Time-Gated Imaging on Live Cancer Cells Using Silicon Quantum Dot Nanoparticles with Long-Lived Fluorescence

Abstract

In this work we demonstrate time-gated confocal fluorescence imaging on live cancer cells immunostained by antibody-conjugated silicon quantum dot nanoparticles (SiQD-NPs) and organic dyes, for simultaneous detection of two biological targets and removal of background autofluorescence. With almost all radiative recombinations occurring through oxide-related defect states located on the SiQD surface, the SiQD-NPs have very long photoluminescence lifetimes of about 25 μs, in contrast to the nanosecondrange lifetimes of other commonly used biological fluorophores. This drastic lifetime difference enables a time-gated imaging method here, in which the time-resolved photon distribution of each pixel of a fluorescence image is measured by using a time-correlated single-photon counting technique. Then, by integrating the photon histogram of each pixel over respective time windows, the long-lived component of the fluorescence image comprising only the fluorescence emitted from the SiQD-NPs is separated from all other short-lived signals resulting from the organic dyes and the cell endogenous luminescence. For instance, the membrane and nucleus of a single cancer cell or two types of cancer cells, immunostained with the SiQD-NPs and the organic dyes, respectively, can be clearly distinguished from each other by time-gating, which otherwise cannot be accomplished by conventional multiplexing due to spectral overlap in the wavelength domain.

Time-gated fluorescence imaging: Advances in technology and biological applications

“…Besides, during the past decade, time-resolved SPAD and PMT array detectors, including commercial ones, were developed and applied in TG imaging. 39,[55][56][57][58][59][60] Comparison of detectors for TG imaging is provided in Table 2. Overall, ICCD/EMCCD has higher sensitivity than CCD, while the latter is cheaper.…”

“…63 Time-correlated single photon counting (TCSPC) technique based on SPAD or PMT detectors was used in TG imaging. 13,29,39,47 In addition, various°uorescence imaging and detection based on TG technique was also demonstrated, including°uorescence lifetime imaging (FLIM), 13,26,43,47,[64][65][66] TG F€ orster resonance energy transfer (FRET), 40,67-69 and°uorescence di®use optical tomography. 33…”

“…Recovery of the concealed micro objects such as large molecules, 11,28 cells, 11,23,29,34,36,39,41,47,49,66,70,72,[74][75][76][77][78][79][80][81][82][83][84] and microorganisms 24,30,32,71,85 from annoying interference (e.g., background AF) in°uorescence imaging with the help of TG technique has attracted increasing attention for years. Marriott et al presented a simple TG microscope and utilized it to visualize the delayed luminescence intensities and lifetime of acridine dyes on polytene chromosomes.…”

Nanoestructuras De Silicio en Biomedicina Y Biotecnología

“…Una manera de mejorar la resolución de estos ensayos es utilizar técnicas de control de tiempo (CT), un ejemplo se ilustra en la figura 2C, dónde los cultivos en conjunto A431 (células de carcinoma epidermoide humano) y SKOV3 (células de carcinoma de ovario humano) fueron inmunoteñidos, usando fluoresceína -isotiocianato (ITC) y NPSis (respectivamente); el resultado fue que no podían ser separados uno de otro por medio de las imágenes de fluorescencia o componente de vida corta, mientras que solo las células SKOV3 (que fueron tratadas con NPSis) mostraron señales de florescencia en componente de vida larga. Estas demostraciones implican que la técnica basada en NPSis -componente de vida larga tiene un gran potencial para contraste y para imagenología de alta sensibilidadóptica, tan precisa como para el discernimiento de márgenes tumorales durante una cirugía sin alterar tejidos aledaños que puedan presentar autofluorescencia o con cromóforos con fluorescencia de vida corta [64].…”

Suppression of Auger Recombination in Nanocrystals via Ligand-Assisted Wave Function Engineering in Reciprocal Space

“…In contrast, τ 4 values measured in CdSe dots of similar sizes are of the order of a few ps, i.e., about two orders of magnitude faster. 4 It should be noted that, since a specific passivant modifies both AR and PL times in the same direction (i.e., F increases both, A decreases both), F-terminated nanocrystals will be ideally suited for applications requiring long carrier lifetimes, such as PV or biolabelling (a new time-gated immunofluorescence imaging method 49 was recently made possible by NQDs with photoluminescence lifetimes in the µs range). On the other hand, as in such nanocrystals the radiative lifetime increases more than AR lifetime, they will exhibit a lower biexciton quantum yield 50 (reduced by a factor of 2 in the smaller structures and nearly by one order of magnitude in GaSb nanocrystals with R = 2.1 nm) than A-capped dots, which will be therefore better suited for applications such as LEDs, single photon sources and lasers.…”