapping regime. And the trapping potential is found to be well approximated by two harmonic functions depending on the displacement regime, instead of a harmonic potential which has been long assumed for OT. This work is published on Optics Express, vol. 16, p.15709-15717 .

If this is not so much interesting to biologists for its application in their research, another recently published paper might attract their eyes. Normally, the bead size for OT experiment is in micrometer range, because they are somehow relatively big in comparison with the biomolecules that are under study such that perturbation to a large extent is expected. Now, good news is that single nano-sized colloidal quantum dots which can emit light of different colours depending upon their sizes can be used for OT experiment. Not only smaller perturbation for the biological system is anticipated, one can also observe the fluorescence signals simultaneously, which no doubt will bring new excitement for biological research. This work is mainly done by Liselotte Jauffred (a PhD student) and Andrew C. Richardson, and published on Nano Letters, vol.8, p.3376-3380.

Hello everybody in nature network. Welcome to my blog! I am Lei, studying biophysics, particularly on single molecule detection. I am originally from China, but having my graduate education in Europe. I had one year experience with optical trapping in Niels Bohr Institute, University of Copenhagen. The research project is about using optical tweezers to study the motion of a single protein in the Escherichia coli outer membrane and its binding stiffness to the cell wall where the receptor is naturally anchored. I am currently visiting Max Planck Institute of Colloids and Interfaces to work on an optical trap setup to allow the position detection of nanometer precision. After this, I will come to Royal Institute of Technology as a PhD student to work presumably on fluorescence imaging and detection. A lot of activities will most probably be engaged in fluorescence correlation spectroscopy (FCS), fluorescence resonance energy transfer (FRET), total internal reflection (TIR), etc. for single molecule detection.
Personally, I am fascinated by what wealth of information can be brought by single molecules. Take FCS as an example, this technique primarily relies on thermal noises, by monitoring the fluorescence intensity fluctuations in a focal volume, one can 1) study the photodynamical properties of fluorescent dyes, such as its triplet state kinetics, photobleaching; 2) the translational and rotational diffusion of target molecules; 3) binding and unbinding kinetics of different molecules that people are interested in; 4) conformational fluctuations of biomolecules and so forth. I think single molecule detection is hot spot for study, and lots of interesting and exciting activities are getting blossomed. It is very crucial to understand molecule-molecule interactions, from which one might get some hint to uncover some fundamental mechanisms in biology. Also, more and more people are interested in understanding the origin of disease, e.g. cancers, from single molecular level, and thus early detection and diagnostics can be possible, and even further ways of curing the disease will be promising. But of course, to achieve such goals, single molecule study itself is not enough, since biology is far more complex, requiring not only the understanding of biology in single molecule level but also in a systematic way. But anyway, undoubtedly single molecule study is one important piece for the whole jigsaw game.
My blog will mainly cover this niche, talking about some interesting papers, new tools for study, and so forth. But I intend not to restrict my topics to these, because I believe any science discipline is not isolated from each other but interconnected, therefore I might also write something foreign but intriguing to me. To make the blog a bit more diverse and vivid, I might also present my personal perception of cultural, and educational differences between China and western countries. So the core of contents of my blog will be single molecule stuff, which will perhaps be coated with a layer of topics in other areas that I have interests and some protruding spikes made of cultural gradients. I am looking forward to your visits and discussions. Welcome to my site!