Single-molecule Fluorescence Spectroscopy^1,2

Single-molecule spectroscopy builds up a statistical macroscopic picture of a population, by spectroscopically probing the behaviour of its constituents. This approach often uncovers additional complexity in the population, which may be obscured and averaged out in bulk measurements. The vast majority of optical single-molecule spectroscopic techniques utilise laser-induced fluorescence to detect the presence of a molecule. In order for there to be, on average, less than one molecule in our laser excitation field, we must optimise several factors including:

• The size of the laser probe region.
• The concentration of the desired fluorescent species in this probe region.
• The signal to noise- maximised using bright, fluorescent organic dyes tagged to the molecules of interest.

Confocal Microscopy

In confocal microscopy, a laser beam is focussed to a diffraction limited spot through a high numerical aperture microscope objective, and unwanted signal from regions outside the laser focus are rejected using a small pinhole. Signal from the resulting probe volume (< 1 fL) is detected using very sensitive detectors capable of counting individual photons, known as avalanche photodiodes (APDs). This allows single-molecule detection to operate in the pM concentration regime, meaning only very small quantities of sample are required.

Two-Colour Coincident Fluorescence Detection (TCCD)^3,4,5

Two-colour coincident fluorescence detection (TCCD), a technique developed in the Klenerman group, detects the fluorescence from red- or blue- labelled species as they diffuse through the overlapped confocal volumes of two spectrally separated laser beams. Temporally coincident photon “bursts” in the red and blue channels give us information about the level of association between the two labelled molecules.

There are few physical techniques for monitoring protein associations, therefore this method has led to a number of ongoing collaborative projects looking at diverse systems and problems, including: the telomerase enzyme, early formation of amyloid fibrils, protein folding, the composition of virus particles, and protein associations in the membranes of live T cells.

Total Internal Reflection Fluorescence (TIRF) Microscopy⁶

Total Internal Reflection Microscopy utilises the evanescent field created when light is reflected off an interface formed between materials of different refractive indices, at angles greater than the critical angle (for glass/water ~ 61^o). This non-propagating evanescent field is of the same wavelength as the reflected radiation, and decays within a distance of < 100 nm from the interface. In conjunction with sensitive CCD cameras, this technique can be used to selectively excite, and monitor, fluorescent particles near the surface, and is of particular use for studying proteins in the membranes of cells.

An ongoing TIRF-based project examines membrane diffusion in boar sperm. A very low number of fluorescent tags are delivered to the desired region of the sperm by nanopipette dosing (see below), so that on average 1 or 2 molecules attach to the surface. Tracking software follows their trajectory, giving information about membrane structure and diffusion.

Studies Using Nanopipettes

Nanopipettes are small glass pipettes with internal tip diameters as low as 10 nm, which find a range of applications in the Klenerman group. When a voltage is created between electrodes placed in the pipette and the bath, a current is formed by ions being channelled towards the pipette tip along their dielectric glass confines. As the pipette tapers, the electric field strength increases, leading to large electric fields near the tip. Charged species experience a number of different forces in this tip region, which can be tuned to control the species’ flow.

Trapping Molecules and Localised Dosing^7,8

Different forces acting at the tip favour flow in different directions, and show different dependencies on the electric field strength. Through carefully tuning the voltage, opposing forces can be made to balance so that charged species are ‘trapped’ at the tip. If voltage pulses are applied away from this value, species can be made to controllably flow from the tip for the duration of the pulse, which is useful for localised delivery of reagents.

The image on the left shows fluorescent molecules trapped and excited at the pipette tip. Changing the voltage allows dosing, demonstrated by the array of fluorescently labelled DNA shown in the central picture. This principle can be extended to create more elaborate pictures such as the University crest (top of poster) and “Degas’ Dancers” above.

Scanning Ion Conductance Microscopy (SICM)⁹

At voltages which allow charged species to flow from the tip, bringing the nanopipette within a short distance of a surface (roughly equal to the inner pipette radius) impedes the flow of current. This change in current can be used as the feedback for a type of scanning probe microscopy called Scanning Ion Conductance Microscopy (SICM). This is capable of creating a topographic image of a cell without distorting the membrane, a problem found in force-based feedback methods such as AFM. Additionally, the nanopipette can be used to apply pressure or suction to cells and monitor their response, or to measure the flow of ions through electrical channels in the membranes of live cells- a technique known as patch clamping.

This type of scanning probe microscopy has been used to image the surface of several cell types, including the image of epithelial cells from toad kidneys displayed above. Much of this work is carried out in conjunction with Prof. Yuri Korchev at Imperial College, London.

References