TRACKING OF SINGLE FLUORESCENT PARTICLES IN 3 DIMENSIONS - USE OF CYLINDRICAL OPTICS TO ENCODE PARTICLE POSITION

We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microsc ope containing a weak cylindrical lens in the detection optics and a m icrostepper-controlled fine focus. Images of small, fluorescent partic les were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through foc us. Particle z position was determined from the image shape and orient ation by applying a peak detection algorithm to image projections alon g the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the opt ical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artif icial solutions and living cells. In a solution of viscosity 30 cP, th e mean squared distance (MSD) traveled by a 264 nm diameter rhodamine- labeled bead was linear with time to 20 s. The measured diffusion coef ficient, 0.0558 +/- 0.001 mu m(2)/s (SE, n = 4), agreed with the theor etical value of 0.0556 mu m(2)/s. Statistical variability of MSD curve s for a freely diffusing bead was in quantitative agreement with Monte Carte simulations of three-dimensional random walks. In a porous glas s matrix, the MSD data was curvilinear and showed reduced bead diffusi on. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restrict ed. The water permeability in individual Chinese Hamster Ovary cells w as measured from the z movement of a fluorescent bead fixed at the cel l surface in response osmotic gradients; water permeability was increa sed by >threefold in cells expressing CHIP28 water channels. The simpl icity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.