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WIREs Nanomed Nanobiotechnol
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Temporal flickering of contrast agents for enhanced optical imaging

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The temporal flickering of contrast agents that labels a biological sample is a unique modality for cellular imaging with single molecule sensitivity. It improves the signal‐to‐noise ratio statistics associated with the noisy in vivo environment and has promising applications in single particle tracking and super‐resolution microscopy techniques. The flickering can be triggered either statistically through the mechanism of temporal fluctuations of the emitter or through external modulation. The enriching toolbox of contrast agents that are feasible for biomedical imaging for the flickering methods will be discussed, with emphasis on the emerging field of flickering gold nanoparticles and the lock‐in detection mechanism. WIREs Nanomed Nanobiotechnol 2016, 8:439–448. doi: 10.1002/wnan.1375 This article is categorized under: Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Diagnostic Tools > In Vivo Nanodiagnostics and Imaging
Temporally sequenced labeling (TSL) experimental results with B16 live mouse melanoma cells loaded with 20 nm gold nanospheres. (a) Sequence of three recorded images of the scattered light from the sample having −25 dB signal‐to‐noise ratio. The irradiation was using a laser beam (at 532 nm) that was modulated using modulation frequency of 3 Hz. The presented images were captured every 20 milliseconds. (b) The Fourier transform of the temporal fluctuations of pixels in the recorded images. The data pixels present a clear peak at the modulation frequency of 3 Hz (red line) and the noise pixel does not contain any peak (black line). (c) Bright field image of the sample. (d) The reconstructed image of the sample using TSL. (Reprinted with permission from Ref . Copyright 2015 NPG)
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Principle of superresolution optical fluctuation imaging (SOFI) and experimental images of cells. (a) Emitter distribution in the object plane. Each emitter exhibits fluorescence intermittency, which is uncorrelated with the others. (b) Magnified detail of the dotted box in (a). The signal from the emitter fluorescence distribution is convolved with the systems point spread function (PSF) and the fluctuations are recorded by a CCD camera as a movie. (c) Each pixel contains a time trace, which is composed of the sum of individual emitter signals, whose PSFs are reaching into the pixel. (d) The second‐order correlation function is calculated from the fluctuations for each pixel. (e) The SOFI intensity value assigned for each pixel is given by the integral over the second‐order correlation function. The second‐order correlation function is proportional to the squared PSF, thus increasing the resolution of the imaging system by a factor of 2. (f) Widefield image of QD625‐labeled 3T3 cells generated by time averaging all frames of the acquired movie (3000 frames, 100 ms per frame). (g) SOFI SR image. The magnified views of the boxed regions in (f) and (g) are presented in the lower left corners, respectively (Scale bars 2 µm). (Reprinted with permission from Ref . Copyright 2009 PNAS)
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Contrast agents used in the flickering methods ranging from 1 to ~100 nm. (a) Fluorescent proteins. (b) Quantum dot. (c) Transmission electron microscopic (TEM) image of gold nanospheres. (d) TEM image of gold nanorods.
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Temporally sequenced labeling (TSL) with added K‐factor algorithm applied to a sample of human embryonic kidney 293 cells loaded with 20 nm gold nanospheres. (a) A single image from the experimental set, where the gold nanoparticles (GNPs) are indistinguishable from the noise. (b) A bright field image of the sample. (c) The reconstructed image of the sample using TSL. (d) The reconstructed image of the sample using both TSL and K‐factor. (e) The superimposing of (b) and (d), where the GNPs are marked in red. (Reprinted with permission from Ref . Copyright 2015 OSA)
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Superresolution temporally sequenced labeling images of human epidermoid carcinoma A431 cell line loaded with both 20 nm gold nanospheres and 15 × 50 gold nanorods. (a) A bright field image of the sample. (b) The reconstructed image of the sample using TSL for each of the two frequencies (marked in green and red). (c) Zoom‐in on an area inside the sample that contains three spots. The image was captured under conditions of continuous illumination of the sample with the two lasers at high power of 50 mW. (d) The same area with the proposed method, where each of the spots contains two different types of gold nanoparticles. (Reprinted with permission from Ref . Copyright 2015 NPG)
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