Optogenetic experiments require light delivery, typically using fiber optics, to light-gated ion channels genetically targeted to specific brain regions. Understanding where light is—and isn’t—in an illuminated brain can be a confounding factor in designing experiments and interpreting results. While the transmission of light, i.e. survival of forward-directed and forward–scattered light, has been extensively measured in vitro, light scattering can be significantly different in vivo due to blood flow and other factors. To measure irradiance in vivo, we constructed a pipette photodetector tipped with fluorescent quantum dots that function as a light transducer. The quantum dot fluorescence is collected by a waveguide and sent to a fiber-coupled spectrometer. The device has a small photo-responsive area (~ 10 um x 15 um), enabling collection of micron-resolution irradiance profiles, and can be calibrated to determine irradiance with detection limits of 0.001 mW/mm2. The photodetector has the footprint of a micro-injection pipette, so can be inserted into almost any brain region with minimal invasiveness. With this detector, we determined transverse and axial irradiance profiles in mice across multiple brain regions at 5 source wavelengths spanning the visible spectrum. This profile data is compared to in vitro measurements obtained on tissue slices, and provides a means to derive scattering coefficients for specific brain regions in vivo. The detector is straightforward to fabricate and calibrate, is stable in air storage > 9 months, and can be easily installed in an electrophysiology setup, thereby enabling direct measurement of light spread under conditions used in optogenetics experiments.
The simultaneous visualization, identification and targeting of neurons during patch clamp-mediated
electrophysiological recordings is a basic technique in neuroscience, yet it is often complicated by the
inability to visualize the pipette tip, particularly in deep brain tissue. Here we demonstrate a novel
approach in which fluorescent quantum dot probes are used to coat pipettes prior to their use. The strong
two-photon absorption cross sections of the quantum dots afford robust contrast at significantly deeper
penetration depths than current methods allow. We demonstrate the utility of this technique in multiple
recording formats both in vitro and in vivo where imaging of the pipettes is achieved at remarkable depths
(up to 800 microns). Notably, minimal perturbation of cellular physiology is observed over the hours-long
time course of neuronal recordings. We discuss our results within the context of the role that quantum dot
nanoprobes may play in understanding neuronal cell physiology.