Cellular imaging using temporally flickering nanoparticles

Abstract

Utilizing the surface plasmon resonance effect in gold nanoparticles enables their use as contrast agents in a variety of applications for compound cellular imaging. However, most techniques suffer from poor signal to noise ratio (SNR) statistics due to high shot noise that is associated with low photon count in addition to high background noise. We demonstrate an effective way to improve the SNR, in particular when the inspected signal is indistinguishable in the given noisy environment. We excite the temporal flickering of the scattered light from gold nanoparticle that labels a biological sample. By preforming temporal spectral analysis of the received spatial image and by inspecting the proper spectral component corresponding to the modulation frequency, we separate the signal from the wide spread spectral noise (lock-in amplification).

Increased localization precision by interference fringe analysis

“…FILM scanning speed was limited by the camera, therefore higher acquisition speeds may be obtained. It was shown in the past that nanoparticles smaller than 60 nm can be imaged with camera integration times shorter than 50 ms 28,29 . Even if the APD scanning step size is 100 nm, this will decrease the acquisition speed by 6.25 times, still slower than the FILM scanning speed.…”

Temporal flickering of contrast agents for enhanced optical imaging

“…As such, it lacks the ability to detect single emitters, thus is not applicable for SR approaches. Recently, an intensity modulation of GNPs was used to invoke their flickering with various applications both in terms of improved SNR and SR through the temporally sequenced labeling (TSL) method …”

“…As the additive background noise is independent from the signal, it does not flicker and thus one is able to extract the desired image data out of the noise and drastically improve the SNR. The flickering can be triggered either by external modulation of the contrast agents through utilization of a specific perturbation that can be a change in temperature, pressure, voltage or laser intensity, and also spontaneously, due to statistical fluctuations in the brightness of the contrast agent, which is depended in the material of choice. An enriching toolbox of contrast agents exists.…”

Superresolved labeling nanoscopy based on temporally flickering nanoparticles and the K-factor image deshadowing

“…The modulated illumination results in a temporal flickering of the scattered light from the GNPs at a known frequency. The mathematical analysis of the technique is presented in [23]. Briefly, the captured intensity images are a temporal sequence of the light scattered from the sample that contains the GNPs.…”

“…The proposed method has two steps, first a temporally sequenced labeling (TSL) routine that was described in a previous work [23] is used to image the sample. The TSL technique increases the SNR, enables the extraction of signal even in poor photon count and improves the localization accuracy of a single particle.…”