I believe that everyone who once has studied biology, no matter what branches of biology, is overwhelmed by exhaustive details of complex pattern of interacting entities. There is no theory in biology in traditional biology textbooks, but only details of collections of phenomena!

With an engineering and physics trained mind, I hardly find it captivating to continue the paths of thoughts that biologists have paved ahead of us. In my views, the current reductionist view points held by the majority of biologists are way-off-track. However, it doesn’t mean that as contemplating the complex world I would argue reductionist view shall be completely abandoned. On the contrary of pealing away reductionist view point entirely, I am strongly convinced that reductionism operating on the integrated complex system will eventually thrive and prosper in the competition to possess the ultimate explaining and predictive power for biology as well as such problem as the origin of life.

I have long been influenced by Konrad Kauffman (who advocates second law of thermodynamics as what he calls “the law before law” and is deeply convinced by the explaining power of it, viewed as a “crazy” physicist by many scientists, and who I knew from the well-known Niels Bohr Institute) and started to hold the view that the non-equilibrium thermodynamics which explains spontaneous order formation together with Darwin’s selection principle is the best candidate to be the ultimate and unified theory of biology. On occasions, I discussed about pieces of my views with my colleague, Yang Yang and my girlfriend. Most fortunately, I recently got to know the book “The origins of order: self organization and selection in evolution” published in 1993 and written by prominent theoretical biologist Stuart Alan Kauffman who holds the same view but of course has more original and more-organized or systematic thoughts.

I have just started to read Stuart A. Kauffman’s book and therefore I will leave my detailed and more comprehensive comments to his thoughts and the book once I finish my reading. However, so far, from my very first impression, I can say it is a very thick book. Personally, I find his way of articulation and presentation of thoughts over-abundant. Anyway, I stop by here and leave my words to later updates on this topic. I welcome anyone interested in searching for a theory of biology join my reading experience.

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 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 and 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.

Anyway, the paper that I would like to introduce comes from Xiaohu Gao’s group in University of Washington, titled “plasmonic fluorescent quantum dots“.

For those who are not so familiar with quantum dots (QDs), I shall say that they are essentially nano-sized semi-conductors which can create charge-carrying electron-hole pairs upon excitation by photon energy which matches the energy band gap and emit photons via reunification of the electron-hole pairs. The good things about QDs compared with chemically synthesized fluorophores include that they are generally more photon-stable (less photon-bleaching), brighter, color tunable by size, and they can be excited by a broad wavelength range but emit light of a confined bandwidth. Of course, disadvantages always lie aside. Apart from blinking (so far as I understand, can be attributed to trapping of electrons at some point), what critically restricts their applications is their bio-incompatibility due to its toxicity arguably because of their sizes being too small to be sufficiently to be sensed by the biological systems and thus the resulted difficulty in excreting them.

Maybe I shouldn’t deviate the lines of story from the particular paper that I want to introduce.  This paper is actually primarily aiming at rendering the QDs some other functionality, i.e. coupling plasmons created by illuminating metal like gold by electromagnetic field with the fluorescence emitted by the QDs so that multiple sensing principles can be used.  Personally I know little about why the oscillation of electron gas density (plasma) is so interesting. What I know is that it is possible to utilize it to enhance some optical signals, or instance raman scattering, like what the surface enhanced raman scattering people are doing. Anyway, there are a lot of people studying plasmon,  and trying to couple it with optical field. I will find some time looking into the point why people got hooked up by plasmon study later on (later on means not in this blog entry). Nevertheless, let’s go back to the story.  Coupling plasmon to optical field is tricky to do.  And the main difficulties of doing this are also summarized in the article .  For my personal understanding, the major aspect is that the mechanism for enhancement of electromagnetic field in presence of plasmon is not fully understood. From fluorescence’s perspective, the plasmon can either quench the fluorescence or enhance it, depending on lots of parameters, like the distance between the gold particles (used to create plasmons by illuminating them with laser) and the fluorescent molecules (QDs in this case), the surface roughness of the gold particles, the polarization of excitation beam, etc.

