Live-cell spectroscopy provides an understanding of the movement of molecules in space and time within different neuronal compartments.

We have established techniques that permit real-time analyses of the diffusion and interaction of biomolecules in living cells.  We primarily use three techniques, all accomplished through multi-photon excitation: fluorescence photobleaching recovery (FPR), fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS).

These techniques enable us to derive diffusion constants by capitalizing on the well-defined focal volume associated with multi-photon excitation.   Multiphoton excitation also minimizes phototoxic damage to tissues.

Our system (Figure 1) utilizes excitation provided by a Mira 900F Ti:Saph laser with broadband optics (750-1000 nm) pumped by a 5 W Verdi laser (Coherent).  The beam is expanded to overfill the back aperture of a 60 X 1.2 NA water immersion lens on an Olympus IX-71 inverted microscope.  Emitted light is detected either with avalanche photodiodes (Perkin-Elmer) or GaAsP photomultiplier tubes (Hamamatsu).  Both provide TTL pulses to a hardware autocorrelator (autocorrelator.com) that provides on-line autocorrelation analysis of spontaneous fluctuations.  We have also integrated a 3-D piezo driven stage (Physiks Instruments) with nanometer resolution that is used                     Figure 1.  Click to enlarge.

to produce a high-resolution scanned image of fluorescently labeled cells. The image target is used to redirect the stage to points of interest for photon-counting measurements.

A further adaptation includes integrating a galvanometer based scanner (Camrbidge Tech.) into the system.  The scanning mirrors are driven by a 3 axis controller board (ISS) controlled through the Globals software (Laboratory of Fluorescence Dynamics, Irvine, CA).  This additional functionality will permit the acquisition of data from live cells and neurons for the analysis of diffusion and interactions using the recently developed techniques of raster image scanning correlation spectroscopy (RICS) and number and brightness (N&B) analysis.