Spectroscopic super-resolution fluorescence cell imaging using ultra-small Ge quantum dots
Mingying SongAli KaratutluIsma AliOsman ErsoyYun ZhouXin YangYuanpeng ZhangWilliam LittleAnn P. WheelerAndrei Sapelkin
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Abstract:
We demonstrate a spectroscopic imaging based super-resolution approach by separating the overlapping diffraction spots into several detectors during a single scanning period and taking advantage of the size-dependent emission wavelength in nanoparticles.This approach has been tested using off-the-shelf quantum dots (Invitrogen Qdot) and inhouse novel ultra-small (~3 nm) Ge QDs.Furthermore, we developed a method-specific Gaussian fitting and maximum likelihood estimation based on a Matlab algorithm for fast QD localisation.This methodology results in a three-fold improvement in the number of localised QDs compared to non-spectroscopic images.With the addition of advanced ultrasmall Ge probes, the number can be improved even further, giving at least 1.5 times improvement when compared to Qdots.Using a standard scanning confocal microscope we achieved a data acquisition rate of 200 ms per image frame.This is an improvement on single molecule localisation super-resolution microscopy where repeated image capture limits the imaging speed, and the size of fluorescence probes limits the possible theoretical localisation resolution.We show that our spectral deconvolution approach has a potential to deliver data acquisition rates on the ms scale thus providing super-resolution in live systems.Keywords:
Fluorescence-lifetime imaging microscopy
The authors measured the absorption and the fluorescence spectra of the quantum dots CdSe/ZnS with 4 nm in size at different concentration with the use of the UV-Vis absorption spectroscopy and fluorescence spectrometer. The effect of quantum dots CdSe/ZnS's concentration on its fluorescence was especially studied and its physical mechanism was analyzed. It was observed that the optimal concentration of the quantum dots CdSe/ZnS for fluorescence is 2 micromole x L(-1). When the quantum dot's concentration is over 2 micromol x L(-1), the fluorescence is decreased with the increase in the concentration. While the quantum dot's concentration is less than 2 micromol x L(-1), the fluorescence is decreased with the decrease in the concentration. There are two main reasons: (1) fluorescence quenching and 2) the competition between absorption and fluorescence. When the quantum dot's concentration is over 2 micromol x L(-1), the distance between quantum dots is so close that the fluorescence quenching is induced. The closer the distance between quantum dots is, the more serious the fluorescence quenching is induced. Also, in this case, the absorption is so large that some of the quantum dots can not be excited because the incident light can not pass through the whole sample. As a result, the fluorescence is decreased with the increase in the quantum dot's concentration. As the quantum dot's concentration is below 2 micromol x L(-1), the distance between quantum dots is far enough that no more fluorescence quenching is induced. In this case, the fluorescence is determined by the particle number per unit volume. More particle number per unit volume produces more fluorescence. Therefore, the fluorescence is decreased with the decrease in the quantum dot's concentration.
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Combination of SRRF and stochastic labeling based on FAST:Fluorogen complexes to achieve super-resolution in 2D, 3D and in time-lapse.
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As a kind of fluorescent nano material with good optical properties and low toxicity, carbon quantum dots have been developed into a new type of fluorescent probe, which can be used for the detection of heavy metal pollution in the environment. At present, the synthesis methods of carbon quantum dots have been mature, but the research reports on the factors affecting the fluorescence properties of carbon quantum dots are few. This paper, starting from the structure of the carbon quantum dots, focuses on the fluorescence characteristics of the dots, discusses the factors affecting the fluorescence performance of the dots, and compares the methods to improve the fluorescence intensity of the dots. The environmental application of carbon quantum dots is analyzed and prospected.
Carbon Quantum Dots
Carbon fibers
Environmental Pollution
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As newly developed fluorescence labels,compare to the traditional organic fluorescent dyes,the semiconductor quantum dots(QDs) are of many advantages,such as broaden and continuous excitation,narrower and more symmetric emission,photochemical stability,longer fluorescence lifetime,higher quantum yield and lower biological toxicity,and so on.These properties make quantum dots(QDs) unique and superior in the applications of cell labeling and tracking and imaging in vivo.It was widely applied in many areas of life science.It breaks through many defects,such as fluorescence performance and biological toxicity,compared to the traditional organ-ic fluorescent dyes.The application of quantum dots(QDs) has greatly promoted the research work of ultra-sensitive,in situ,real-time and dynamic bio-tracking and bio-imaging in life sciences.In this paper,the fluorescent properties of quantum dots and the application of cell labeling and tracking imaging in vivo are reviewed,and the prospects in Fluorescence in situ hybridization,flow cytometry,Re-al-time quantitative fluorescence PCR are envisioned.
Quantum yield
Fluorescence-lifetime imaging microscopy
Live cell imaging
Tracking (education)
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We present a new imaging technique optimizing the spatio-temporal resolution in fluorescence microscopy. This method achieves short integration time as SOFI, with high spatial resolution comparable to STORM, leading towards super-resolution imaging within living cells.
Temporal resolution
Fluorescence-lifetime imaging microscopy
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Quantum dots (QDs) are semiconductor particles of a few nanometers in size that have unique optical and electronic properties. These properties include stable high‐intensity fluorescence and strong resistance to light bleaching, making them attractive as imaging agents. Mitochondria play many extremely important roles in cells. During apoptosis, several characteristics of mitochondria change that are exploited using fluorescent imaging probing including QDs. Herein, a mitochondria‐targetable fluorescence probe (CdSe/ZnS@PEI‐TPP) based on QDs is developed by covalently binding low‐molecular‐weight polyethyleneimine (PEI)‐modified CdSe/ZnS QDs with the small molecule (3‐carboxypropyl) triphenylphosphine (TPP). CdSe/ZnS@PEI‐TPP QDs have excellent optical emission properties. The ultraviolet and visible spectrophotometry (UV–vis) and fluorescence spectra of QDs show that TPP is successfully integrated into the QD structure. Cell proliferation measurements from 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assays demonstrate that the modified QDs are less cytotoxic. The modified QDs show high fluorescence in cancer cells in vitro. These data demonstrate that CdSe/ZnS@PEI‐TPP QDs successfully target mitochondria and are used as fluorescence imaging probes.
HeLa
Fluorescence-lifetime imaging microscopy
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Fluorescence lifetime microscopy (FLIM) is restricted by the modest pixel count of singlephoton-avalanche-diode (SPAD) arrays. We report micro-scanning and super-resolution of a SPAD array, yielding 32x improvement in resolution and enabling high-frame-rate, wide- field FLIM.
Fluorescence-lifetime imaging microscopy
Single-photon avalanche diode
Frame rate
Avalanche diode
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Fluorescence-lifetime imaging microscopy
Temporal resolution
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Most super-resolution microscopy thus far has exploited the photophysics of fluorescent molecules. And, though several techniques for super-resolution fluorescence microscopy have found wide use in routine imaging of cells, no approaches have emerged that demonstrate super-resolution imaging in scattering tissues. Recently, we introduced a super-resolution-imaging method, multiphoton spatial-frequency-modulated imaging (MP-SPIFI), that provides enhanced resolution for both luminescent and coherent nonlinear interactions, and that opens a path for super-resolution imaging deep inside of scattering tissues.
Fluorescence-lifetime imaging microscopy
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The preparation and improvement of fluorescence property of CdSe quantum dots were presented.CdSe quantum dots were fabricated in water,and were characterized by XRD and PL.The results showed that fluorescence property of CdSe quantum dots could be improved by adjusting the processing temperature and wrapping with ZnS shell.
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