Automated microscope-independent fluorescence-guided micropipette

Abstract

Glass micropipette electrodes are commonly used to provide high resolution recordings of neurons. Although it is the gold standard for single cell recordings, it is highly dependent on the skill of the electrophysiologist. Here, we demonstrate a method of guiding micropipette electrodes to neurons by collecting fluorescence at the aperture, using an intra-electrode tapered optical fiber. The use of a tapered fiber for excitation and collection of fluorescence at the micropipette tip couples the feedback mechanism directly to the distance between the target and electrode. In this study, intra-electrode tapered optical fibers provide a targeted robotic approach to labeled neurons that is independent of microscopy.

Fig. 1.

(a) System architecture. Excitation light from 405 nm laser is spatially filtered and sampled using a beam splitter. It is then coupled to the optical fiber. Fluorescence light follows the optical detection path where it is spatially filtered and focused onto a detector. (b) The optical fiber is attached to a linear actuator (yellow box) and enters the electrode holder through a ferrule (blue box). To visualize the beam profile, light exiting the tapered optical fiber is imaged in fluorescein under a FITC filter (red box). 50 $\mu$m scale bar. (c) Resistance through the micropipette is monitored while the linear actuator moves the tapered optical fiber towards the micropipette tip (left). During automated approach, the micropipette is raster scanned in a 2D plane and descends after each scan (right).

Fig. 2.

(a) Fluorescent bead with a 2 $\mu$m diameter illuminated by the fluorescence guided electrophysiology system. Scale bar represents 25 $\mu$m. (b) Reconstruction of bead using internal illumination. (c) Full width half maximum measurement of (a) and (b) using a Gaussian fit of microscope data (blue trace) and micropipette data (red trace).

Fig. 3.

(a) Overlayed image of brightfield and fluorescence (DAPI Filter Set). Scale bar is 25 $\mu$m. (b) Reconstruction of center neuron in (a). Z axis represents photon counts above the minimum count. (c) Transverse measurement of (b).

Fig. 4.

(a) Peak count positions during ROI scans to a $\sim$ 10 $\mu$m aggregation of fluorescent beads. (b) End points for the three trajectories. (c) Fluorescence as a function of descent.

Fig. 5.

(a) Final position after automated approach of stained B35 neuroblastoma cell. Scale bar represents 25 $\mu$m. (b) Max photon count/ms per scan during descent. (c) Trajectory of micropipette tip plotted over composite image of brightfield and fluorescence (DAPI Filter Set).