Subdiffraction, Luminescence-Depletion Imaging of Isolated, Giant, CdSe/CdS Nanocrystal Quantum Dots

Abstract

Subdiffraction spatial resolution luminescence depletion imaging was performed with giant CdSe/14CdS nanocrystal quantum dots (g-NQDs) dispersed on a glass slide. Luminescence depletion imaging used a Gaussian shaped excitation laser pulse overlapped with a depletion pulse, shaped into a doughnut profile, with zero intensity in the center. Luminescence from a subdiffraction volume is collected from the central portion of the excitation spot, where no depletion takes place. Up to 92% depletion of the luminescence signal was achieved. An average full width at half-maximum of 40 ± 10 nm was measured in the lateral direction for isolated g-NQDs at an air interface using luminescence depletion imaging, whereas the average full width at half-maximum was 450 ± 90 nm using diffraction-limited, confocal luminescence imaging. Time-gating of the luminescence depletion data was required to achieve the stated spatial resolution. No observable photobleaching of the g-NQDs was present in the measurements, which allowed imaging with a dwell time of 250 ms per pixel to obtain images with a high signal-to-noise ratio. The mechanism for luminescence depletion is likely stimulated emission, stimulated absorption, or a combination of the two. The g-NQDs fulfill a need for versatile, photostable tags for subdiffraction imaging schemes where high laser powers or long exposure times are used.

Photon Counting Data Analysis: Application of the Maximum Likelihood and Related Methods for the Determination of Lifetimes in Mixtures of Rose Bengal and Rhodamine B

“…[3][4][5][6] Typically, in a TCSPC experiment, a fluorescence lifetime is determined by acquiring a histogram of arrival time differences between an excitation pulse and the pulse resulting from a detected photon. As we have noted, 3,4 when a histogram of sufficient quality cannot be obtained to provide a good fit by means of minimizing the residuals (RM) between the experimental data and a given functional form, the maximum likelihood (ML) technique is particularly effective, namely when the total number of counts is very low.…”

“…In other words, the total number of predicted counts must be equal to the total number of observed photon counts. The number, therefore, of predicted counts in the jth bin is given by: (5) where = ∑ . It should be noted that in the above equation we allowed the shift parameter, b, to assume continuous values.…”

The Number of Accumulated Photons and the Quality of Stimulated Emission Depletion Lifetime Images

“…Suppression of excited molecules with the depletion beam was not efficient at early time points in the fluorescence decay curve. The signal from the first ~500 ps was discarded by time gating to achieve the best spatial resolution . 64‐time channel fluorescence decay curves for 262 144 pixels from the image were extracted using SPCImage.…”

Review of recent developments in stimulated emission depletion microscopy: applications on cell imaging

“…Despite the current existence of STED dyes acceptable for use with fixed cells, 88 continual efforts are made toward discovering new probes for use in STED microscopy. 42,89 The feasibility of using CdSe/CdS quantum dots as a photostable label has also been recently explored; 90 lateral resolution of ∼40 nm was reported on air-glass interface but cell studies and cytotoxicity were not evaluated.…”