Quantum Dot Blueing and Blinking Enables Flourescence Nanoscopy

7/31/2019 Quantum Dot Blueing and Blinking Enables Flourescence Nanoscopy

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†German Cancer Research Center (DKFZ), Optical Nanoscopy Division, ImNeuenheimer Feld 280, 69120 Heidelberg, Germany,‡BioQuant Center, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany, and§Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Go¨ttingen, Germany

Presented by: Harry Benjamin Russell



Overview Background Concepts

The Experiment

Results Conclusions



Superresolution flourescence imaging of cellswas achieved using bioconjugated CdSe/ZnSquantum dot markers.

Fluorescence blueing and blinking of quantumdot cores facilitates separation of blinkingmarkers residing closer than the diffraction

barrier. Ground state depletion followed by individual

molecule return microscopy was enabled by thehigh number of successive photons emittedyielding a resolution size equal to that of the size

of a single quantum dot (∼12 nm). The experimental set-up utilized drastically

lowers the entry cost to do such nanoscopicimaging



STED - Stimulated Emission Depletionthe fluorescence ability of the dye is switched using ade-excitation beam causing a stimulated emissionbefore it can spontaneously fluoresce.

GSD - Ground state depletion microscopy: thefluorophores are switched back and forth from longlived dark triplet state to the excited state.

GSDIM – Ground state depletion with single moleculereturn microscopy: All flourophores are switched to thedark triplet state, single molecules will spontaneouslyreturn to their fluorescent states.



A Quantum Dot is a semiconductor whosecharacteristic length is bound within the exciton Bohrradius thus binding the excitons of thesemiconductor in all three spatial dimensions.

Constraining further than the Bohr radius causes an

effect called Quantum Confinement and changes theproperties of the semiconductor.

For example, the absorption and emissionwavelength of light shifts towards smaller

wavelengths.

Typical dots are made of binary alloys such ascadmium selecnide, cadmium sulfide, indiumarsenide, indium phosphide and more.



Blinking – large intensity fluctuations inquantum dots providing stochastic ON andOFF events.

Blueing – A continuous shift toward shorterwavelengths upon steady illumination.Attributed to a continuous size reduction of the CdSe core due to photooxidation.

Bleaching – Photochemical destruction of afluorophore



Due to short off times inherent to quantum dots,signals within the subdiffraction range overlapand localization fails. These signals are omittedand the structure appears interrupted.

Scale bars = 2 microns



Due to stochastic blueing of quantum dots,two quantum dots within the subdiffractionrange will emit different fluorescence signalsallowing distinct localization of each quantum

dot.

Stochastic photooxidation during themeasurement finally opens the door to acontinuous space of separable states andenables diffraction-unlimited imaging.



Mammalian PtK2 cells were labeled withcommercially available quantum dots (Qdot 705) and placed in the experimental setup below:



Scale bars = 1 micron



Due to the high amount of photons exhibited during illumination…

Electron multplying is not needed and an ordinary ccd such as the onein a simple webcam can be used.



Scale bars = 2 microns



Labeling of cells with quantum dots andholding under constant illumination enablesGSDIM imaging only limited by the size of thequantum dots.

This is caused by the stochastic blueing of quantum dots, allowing separation of blinking markers in regions below thediffraction limit.

Due to the high number of photons emittedby the process, the use of an EM CCD is nolonger required and can be observed using asimple webcam.

Quantum Dot Blueing and Blinking Enables Flourescence Nanoscopy

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