Simultaneous fluorescence and phosphorescence lifetime imaging

Abstract

We present a lifetime imaging technique that simultaneously records fluorescence and phosphorescence lifetime images in laser scanning systems. It is based on modulating a high-frequency pulsed laser by a signal synchronous with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multi-dimensional TCSPC. Fluorescence is recorded during the on-phase of the laser, phosphorescence during the off-phase. The technique does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using excessively high pulse power for phosphorescence excitation. Laser modulation is achieved either by electrically modulating picosecond diode lasers, or be controlling the lasers via the AOM of a standard confocal or multiphoton laser scanning microscope. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 7903 790320-5 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 7903 790320-6 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms

Time-Resolved Emission Imaging Microscopy Using Phosphorescent Metal Complexes: Taking FLIM and PLIM to New Lengths

“…This technique was validated using yeast cells stained with [Ru(bpy) 3 ]Cl 2 (τ ¼ 670 ns) and zinc oxide nanoparticles in a zinc ointment, which displayed both fluorescent (τ ¼ 1 ns) and phosphorescent (τ ¼ 2 μs) emission [90]. FLIM/ PLIM data were recorded on a Zeiss LSM 710 microscope connected to Becker and Hickl hardware (DCS-120 confocal scanner, DDG-210, SPC-150 TCSPC module) (Fig.…”

“…This FLIM/PLIM technique is also based on a multi-dimensional time-correlated single photon counting [106]. Published by The Royal Society of Chemistry (TCSPC) process in combination with either confocal or multiphoton laser scanning [90], and thus can be used with commercial microscopes (Fig. 31).…”

“…This is followed by a "laser off" period, during which time the excitation beam is blocked by an electronic shutter, allowing for photon detection via TCSPC and subsequent generation of a phosphorescence decay curve at every pixel. The confocal scanner synchronises the "on"/"off" modulation of the excitation beam with the pixel clock of the microscope scan head [90], as shown in Fig. 18, correlating pixel dwell time with excitation and photon collection.…”

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…TCSPC FLIM is then used to determine photon times both within the laser pulse period and within the modulation period. These times are used to build up fluorescence and phosphorescence lifetime images (FLIM and PLIM) simultaneously (Becker et al, 2011a).…”

Fluorescence lifetime imaging with near-infrared dyes

“…The result can be interpreted as an array of pixels, each containing a fluorescence decay function, in a large number of consecutive time channels. The photon distribution can be extended by additional parameters [19,21], such as the wavelength of the photons [22], the time from a single [23,24] or periodic [25] stimulation of the sample, the depth in the sample during z-stack recording [24], or the time in an additional modulation period of the laser for phosphorescence lifetime imaging [26].…”