Two-channel fluorescence lifetime microscope with two colour laser excitation, single-molecule sensitivity, and submicrometer resolution

Abstract

We present results from a two channel confocal microscope set-up allowing one to efficiently record two-colour as well as polarization resolved time-correlated single molecule fluorescence data. In addition to their spectral characteristics, single molecules can be distinguished by their fluorescence lifetime and polarization. This provides independent distinctive information and results in enhanced detection sensitivity. The set-up we present uses two picosecond diode lasers (440nm and 635 nm) for fluorescence excitation and a piezo scanner for sample movement. A learning scan algorithm permits very fast piezo scanner movement and offers a superior positioning accuracy on single molecules. The time-correlated photon counting system uses Time-Tagged Time-Resolved (TTTR) data aquisition, in which each photon is recorded individually. This method allows for the reconstruction not only fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function on a single spot of interest. Cross-correlation between two channels can be used to eliminate detector artifacts. Finally, fluorescence antibunching can also be analyzed. We show results obtained with immobilized and diffusing red and blue excited fluorescently labelled latex microspheres, as well as from single fluorophore molecules.

Enhanced Extraction of Silicon-Vacancy Centers Light Emission Using Bottom-Up Engineered Polycrystalline Diamond Photonic Crystal Slabs

“…We used the Micro-Time200 (PicoQuant) confocal time-resolved microscope platform. 60 The sample was excited with a 2 μW average power in the diffraction limited confocal observation spot by 470 nm diode laser through 100× magnification air spaced objective (Olympus, NA = 0.8). The PL was collected in epifluorescence observation mode by the same objective, passed through a 75 μm diameter pinhole and detected by a Siavalanche photodiode (PerkinElmer SPCM-AQR-14) through an optical filter transmitting 740 ± 13 nm (Semrock).…”

“…We used the MicroTime200 (PicoQuant) confocal time-resolved microscope platform . The sample was excited with a 2 μW average power in the diffraction limited confocal observation spot by 470 nm diode laser through 100× magnification air spaced objective (Olympus, NA = 0.8).…”

Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels

“…To solve the problem we previously extended the TTTR data stream concept to contain markers for synchronization information from, e.g., a piezoscanner. 15 The same concept, only with more marker signals, was implemented in the present instrument. This makes possible to reconstruct two-dimensional ͑2D͒ or threedimensional ͑3D͒ images from the stream of T3 records, since the position of the scanner can be determined during data analysis.…”

Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics

“…Specifically, the external TCSPC equipment consisted of picosecond pulsed laser diodes of wavelength of 485 nm for excitation of Cy2 or 640 nm for excitation of Cy5 (PDL 800-D with LDH-P-C-485 or LDH-P-C-640B; PicoQuant GmbH, Berlin) with a pulse rate of 40 MHz, and the TimeHarp 200 TCSPC board (PicoQuant GmbH). Synchronization of the data acquisition with the scanner movement was controlled by SymPhoTime software using the time-tagged time-resolved (TTTR) measurement mode in which each photon is stored separately. − A PMA-182-N-M photomultiplier tube (PMT) detector (PicoQuant GmbH) with a low photocathode quantum efficiency (15% at 450 nm and 9% at 550 nm) along with band pass emission filters HQ520/35 for Cy2, HQ580/70 for Cy3, or HQ685/70 for Cy5 (AHF Analysentechnik, Tübingen, Germany) were used for measuring the fluorescent emission. The instrument response function (IRF) of the whole setup was measured to be approximately 250 ps.…”