In a recent article in the journal Nanotechnology, researchers used a single-particle imaging method for excitation of moderate-intensity fluorescence to achieve spatial resolution. Semiconductor nanocrystals were accessed here, the emission life of which depended on the excitation intensity.
Study: Modulation of the shelf life of the excited state in semiconductor nanocrystals for superresolution images. Image credit: GiroScience / Shutterstock.com
Nanocrystals
The rate of excitation of the excited fluorophore with optical modulation is an important tool for achieving an overresolution in microscopy techniques, including ground state depletion microscopy, stimulated emission depletion, or fluorescence transition microscopy. reversible saturated optics.
In organic dyes or fluorescent proteins, excitation methods involve the adaptation of the transition rate from an excited state to a ground state. However, semiconductor nanocrystals intrinsically have multiple excited, gray, or charged states.
Excessive energy stored in the core of the nanocrystal or trapped in most of the nanocrystal or on its surface gives rise to the origin of multiple states. Overlapping the wave function of an exciter with trapped charges can increase the probability of non-radiative energy transfer from the exciter to the charge. Thus, the excitation life of the excited state decreases depending on the location and the amount of charge trapped within the particle.
Semiconductor nanocrystals for high resolution images
In the present study, the researchers used the useful life depending on the excitation intensity of the semiconductor nanocrystal to increase the spatial image resolution beyond the diffraction limit. A single semiconductor nanocrystall was scanned through a confocal point with a diffraction limit to modulate the excitation power density, which is maximum at the center and minimized toward the edges. Thus, several excess photogenerated charges in a particle can be modulated; consequently, its shelf life is also modulated in the excited state.
The modulation of the useful life relative to the position of a particle according to the focused laser allowed to improve the spatial resolution of the scanned image of the nanocrystal. This was demonstrated by the separation of two nearby emitting images, which remained unresolved in the intensity image. This method combines the simplicity and robustness of the measurement technique that combines the confocal laser scanning microscope with the lifetime fluorescence measurement capability.
Research results
The researchers used cadmium selenium (CdSe) / cadmium sulfur (CdS) / thick shell quantum dots (QD); the thickness of the shell helped reduce the flicker of the fluorescence. Transmission electron microscopy (TEM) imaging showed that the average particle size is in the range of 18 ± 2 nanometers, which included a CdS shell consisting of approximately 15 atomic layers. In this type of giant QD, the interaction between the excited state carriers and the surface trap states was weakened, further suppressing non-radiative Auger recombination. Fluorescence flicker was suppressed by reducing Auger efficiency and hot carrier capture. If not removed, flicker could lead to single-particle confocal scan images, which reduce the accuracy of particle location.
The radially polarized laser beam was used to scan semiconductor nanocrystals with the focal point limited by diffraction. Here, the radial polarization mainly caused an axially polarized excitation of the approximate diameter of 180 nanometers to a wavelength of 640 nanometers. This improved the diffraction limit resolution by about 1.4 times compared to a linearly polarized Gaussian beam.
A confocal microscope equipped with a multichannel picosecond event timer was used for all fluorescence measurements, enabling lifetime fluorescence imaging. To focus the excitation light and collect the fluorescence light, a high numerical aperture lens was fitted for the system. As a source of excitation, a white light laser system with an adjustable acoustic-optical filter was used. A non-polarizing beam splitter reflected the excitation light toward the target. Calculations for the half-life of the excited state were performed by adjusting the fluorescence decay curves with a multiexponential decay model.
The characteristic intensity pattern, shown from the recorded scanning image of the single quantum emitter, depended on the dipole moment orientation of the fluorophore excitation. However, the three-dimensional (3D) excitation isotropy of spherical semiconductor nanocrystals avoided dependence on the excitation pattern of the orientation.
The fluorescence life of the semiconductor nanocrystal that depended on the excitation power allowed the image of the particle fluorescence lifetime image to be reduced compared to the diffraction-limited intensity image.
The non-additivity of lives depended on the inclination of the sigmoid curve at its inflection point and the number of photons detected that determined the fit quality of the fluorescence decay curve. In addition, the non-additivity of lives led to improved resolution. However, both the inclination of the curve and the amount of photons depended on the experimental conditions, including the optical properties of QD and the parameters of the microscope.
Conclusion
In conclusion, the researchers presented the principle test measures for an overresolution imaging method based on the modulation of the excitation life of the excitation of semiconductor nanocrystals. They showed that this method could achieve a resolution ten times at moderate fluorescence intensities, which could be further improved by collecting more photons.
This method used a conventional fluorescence confocal microscope based on lifetime fluorescence images of semiconductor nanocrystals to access a broad community of fluorescence microscopy. Although the present work involved excitation using a radially polarized laser beam, a linearly polarized Gaussian beam can also be used for excitation with a minimized focus diameter.
Reference
Ghosh, S., Hollingsworth, J., Gallea, JI, Majumder, S., Enderlein, J. and Chizhik, A. (2022). Modulation of the shelf life of the excited state in semiconductor nanocrystals for superresolution images. Nanotechnology. https://iopscience.iop.org/article/10.1088/1361-6528/ac73a2
Disclaimer: The views expressed herein are those of the author expressed in private and do not necessarily represent the views of AZoM.com Limited T / A AZoNetwork the owner and operator of this website. This disclaimer is part of the Terms and Conditions of Use of this website.