Caglar M et al. (2019), All-Optical Detection of Neuronal Membrane Depo...

XB-ART-57110

Nano Lett 2019 Dec 11;1912:8539-8549. doi: 10.1021/acs.nanolett.9b03026.

Show Gene links Show Anatomy links

All-Optical Detection of Neuronal Membrane Depolarization in Live Cells Using Colloidal Quantum Dots.

Caglar M , Pandya R , Xiao J , Foster SK , Divitini G , Chen RYS , Greenham NC , Franze K , Rao A , Keyser UF .

???displayArticle.abstract???
Luminescent semiconductor quantum dots (QDs) have recently been suggested as novel probes for imaging and sensing cell membrane voltages. However, a key bottleneck for their development is a lack of techniques to assess QD responses to voltages generated in the aqueous electrolytic environments typical of biological systems. Even more generally, there have been relatively few efforts to assess the response of QDs to voltage changes in live cells. Here, we develop a platform for monitoring the photoluminescence (PL) response of QDs under AC and DC voltage changes within aqueous ionic environments. We evaluate both traditional CdSe/CdS and more biologically compatible InP/ZnS QDs at a range of ion concentrations to establish their PL/voltage characteristics on chip. Wide-field, few-particle PL measurements with neuronal cells show the QDs can be used to track local voltage changes with greater sensitivity (ΔPL up to twice as large) than state-of-the-art calcium imaging dyes, making them particularly appealing for tracking subthreshold events. Additional physiological observation studies showed that while CdSe/CdS dots have greater PL responses on membrane depolarization, their lower cytotoxicity makes InP/ZnS far more suitable for voltage sensing in living systems. Our results provide a methodology for the rational development of QD voltage sensors and highlight their potential for imaging changes in cell membrane voltage.

???displayArticle.pubmedLink??? 31686516
???displayArticle.pmcLink??? PMC7007274
???displayArticle.link??? Nano Lett

???attribute.lit??? ???displayArticles.show???

	Figure 1. Electrochemical setup used to apply voltage and monitor PL from QDs. (a) Confocal microscope and electrochemical cell used to evaluate a potential voltage sensor. QDs are deposited at the working electrode/electrolyte interface of an electrochemical cell with voltage applied between the ITO and Pt electrodes, using a potentiostat. Simultaneously, samples are excited with a 532 nm laser (∼1 μm beam size) with emission collected through the ITO back surface. (b) Cartoon schematic of device cross-section shown alongside COMSOL simulation depicting the variation in potential across the device for a positive voltage applied to the ITO. Shown in the case of 1 mM KNO3, the potential is shown to drop predominately across the QD and extended double layer with the Ta2O5 layer (dielectric) experiencing little to no potential drop. (c) Cross-sectional TEM image of chip used for applying a voltage to QDs. The electrochemical device consists of an ITO bottom electrode atop which is evaporated ∼300 nm of high-κ dielectric (Ta2O5); QDs sit smoothly at the electrolyte (KNO3) dielectric interface as shown above.
	Figure 2. Voltage dependence of steady-state PL spectra. (a) CdSe/CdS QD PL spectrum on the application of an electric field of up to 0.9 MV/cm. The spectra red shift (∼3 nm) and broaden (Δfwhm ∼7 nm) as the field is increased, in line with previous studies.28 (b) Dependence of the change in PL intensity on the electric field strength. Between 0.5 and 1 MV/cm, as the field is increased, \|ΔPL/PL\| increases approximately linearly; at the extrema of fields (<0.25 MV/cm and 1 MV/cm>), there is a tail off in the response. The error bars are derived from a minimum of 20 repeat experiments. (c) PL spectrum of InP/ZnS QDs on the application of an electric field of up to 0.9 MV/cm. In this case, the spectrum blue shifts (1.5 nm) with a maximum \|ΔPL/PL\| of ∼70%. (d) \|ΔPL/PL\| change of CdSe/CdS QDs at varying salt concentrations and constant applied voltage (1 V). \|ΔPL/PL\| decreases approximately linearly, in line with an increase in the Debye screening length when salt concentration is lowered. Repeating the experiments with deionized water produces no PL change on application of a voltage (SI, S7). (e) Normalized PL response under 1 Hz AC voltage sweep (top blue). The response of both CdSe/CdS (green) and InP/ZnS (maroon) QDs as well as a perylene diimide dye (purple) cast on the chip mirrors the voltage sweep with minimal lag, demonstrating the platform can be used to calibrate the response of a variety of materials to a changing electric field.
	Figure 3. PL response of QDs in live Xenopus laevis retinal ganglion cell axons to cell depolarization. (a) Cartoon schematic of the setup, showing neurons illuminated with a 532 nm laser with PL from the QDs imaged in reflection. Stimulated membrane depolarization via osmotic shock causes a drop in the intracellular field, resulting in an increase in the PL. (b) Example traces of QD PL following depolarization under various conditions (top left): CdSe/CdS QDs loaded at 0.5 mg/mL into the embryo show a sharp 66% increase in PL on depolarization. In other cases, the PL increase is smaller (32% for CdSe loaded at 0.1 mg/mL into the embryo). InP/ZnS QDs loaded at 0.5 mg/mL into the embryo show a sharp ∼11% increase in PL on depolarization. QDs also respond to depolarization created by addition of potassium gluconate. The weak oscillations in PL that can be seen after the initial sharp PL change may correspond to additional depolarization events. We note that the laser power and spectrometer acquisition time were varied between experiments, explaining the difference in noise and absolute counts (see Experimental Methods for further discussion).
	Figure 4. Using QD voltage reporters to detect neuronal membrane depolarization with wide-field PL imaging. (a) CdSe/CdS QDs are injected on top of the RGC axons expressing a calcium indicator (jGCaMP7f). QDs remaining in suspension are removed by washing. The image (left panel) shows a wide-field PL image of neurons in the dye emission channel. Individual or few QDs are identified (white boxes; SI, S15) and their PL intensity tracked over time (central panel). As the neuronal membrane potential changes, the total QD PL initially increases before dropping following cell death or repolarization (right panel; integrated over all identified QDs). t0 shows the intensity of QDs before addition of H2O, t1 on cell depolarization, and t2 following cell death/repolarization (b, i–iv). The PL of individual QDs (red) and the dye (black) can be simultaneously tracked in the locations marked. The PL changes are correlated between the two emission channels allowing them to be distinguished from blinking/bleaching events. The magnitude of PL increase on depolarization is typically larger for QDs compared to the dye, as indicated by the arrows.

References [+] :

Abdelfattah, A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices. 2016, Pubmed