A position-sensitive photon event counting detector applied to fluorescence imaging of dyes in sol-gel matrices

Abstract

A photon event counting imaging detector, originally developed for astronomical applications, has been adapted for use on a low light level fluorescence microscope. It is based on a 40 mm diameter, three-microchannel plate image intensifier with a photon gain of 107 when operating in the photon event counting mode. The intensifier's output screen is lens coupled to a full frame progressive scan CCD camera which transfers the photon event data into a PC via a framegrabber. The image is built up from the photon events in the frames using software-based event processing. The high sensitivity of the photon counting approach means that low excitation light levels and/or a low probe concentration can be used. Moreover, the inherent linearity between incident intensity and the number of detected events provides intrinsic photometric accuracy independent of camera gain and offset. To demonstrate the viability of photon event counting imaging applied to fluorescence studies, we have imaged 4-dicyanomethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran- (DCM-) and rhodamine 6G-doped thin films produced by the sol-gel technique using tetraethylorthosilicate (TEOS) as a precursor. We show that fluorescence imaging is a feasible tool for finding cracks, streaks or other defects and for assessing the uniform thickness of the finished films. In addition, we image the fluorescence in response to an excitation beam illuminating rhodamine 6G-doped bulk monolith sol-gel samples. The results are analysed using the Lambert-Beer law.

Time-resolved fluorescence microscopy

“…45,46 Linearity, high dynamic range, high sensitivity, zero read-out noise, large area, well-defined Poisson statistics and good spectral response in the UV are particular strengths of the photon counting imaging approach. Applications of photon counting imaging to diverse fields such as autoradiography, 47 bioluminescence 48 and fluorescence imaging 49 have also been reported.…”

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

“…For lifetime measurements the laser was triggered once during each exposure with 5 μs delay after the start of the frame exposure. A 40 mm diameter dual proximityfocused, three-microchannel plate image intensifier (Photek, U.K.) operating in photon counting mode [9][10][11] was mounted on the side port of an inverted microscope (Eclipse TE2000-E, Nikon, Japan). The laser was focused into the sample either through a 4 Â 0.13NA air objective (Nikon, Japan; for Ru solutions) or a 100 Â 1.4NA oil immersion objective (Leica, Germany; for cell samples), and the detected light was collected with the same objective.…”

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

“…The Hubble Space Telescope's Faint Object Camera (Kröger et al, 1992), and the European Space Agency's X-ray Multi-Mirror satellite (the most powerful X-ray telescope ever placed in orbit, launched in 1999) were fitted with a photon counting imaging optical monitor (Mason et al, 2001). The technique has also been used in autoradiography (Tarhoni et al, 2011), bioluminescence (Rembold et al, 1997) and fluorescence imaging (Hirvonen et al, 2014a,b;Read et al, 1997;Sergent et al, 2010;Suhling et al, 2001). Wide-field photon counting imaging has some distinct advantages over direct CCD-based imaging, in particular the ability to time the arrival of photons.…”