Fluorescence Correlation Spectroscopy imaging
Major collaborators from both sides:
Guoxing Wang (SJTU), Jicun Ren (SJTU),
Jerker Widengren (KTH), Ozan Öktem (KTH)
Introduction, proposed research and major aims:
In Fluorescence Correlation Spectroscopy (FCS), intensity fluctuations are analysed, which
originate from a low number of fluorescent molecules excited by a focused laser beam. The
fluorescence intensity fluctuations reflect molecular fluctuations at thermo-dynamic
equilibrium. No external synchronization in the form of a perturbation is needed. The
technique can in principle offer information about any molecular dynamic process in the
nanosecond time range and longer that manifests itself as a change in fluorescence intensity.
The break-through of the FCS technique in the early 90´s [1] has inspired many research
groups to start using FCS, and pharmaceutical companies to use FCS and related techniques
for drug screening purposes. FCS is a very versatile technique in that many different
molecular dynamic processes, such as translational and rotational diffusion, chemical
kinetics, and binding reactions, as well as concentration and density of molecules, can be
followed in a wide range of environments, and down to a single molecule level.
Although the FCS technique by now is a relatively old and established single molecule
technique, it is still a technique under development. The possibilities to exploit fluorescence
fluctuations to retrieve molecular dynamics information are far from exhausted, and many
interesting fluorescence fluctuation approaches still remain to be established and applied. In
the last few years, much effort has been directed towards extending point-wise fluorescence
fluctuation analyses towards massively parallel readouts, in a matrix of detection points,
thereby providing both temporal and spatial fluorescence fluctuation data.
The biological relevance of such spatio-temporal fluctuation data is very large. A range of
cellular processes, such as nuclear organization, signaling in cell division, nerve cell signal
propagation, differentiation and migration all rely on diffusion processes, local dynamic
equilibria inside cells and non-uniform spatial distributions of molecules, which eventually
are all linked to each other. Current fluorescence imaging techniques can spatially resolve
protein abundance and to some extent molecular interactions, but the more or less static
views provided give far from the full information. Very recently, the first steps have been
taken towards merging spatial and temporal fluorescence fluctuation analyses for quantitative
imaging of protein diffusion and interaction in living cells [2]. However, the abilities to study
dynamic parameters are still relatively limited, and many molecular dynamic processes of
interest can not be resolved with the current time resolution.
In this project, we want to develop the necessary instrumentation, algorithms, software and
data processing tools to perform FCS on a massively parallel scale and with a further
improved time resolution. To reach these goals, several hurdles need to be overcome:
- An instrument has to be designed which can perform both excitation and detection in a
highly parallelized manner, offering a high signal-to-background measurement
capability, without causing significant photo-degradation of the sample. A prototype for
massive parallel FCS measurements is under construction in the Widengren lab at
KTH, which can meet these requirements.
- A detector array is needed, offering both high time resolution (frame-rate in the
microsecond time range) and high detection quantum yield. Latest generation cMOS
detector arrays can offer the required combination of time-resolution and detection
sensitivity. A detector array of this type can be offered to the project via collaboration
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with a research group at Delft University, Holland, and will be implemented in the
setup mentioned above.
With a detector array with up to a million of pixels, where for each pixel a correlation
function has to be calculated at a time resolution down to microseconds, and where for
each pixel it is also of interest to calculate the cross-correlation of the detected
fluorescence fluctuations of that pixel with the detected fluorescence fluctuations in the
other pixels, one understands that there is a huge amount of data to handle. Normal
readouts and programming procedures would be too slow. However, low-level
programming using field-programmable gated arrays (FPGA) can offer a remedy to this
problem. Expertise in FPGA programming will be provided by the Guoxing Wang
group at SJTU.
FCS analyses rely on single-molecule fluorescence fluctuations. The measurements
thus ultimately depends on how much fluorescence information that can be extracted
out of each fluorescent molecule per time unit. The research groups of Jicun Ren
(SJTU) and Jerker Widengren (KTH) have extensive experience of FCS measurements
using a wide range of different fluorophore marker molecules and strategies to optimize
this information flow. This experience will come well in place in this project, where the
demands on the fluorescence signal optimization will be high.
The huge amount of data to be handled calls for algorithms to minimize the load on
data processing. At the same time, single molecule fluorescence fluctuation data are
limited by the fluorescence emission rate of the individual molecules. The data can thus
be sparse both in the temporal and spatial domains. Ozan Öktem (KTH) and co-workers
at the Mathematics department at KTH have the expertise in devising algorithms to
handle and extract out the maximum information content out of sparse data, and will as
such provide an important contribution to this project.
Research plan and involvement of a CSC funded student:
In the proposed project, the hurdles above can to a large extent be addressed in parallel by
the respective research groups, given a close interaction and feed-back between the groups.
When the project is started, a CSC-funded PhD student can relatively soon be engaged. This
student would preferentially first work for two years with Guoxing Wang at SJTU,
developing the FPGA programming. After two years the student would go to KTH and work
for the second half of the PhD period in the group of Jerker Widengren, with the
experimental implementation, and then also being involved in some biomedical application
project using the setup developed. This will be done in collaboration with research groups at
the Karolinska Institute, which is closely located to KTH and with which close collaboration
already exists.
Taken together, fluorescence fluctuation imaging with high time resolution has a huge
potential in cellular biology and biomedicine. To realize an imaging tool with the required
performance, a combination of expertise in fluorescence spectroscopy/imaging, data
processing and image analysis is required. This is a very demanding task, but by combining
our expertise we have the prerequisites to bring this development to a successful end.
REFERENCES:
1. Rigler R, Mets Ü, Widengren J, Kask P “Fluorescence Correlation Spectroscopy with high
count rate and low background: analysis of translational diffusion” Eur Biophys J (1993),
22:169-175
2. Capoulade J, Wachsmuth M, Hufnagel L, Knop M “Quantitative fluorescence imaging of
protein diffusion and interaction in living cells” Nature Biotechnol (2011), 29(9): 835-842