They found the spacing of QDs to gold coating (or shell) is very crucial to ensure the fluorescence enhancement due to surface plasmon resonance.  This is done by controlling the thickness of lipid-PEG-COOH conjugate and fine tuned by some polyelectrolyte bilayers, namely cationic polyallylamine hydrochloride and anionic polystyrene sulphonate via layer-by-layer assembly, encapsulating  the QDs before coating them with gold. The lipid-PEG-COOH conjugate is needed to allow water-solubility to be used for biological studies.  In reality, what they did is more complicated than what I described here. Please refer to the paper for more detailed explanation.

Something nice about their product is that the optical properties are satisfactorily preserved. For instance, the fluorescent peak is not shifted, the quantum yield is not too much compromised compared with originally synthesized QDs, and the photon stability seems quite good. I should also not forgot mentioning that the polyelectrolyte bilayers help somehow with the quantum efficiency. According to the authors’ explanation, this  is mainly due to reducing the diffusion of gold ions on to the QD core surfaces.

What else should I say in the end? Well, it is nice to update my knowledge concerning anything relevant to my future career, though personally the paper I talked about is not super interesting. It is a nice work, anyway.

Here comes the main content for this blog entry:

I read this article (Biophy. J. Vol.93, 684-698, 2007, doi:10.1529/biophysj.106.102087) perhaps 3 or 4 months before. I think it is worth reading, if you have working on fluorescence cross correlation spectroscopy. And I have some personal comments on it, which I would like to share with those who have the interests.

Good points:
•  The use of single laser to excite two fluorescent proteins (FPs) with distinct emission wavelengths for cross correlation read out  inherently avoid problems associated with miss-alignment of focal planes when using two lasers of different wavelength.
• Using large stokes shift FPs or dyes diminishes the concern for photons bleeding through different channels (of different wavelength bandpass)
Bad points:
• It claims studying EGFR in physiological conditions without over-expressing the EGFR and without ligand stimulation, but they used chemicals like sodium azide, sodium fluoride (both of which are poisons) and 2-deoxy-D-glucose (which can inhibit cell growth because it can not participate in glycolysis) to inhibit endocytosis of EGFR (a type of membrane receptor).
Argument:
• Mathematically treating the count rates of labeled dimers as twice of monomers may not be physically right, due to self quenching effect which can normally take place between adjacent fluorophores.  But of course, for simplification purpose, it is not wrong.

Blogging is not new to me, since I have years of posting blog entries on the internet. However, it is the first time to run a personal science blog in English. I have been writing a similar blog with the same name in my mother tongue, Chinese,  from September 1st, 2007 (http://www.sciencenet.cn/u/AlecXu/).  With time passing by, I gradually get a more and more intensive feeling that writing science in Chinese is not an optimal option.  Since it inherently restricts the scope with whom I can share my thoughts, to only the people who are acquainted with Chinese language .  Now,  though this transition I am making could risk losing my pools of Chinese science readers (by just looking at the amazing number of hits, more than 170,000, which I have got so far ), I also have the chance to potentially interact with a broad audience and discuss with people of common interests.  Shameful to say that that amount of hits from my Chinese science blog  seldom generate meaningful comments because the visitors are generally not people from my field. Strangling in the dilemma of choosing more hits or more interesting comments, I lean to the later. Anyhow, I am happy to start from the scratch again.

My name is Lei Xu, currently doing my PhD with Prof. Jerker Widengren from Royal Institute of Technology in Stockholm. Our lab activities are mainly focusing on developing and applying fluorescence correlation spectroscopy and related methods to biological study.  I am involved in some projects of using various single molecule techniques to biomedical investigations, like breast cancer, Alzheimer’s disease, NK immunity, etc. My original background comes from Biological Engineering, but then I change to Biological Physics for my master and doctoral study to seek the underlying principles of  life. A bit irony is that perhaps more people are keen to directly see the values of their research which engineering is more blessed with.  I won’t hold resistance to it though. Simply understanding is more like the cup of tea of mine.