Spcm cd3516h

The hydrodynamic diameter (D_H) of fluorescent-labeled polymers [i.e., FITC-dextran (40, 70, or 150 kDa) and FITC-PEG (5 and 40 kDa)] was determined by fluorescence correlation spectroscopy. An Olympus IX73 inverted microscope (Olympus, Tokyo, Japan) using an immersion Super Apochromat objective (1.2 numerical aperture, ×60, UplanSApo, Olympus) was used to perform the measurements. Emitted photons were filtered with a bandpass filter (512 nm) before detection with a single-photon avalanche diode (SPCM CD3516H, Excelitas). The free dye Atto 488 carboxylic acid (D = 400 μm/s² at 298 K, Thermo Fisher Scientific) was dissolved at a concentration of 10 nM in double distilled water and used to calibrate the confocal volume of the excitation channel at 481 nm. Intensity fluctuations were recorded over 60 s. The experimental autocorrelation curves of the calibration dye Atto 488 and the fluorophore coupled samples were fitted with a one-component triplet state model, as previously described (20 ). Data were processed using PicoQuant Software (Berlin, Germany).

Pulsed Interleaved Excitation (PIE) and solution-based smFRET experiments were performed on a MicroTime 200 confocal microscope (PicoQuant, Berlin, Germany). Prior to the recording, microscope slides (170 µm thickness, No. 1.5 H precision cover slides, VWR Marienfeld, Leicestershire, Great Britain; LH26.1) were coated for at least one min with 1 mg/mL filtered (0.2 µm) bovine serum albumin (BSA) in 50 mM HEPES-K pH 7.0, after which the BSA solution was removed by pipetting and replaced by 150–200 µL of the sample.
The laser pulse rate was set at 40 MHz. Fluorophores were alternately excited, using a 532 nm (LDH-P-FA-530-B; PicoQuant, Berlin, Germany) and 638 nm (LDH-D-C-640; PicoQuant, Berlin, Germany) laser. The laser beam was focused 7 µm away from the glass-solution interface in the z-direction, by means of an oil-immersed objective lens (UPlanSApo 100x1.40 NA; Olympus, Tokyo, Japan). The emitted photons from the sample were coordinated through a 100 µm pinhole, separated through a laser beam-splitter (ZT640RDC; Chroma Technology, Bellows Falls, Vermont), filtered by either a HQ690/70 (Chroma Technology, Bellows Falls, Vermont) or a 582/75 (Semrock, Rochester, New York) emission filter, and recorded by two photon counting modules (donor photons: SPCM-AQRH-14-TR, acceptor photons: SPCM-CD-3516-H; Excelitas Technologies, Waltham, Massachusetts).

Single molecule FRET measurements were acquired using a custom-built PicoQuant MicroTime 200 Fluorescence Lifetime Microscope. smFRET data acquisitions were conducted using pulsed interleaved excitation at 80 MHz. Both 532 nm (LDH-D-TA-530; Picoquant) and 637 nm (LDH-D-C-640; Picoquant) lasers were simultaneously used to characterize the fluorescent behavior of both fluorophores and the efficiency of energy transfer between molecules potentially showing FRET. The sample slide was immobilized on a scanning x-y-z piezo stage (P-733.2CD; Physik Instrumente) while being excited and observed through a 100x oil immersed lens (100 × 1.4 NA; Olympus). The photons emitted from the sample post-excitation were collected back through the objective, separated through a dual band dichroic beam splitter (Zt532/640rpc-UF3; AHF/Chroma) and sent to two SPAD photodiodes (SPCM CD3516H; Excelitas technologies) preceded by excitation filters. A 550 nm (FF01-582/64;AHF/Semrock) and 650 nm (2XH690/70;AHF) emission filter were used for the donor and acceptor channels, respectively. All acquisitions were performed in the presence of a photo-stabilizer and oxygen scavenging solution buffer system (ROXS).