Coatings of semiconductor quantum dots for improved visibility of electrodes and pipettes
Inventors
Medintz, Igor L. • Andrasfalvy, Bertalan K. • Susumu, Kimihiro • Delehanty, James B. • Huston, Alan L. • Macklin, John J. • Barbic, Mladen
Assignees
Publication Number
US-9880149-B2
Publication Date
2018-01-30
Expiration Date
2034-02-05
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
A glass pipette such as an electrode for electrophysiological recording is coated with quantum dots. This greatly aids the ability to observe the glass pipette, particular in tissue as the quantum dots provide an excellent performance under two-photon illumination used to visualize objects at depths of hundreds of microns.
Core Innovation
The invention provides a method and device wherein a glass pipette, suitable for electrophysiological recording, is coated with luminescent semiconductor quantum dots (QDs). This coating enables visualization of the pipette tip under two-photon microscopic illumination, facilitating its use for electrophysiological recording and calcium sensing. The QD coating produces a bright fluorescence signal at the pipette tip, allowing observation of the pipette even at depths of several hundred microns in brain tissue, which enhances visibility compared to conventional methods using fluorescent dyes inside the pipette.
The core problem addressed is the difficulty in visualizing very small glass pipettes during electrophysiological recording, particularly during in vitro or in vivo recordings in brain tissue at depths of several hundred microns. Traditional methods rely on filling the pipette with fluorescent dyes which are ejected and accumulate, reducing visibility and causing complications. Fluorescent dyes also have small two-photon action cross sections, require high laser power, are prone to photobleaching, and may cause tissue damage. Furthermore, dye use is problematic when cells already express fluorescent proteins. Hence, there has been a need for a brightly fluorescent glass pipette visible under two-photon illumination without the drawbacks of dye ejection.
This invention overcomes these challenges by coating the pipette tip with hydrophobic quantum dots that strongly adhere to glass, optionally embedding the QDs in a polymer matrix dissolved in an organic solvent. The QD-coated pipette allows for continuous and bright visualization under two-photon excitation at reduced laser power, shows enhanced durability and stability, and does not impair electrophysiological measurements or cell viability. This provides a robust and reusable visualization technique for patch clamp electrodes and related probes during deep tissue recordings.
Claims Coverage
The patent includes two independent claims focusing on methods of visualizing biological samples using glass pipettes coated with quantum dots.
Method of coating and visualizing a glass pipette tip with quantum dots under two-photon illumination
The method includes providing a glass pipette with a tip and back, contacting the tip with hexane while applying positive pressure at the back, then contacting the tip with quantum dots suspended in an organic solvent under positive pressure at the back to coat the tip with quantum dots, confirming the tip is not clogged, applying the coated tip to cells or tissue, visualizing the tip under two-photon microscopic illumination to cause the quantum dots to fluoresce, positioning the tip to avoid compromising cells, and performing electrophysiological recording.
Method of coating and visualizing a glass pipette tip with quantum dots for calcium sensing
Similar to the first method, this includes providing a glass pipette, contacting the tip with hexane under positive pressure, coating the tip with quantum dots in an organic solvent under positive pressure, confirming the tip remains unclogged, applying to cells or tissue, visualizing the tip under two-photon excitation to generate fluorescence, positioning to avoid cell damage, and using the pipette to perform calcium sensing.
The independent claims cover methods of preparing a glass pipette coated with quantum dots using organic solvents and positive pressure, visualizing this pipette under two-photon microscopy to obtain fluorescence signals, and applying the pipette for electrophysiological or calcium recording in biological samples.
Stated Advantages
Quantum dots provide high quantum yields and large two-photon absorption cross-sections, enabling bright fluorescence signals at sub-milliwatt excitation powers.
Quantum dot coatings are highly resistant to photobleaching and chemical degradation, ensuring durable and stable probe visualization over extended periods.
The QD coating strongly adheres to the glass pipette surface, resisting detachment even in vivo, and can be embedded in polymers for enhanced robustness.
The fluorescent signal from QDs is substantially brighter than conventional fluorescent dyes used inside pipettes, improving visibility deep in tissue with lower laser powers.
QD coatings do not interfere with seal formation or electrophysiological recordings, ensuring that neuronal electrical activity and calcium sensing remain uncompromised.
QD-coated pipettes can be prepared rapidly, easily, in multiple emissive colors, and stored without special conditions for long periods.
Documented Applications
Use of QD-coated glass pipettes for electrophysiological patch clamp recordings of neuronal cells in vitro, ex vivo brain slices, and in vivo brain tissue.
Visualization of pipette tips during two-photon microscopic imaging while performing electrophysiological measurements and calcium ion sensing in neuronal cells.
Simultaneous multi-color imaging using pipettes coated with different quantum dot emissions to monitor multiple cells concurrently.
Coating and visualization of various probes and pipettes such as extracellular probes, sharp electrodes, viral injection pipettes, juxta-cellular and extracellular probes, and probes for single cell electroporation where probe visibility is beneficial.
Interested in licensing this patent?