Description of specimen: The specimen is the photosynthetic antenna protein, Allophycocyanin, APC.

Description of measurement: Single molecules of solution-phase APC were studied for periods of over one second. Every detected photon was used three different ways. The macrotime of the detected photon was used to measure APC fluorescence intensity dynamics (Fig. 1, red and blue traces). The microtime of the detected photon, measured relative to an electronic synchronization pulse from our pulsed excitation source, was used to measure the time-varying fluorescence lifetime (Fig. 1, green trace). Finally, every detection event was used to gate an electrokinetic restoring force that confines the molecule to the confocal detection volume by canceling out Brownian Motion. The result is that intensity and lifetime dynamics of single solution-phase molecules can be measured for prolonged periods of time without having to immobilize and consequently perturb the molecule.

Interpretation: Our data and interpretation were published in Nature Chemistry, 2010, 2, 179. This paper was also honored as the featured cover article, (http://www.nature.com/nchem/journal/v2/n3/index.html). The results showed a diversity of lifetimes and intensities (Fig. 2 a, blue dots) with a concentrated grouping of points at high lifetime and high intensity (Fig. 2 b). By being able to measure long traces of solution-phase APC (Fig. 1a), we were able to show that most molecules started out in this concentrated group of points, but that photoinduced denaturation of the protein resulted in accessing a more analog distribution of intensities and lifetimes at later times (Fig. 2 a, red trace, corresponding to the trajectory in Fig. 1 d). Finally, the different correlations between changes in lifetime and intensity (Fig. 2 c), provided evidence that the radiative and non-radiative lifetime were both fluctuating.

System configuration: Excitation was provided by the spectrally filtered supercontinuum output of a nonlinear photonic crystal fibre (FemtoWhite 800, Newport) pumped by a mode-locked Ti:Sapphire laser (790 nm, 200 fs pulse length, 76 MHz repetition rate, Mira 900, Coherent) yielding pulses centred at 596 nm with a FWHM of 3 nm and length of 3 ps. The laser light was steered into the epi-port of a Nikon TE-3000 microscope and focused with an oil objective (numerical aperture NA=1.0) to provide an average power of 1.7 kW cm2 at the sample. Sample fluorescence is collected back through the same objective and passed through dichroic (620DCLP) and long pass (HQ620LP) filters, a 200 mm pinhole and focused onto an APC (Perkin Elmer, SPCM-CD 2801). Time-correlated single photon counting (TCSPC) is achieved using the PicoHarp 300 timing module. A total instrument response function (IRF) of 0.3 ns was measured from scatter from a glass coverslip. Feedback direction and magnitude are calculated for each 25 ms cycle by phase-sensitive integration of photons detected in the previous cycle. A single detected photon produces a feedback pulse of 24 V vector magnitude.

Comments: This work was performed at the Moerner Lab at Stanford University.

Description of specimen: Single Atto655 molecule on glass in air with a silicon nitride AFM tip

Description of measurement: Fluorescence lifetime imaging combined with atomic force microscopy. An AFM tip is kept fixed in the laser focus while the sample is scanned through the focus. Thus, the influence of the AFM tip on the fluorescence of a single molecule can be probed with high spatial accuracy. "Simultaneous single molecule atomic force and fluorescence lifetime imaging" Proc. SPIE 7571, 757109 (2010).

Interpretation: The spatial distribution of the change in fluorescence lifetime and the quenching of the fluorescence depend on the geometry of the AFM tip and the orientation of the fluorophore's polarizability. The high localization of the quenching can be applied to super-resolution fluorescence imaging.

System configuration: The measurement was taken with a MicroTime 200 system combined with an Asylum MFP-3D AFM. The sample was excited with linearly polarized light at 640nm through an Olympus 100x, 1.45NA oil immersion objective. Fluorescence was collected through the same objective and detected with an MPD avalanche photodiode (no pinhole). The dichroic and emission filter used were from Chroma Technology (z467/638rpc and HQ690/70m) The scan size is 64nm with 64x64 pixel resolution with 7.8 ms/pixel.

Comments: The original image was created using the SymphoTime Software (version 5.13). The intensity is encoded as the brightness of a pixel while the average fluorescence lifetime is represented as the pixel's color. The original image was modified in ImageJ for better visual contrast. The pixel resolution was enhanced to 512x512 pixels and a median filter with 15 pixel radius (approx. 1.8 pixel in the original image) was applied.

Description of specimen: Macrophage cells incubated with CdSe/ZnS quantum dots of two sizes (3 and 3.5 nm)

Description of measurement: FLIM

Interpretation: Size matters. Semiconductor quantum dots exploit a cell's active transport machinery delivering them to specific nuclear and cytoplasmic compartments. Smaller quantum dots localize into the nucleus demonstrating very short photoluminescence lifetime. Bigger quantum dots do not exhibit nuclear localization and are present at the membrane on the cell surface only. Related publication: I. Nabiev et al, Nano Letters 7(11) 3452 (2007)

System configuration: Excitation: PDL 800-B, LDH-P-C-470 nm, Objective: PLANO 100x Planapochromat, NA 1.4, oil immersion Image size: 27.20 µm × 27.20 µm Image pixels: 300x300 Recording time: 8.95min

Description of specimen: Complementary Metal Oxide Semiconductor Active Pixel Sensor chip layout (radiation sensor).

Description of measurement: Scan of a subset of pixels (10x10 squared micrometers) with a micro-focused laser spot with sub-micron positioning capabilities.

Interpretation: The colour map shows in a colour plot the response in terms of a voltage drop of a subset of pixels as a function of the laser spot position.

System configuration: Laser driver PDL 800-B+ laser head LDH-P-780.