Non-Imaging Optical Techniques
A variety of optical techniques have been developed over the years for experimental intervention into living specimens. These techniques have the advantage over more traditional experimental approaches, such as micromanipulation, that there is less chance of unwanted collateral damage and that probes can reach deep within specimens (provided the specimens are reasonably transparent).
Some years ago we developed cell ablation system for use with embryos, in particular C. elegans (Sulston and White, 1980 Dev Biol 78(2):577). Derivatives of this system are now widely used in many laboratories to study inductive cell-cell interactions in developing embryos. It was found that the optimum strategy for a cell ablation system that has high precision yet produces a minimum amount of collateral damage, was to use a pulsed source of around 1ns duration and a wavelength around 440nm. A nitrogen laser pumping a dye laser is currently the most favored irradiation source.
Fluorescent recovery after photobleaching
This technique has been used to measure intracellular movements such as the diffusion of proteins in membranes (Ladha et al., 1997 J Cell Sci 1997 110(9):1041) and the movements of microtubules during mitosis (Centonze and Borisy, 1991 J Cell Sci 100( Pt 1):205). Fluorescently-labeled target structures are illuminated by a high-intensity patterned source (typically a laser illuminating a line) for a short time in order to produce a bleached pattern. The structures are then imaged using low levels of irradiation to visualize the dynamic changes in the bleached pattern. From these data diffusion rates or movement velocities can be calculated.
Photoactivation of caged compounds
Several fluorophores (fluorescein, rhodamine green) and bioactive agents (calcium, ATP, neurotransmitters, calmodulin inhibitors) can be rendered inert by the addition of a molecular cage. The cage is designed such that it can be photolyzed by short-wavelength irradiation thereby releasing the fluorophore or bioactive agent (Theriot and Mitchison, 1992 J. Cell Biol. 119:367). Irradiation by a focused, transient beam of light allows the bioactive agent (e.g. a signaling molecule) to be released within a cell with high spatial and temporal precision. This technique is becoming a very powerful experimental tool for the cell and neuro biologist, particularly as more caged molecules are becoming available. Most currently favored caging techniques require irradiation at around 320nm for photolysis of the cage. There is considerable interest in the possibilities for multiphoton uncaging (Denk, 1994 PNAS 91(14):6629). As in the case of multiphoton excitation fluorescence imaging, multiphoton events only occur in significant abundance within the focal volume of an objective which is directing light derived from a high-peak power laser source into the sample. This gives the technique exquisite spatial localization as photolysis (and hence the release of a bioactive agent) will only occur in the focal volume of the objective.
Small particles (0.5m - 10m) may be trapped by radiation pressure in the focal volume of a high-intensity, focussed beam of light. This technique may be used to move small cells or sub-cellular organelles around at will by the use of a guided, focussed beam (Askin et al., 1987 Nature 330: 769). Ingenious systems, using optical trapping in conjunction with interferometry to measure small displacements, have been used to measure the force exerted by individual motor proteins (Kojima et al., 1997 Biophys J 73(4):2012). Optical trapping offers a variety of experimental possibilities. For example, a bead coated with an immobilized caged bioactive probe could be inserted into a tissue or even a cell and moved around to a strategic location by an optical trapping system. The cage could then be photolyzed by multiphoton uncaging. This would provide a non-diffusable localization of the bioactive probe at a time and place determined by the experimenter. The optimal wavelengths for optical trapping are in the 800nm -1100nm range. Typically powers of around 100mw are used.