Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations

Abstract

Abstract. Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate ½RuðbipyÞ 3 2þ , was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. ½RuðbipyÞ 3 2þ characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors

“…A false rainbow color scale was assigned to each fluorescence lifetime value (blue for a short lifetime and red for a long lifetime) to provide lifetime maps. Average microbubble lifetimes were calculated by masking the bubble of interest and fitting a monoexponential decay to all of the binned pixels (19). Further image analysis was completed by exporting fitted data to Origin Scientific solutions (OriginPro 8.5, OriginLab Corporation) and Matlab (R2009b, MathWorks Inc.) (Figs.…”

Wide-field time-correlated single-photon counting (TCSPC) lifetime microscopy with microsecond time resolution

“…This method allows submicrowatt excitation powers for the imaging of long-lifetime probes such as lanthanides compounds [2,3], which are widely used for monitoring drug delivery, analyte sensing, and tissue and cell imaging, where time-resolved acquisition allows the discrimination between short-lived autofluorescence of the sample and the long lifetime signal from the probe. Moreover, transition metal complexes can be used to probe the microenvironment, e.g., oxygen concentration, in living cells where a low excitation power is preferable [4,5]. We have employed our wide-field TCSPC system to image the microsecond decay times of an oxygen sensor Ru (dpp) with a 1 MHz camera frame rate and 16 × 64 pixels image size, and europium-containing beads in living cells with 20 kHz frame rate, which allows a bigger image size of 320 × 256 pixels.…”

Wide-field time-correlated single photon counting (TCSPC) microscopy with time resolution below the frame exposure time

“…This fast acquisition time combined with single photon detection sensitivity and low excitation power is advantageous for live cell imaging (see Fig. 5), oxygen sensing [13], auto-fluorescence free imaging or Förster Resonance Energy Transfer (FRET) with long lifetime probes [16] to identify molecular interactions.…”

“…4c) show mean lifetimes of $1 μs and 5 μs for Ru(dpp) in water and glycerol, respectively. The Ru(dpp) lifetime increases with viscosity due to slower oxygen diffusion rate [13]. Each data set consists of 70,000 images acquired in 1.3 s, with 313,000 and 231,000 photons for the water and glycerol data sets, respectively.…”