Timeharp200

spFRET experiments were performed on a home-built confocal microscope, as previously described (34 (link)). Reactions were performed in SigmaCote™ treated 384-well microplates, as described above in 40 μl total reaction volume. DNA was diluted to 30–70 pM and subsequently equilibrated with the specified TBP concentrations in binding buffer (0.5 mM MgCl₂) containing the indicated KCl concentration for 20 min; samples additionally contained 0.5–1 mM ascorbic acid to minimize photobleaching. Samples were illuminated with 488 nm laser light with donor (535AF45 filter) and acceptor (HQ705/90 filter) emission signals collected on avalanche photodiodes (SPCM-AQ-14, PerkinElmer) for 30 min. Single photon data from a time-correlated-single-photon-counting board (TimeHarp200, PicoQuant) were analyzed using in-house software; single-molecule events were discriminated against background following a burst selection protocol that is described elsewhere (34 (link)). Proximity ratios and histograms were then generated in IGOR software (Wave Metrics) with data fit using the integrated Gaussian fitting function; Gaussian curve-fits in Figure 2 excluded the donor-only peak.

FCS measurements were performed on a home-built system, which was based on an inverted Olympus IX73 microscope. A NKT supercontinuum white-light pulse laser was used as the excitation laser. The repeat frequency, excitation wavelength, and laser power were 3.123 MHz, 592 nm, and 60 μW, respectively. The excitation light was focused by a 60× water immersion objective. The fluorescence emitted by the sample was collected by the same objective and detected by an Excelis Detector (SPCM-AQRH-16). Timeharp 200 (PicoQuant, Berlin, Germany) was used for photon-counting. The coverslip used in the cuvette was coated with a layer of PEG (2 k) to avoid adsorption of fluorescent molecule. The temperature was controlled by an mK1000 series temperature controller (INSTEC, Inc., USA).
QD solutions (200 µL, 8 nM) were mixed with the equal volume BSA solutions with different concentrations (50 nM to 100 µM) diluted in different phosphate buffers (pH = 6.0, 7.4, and 9.0). The solution was incubated at different temperatures (298 K, 300 K, 303 K, and 308 K) for 1 h before FCS measurements. Each sample was measured at least three times and then the average was taken to reduce the error.

FCS traces were collected in live cells expressing GFP-tagged proteins. For each cell, triplicate measurements were recorded in the nucleus and the corresponding diffusion times were averaged. FCS measurements were performed on a customized system comprised of an Olympus IX71 microscope and a scanning confocal module (Microtime 200, PicoQuant GmbH) for time-correlated single photon counting time-tagged time-resolved measurements (Time Harp 200, PicoQuant GmbH). A picosecond pulsed 467 nm laser line was used as excitation source for GFP via a water immersion objective (60×, NA = 1.2). Emitted fluorescence was collected using the same objective and filtered from the excitation light by a dichroic mirror (z467/638rpc, Chroma). Fluorescence signals were selected through a 50 μm pinhole to exclude the background noise and out-of-focus fluorescence, and finally recorded by a single photon avalanche photodiodes (SPCM-AQR-14, PerkinElmer Inc.) after passing the 520–40 (Chroma) band pass filter. Collected fluorescence fluctuation was autocorrelated using the software SymPhoTime (PicoQuant GmbH) and fitted with a least-square algorithm.