Ultrasensitive imaging: iSCAT

We have been developing interferometric scattering microscopy (iSCAT) to fundamentally question the supposedly established limits in sensitivity, speed and precision for light microscopy beyond fluorescence. We currently exploit the unique capabilities of iSCAT to study:

1. Motor proteins and membrane dynamics

Biological function frequently relies not only on intramolecular dynamics, but also on the absolute and relative motion of nanoscopic objects such as virions or individual proteins. The function of the cell membrane requires at least temporary inhomogeneity while molecular motors continuously fight Brownian motion. All of these and many other systems have in common that an ideal way to study them is to observe them in action, ideally in real time. The difficulty has been that established single molecule imaging techniques based on fluorescence detection are orders of magnitude too slow to capture these dynamics with sufficient precision. Using light scattering, we can surpass these limits by many orders of magnitude while using label sizes comparable to single quantum dots.

An excellent example where these capabilities are of particular advantage is the study of molecular motors. Directly observing the stepping mechanism has been thought to be beyond the capabilities of optical microscopes, yet we have recently shown that iSCAT can record motion with simultaneous nanometer spatial and sub-ms temporal precision. This has allowed us to directly measure the stepping mechanism of myosin 5, revealing a highly constrained diffusional search that helps us to understand the basis of highly efficient processive motion on the nanoscale. 

For more details see our recent publications in eLife and Nano Letters.

2. Label-free sensing down to single molecules

Single molecule detection is predominantly performed using fluorescence as a contrast mechanism. Most biological species, however, are intrinsically non-fluorescent, requiring labelling. Together with limited photophysics, there is much motivation to develop optical sensors operating at the single molecule level without any labels.

We have recently shown that iSCAT is capable of detecting, imaging and even tracking individual, unlabelled proteins through light scattering alone. This proof-of-principle study paves the way for a wide variety of applications not only in biosensing, but also for completely new avenues for studying protein-protein and protein-substrate interactions.

For more details, see our recent publication in Nano Letters.

3. Label-free imaging of nanoscale dynamics in condensed matter physics

The high sensitivity of iSCAT to refractive index perturbations - in the extreme a single protein in water as discussed in (2) can also be used to study nanoscale dynamics in situ. We are interested in using these capabilities to study phase transitions that are otherwise unobservable, such as lipid bilayer formation, or nanoscopic phase separation in bilayer membranes.

For more details, see our recent publication in ACS Nano.

Ultrafast Imaging: Time-domain Raman

1. Energy flow in molecules: vibronic coupling, singlet fission and charge transfer

Light-induced processes require the efficient conversion of photon energy into nuclear or electronic motion. Although the first step almost invariably involves near instantaneous movement of an electron from a ground to an excited electronic state, the desired function usually only happens after a considerable time-delay and is subject to competition with other relaxation processes. We are particularly interested in using vibrational coherence as a probe to study the origins of efficient energy flow in molecules. To achieve this, we have been developing time-domain Raman spectroscopy to monitor nuclear wave packet motion directly. 

For more information, see our recent publications in Nature Physics (singlet fission), PRL (internal conversion) and JACS (conical intersections).

2. Energy flow on nanometer length and femtosecond time scales

We use ultrashort visible pulses of light (<10 fs) to study electron motion on the nanoscale. We are particularly interested in what happens on timescales before electronic dephasing has taken place (<50 fs), which has been traditionally too short to observe experimentally, in particular in combination with direct optical imaging. 

3. Time-domain Raman microscopy: label-free imaging

We aim to transfer our experience with time-domain Raman spectroscopy into an imaging modality with unprecedented imaging speed, ease and sensitivity.

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