Picosecond wide-field time-correlated single photon counting fluorescence microscopy with a delay line anode detector

Hirvonen, L; Becker, Wolfgang; Milnes, J.; Conneely, T.; Smietana, Stefan; Marois, Alix Marie Le; Jagutzki, O.; Suhling, Klaus

doi:10.1063/1.4961054

Cited by 23 publications

(18 citation statements)

References 32 publications

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“…38,39 SPAD arrays can detect individual photons in each pixel with picosecond resolution without an image intensifier, and 256 × 256 pixel devices have been developed. 40 They allow enormous count rates 41 and are very promising single photon sensitive cameras for the future.…”

Section: Discussionmentioning

confidence: 99%

Photon counting phosphorescence lifetime imaging with TimepixCam

Hirvonen

Fisher-Levine

Suhling

et al. 2017

Review of Scientific Instruments

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TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window and read out by a Timepix Application Specific Integrated Circuit. The 256 × 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting imaging. We have characterised the photon detection capabilities of this detector system and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200 μm diameter polystyrene beads.

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Section: Discussionmentioning

confidence: 99%

Photon counting phosphorescence lifetime imaging with TimepixCam

Hirvonen

Fisher-Levine

Suhling

et al. 2017

Review of Scientific Instruments

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“…Objectivebased TIRF and supercritical angle fluorescence would also be feasible with this approach. 51 The data acquisition does not pose major problems for the SPCM software. Either triggered sequential recording with the "autosave" function or "spatial mosaic" imaging can be used.…”

Section: Discussionmentioning

confidence: 99%

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

Becker

Hirvonen

Milnes

et al. 2016

Review of Scientific Instruments

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We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the “start” input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the “stop” input. The time between start and stop is directly converted into position, with a resolution of 200–250 μm. The data acquisition software builds up the distribution of the photons over their spatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.

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“…In objectivebased TIRF, the excitation light is reflected back from the coverslip, and the sample is excited by an evanescent wave only near (up to ∼100 nm) the coverslip. TIRF-FLIM has been demonstrated with quadrant anodes [172], SPAD array detectors [173], and delay line anodes (see figure 8) [174]. A similar technique is supercritical angle fluorescence (SAF) microscopy where whole sample is excited but the light is collected only from high angles near the coverslip [175,176].…”

Section: Specialised Microscopy Techniquesmentioning

confidence: 99%