Beyond Range: Innovating Fluorescence Microscopy

Abstract

Time-of-Flight (ToF) technologies are developed mainly for range estimations in industrial applications or consumer products. Recently, it was realized that ToF sensors could also be used for the detection of fluorescence and of the minute changes in the nanosecond-lived electronic states of fluorescent molecules. This capability can be exploited to report on the biochemical processes occurring within living organisms. ToF technologies, therefore, provide new opportunities in molecular and cell biology, diagnostics, and drug discovery. In this short communication, the convergence of the engineering and biomedical communities onto ToF technologies and its potential impact on basic, applied and translational sciences are discussed.Keywords: Time-of-Flight (ToF); fluorescence lifetime; microscopy Converging CommunitiesIt was the summer of 2005 when in the laboratory of Fred Wouters we started the first autonomous runs of our new in-house developed high throughput fluorescence lifetime imaging (FLIM) system. However successful that development was, one key element in allowing the biomedical community to benefit from FLIM systems, to enable the screening technologies we were pioneering [1] and to get closer to the routine application of FLIM, was still missing: replacing the expensive, comparatively fragile, and not very user-friendly, multi-channel plates used in FLIM with better technologies. The basic idea was to find a silicon-based detector capable of demodulating fluorescence signals on a bi-dimensional array of pixels. Frequent searches of primary literature returned only a few, though interesting, papers including: Nishikata et al. [2] on the development of a dedicated lock-in imager in CCD technology and, Mitchell et al. [3,4] on the adaptation of conventional CCD cameras to achieve demodulation. Unfortunately, ad hoc technologies for lock-in imaging appeared to be operating at OPEN ACCESS

Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay

“…In the last decade a number of SPAD array image sensors have emerged, which simultaneously deliver single photon sensitivity, megapixel spatial resolution and picosecond timing resolution [7][8][9]. These SPAD arrays hold great promise for the advancement of time-resolved fluorescence microscopy, but they currently have a low fill factor and much higher noise levels than MCP-based detectors.…”

“…However, direct detection of individual photons is not possible with these particular cameras. In this context, we note CMOS single photon avalanche diode (SPAD) array image sensors have been developed, incorporating picosecond timing circuitry in each pixel or chip [7][8][9].…”

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

“…The pixel integrates both phases of the modulated fluorescence simultaneously by directing accumulated photocharge via dual transfer gates from the photogate to storage gates. The operating principle of this imager is very similar to that of various time-of-flight image sensors which have been demonstrated in CMOS implementations (Kawahito et al, 2007;Lange and Seitz, 2001;Oggier et al, 2004) and demonstrated to be suitable for FLIM (Esposito, 2012).…”

“…Although many FLIM experiments are performed by photoelectronic vacuum tube or image intensifier-based technology, and this technology continues to be developed (Hirvonen et al, 2014a,b;Rinnenthal et al, 2013;Sergent et al, 2010), in the last decade a number of complementary metal oxide semiconductor (CMOS) or CCD image sensors have emerged, which simultaneously deliver single photon sensitivity, megapixel spatial resolution and picosecond timing resolution (Esposito, 2012). While solid state detector arrays have been used for frequency-domain FLIM (Esposito, 2012), the recent development of single-photon avalanche diode (SPAD) array detectors with picosecond timing capabilities hold great promise for the advancement of time-resolved fluorescence microscopy -especially in view of the limitations of the variety of current FLIM implementations (Charbon et al, 2013;Charbon, 2014).…”

“…However, applications in diverse areas such as forensic science (Bird et al, 2007), combustion research (Ehn et al, 2011;Ni and Melton, 1996), luminescence lifetime mapping in diamond (Liaugaudas et al, 2009(Liaugaudas et al, , 2012, microfluidic systems (Benninger et al, 2005a(Benninger et al, , 2006Elder et al, 2006;Graham et al, 2010;Magennis et al, 2005;Robinson et al, 2008), art conservation (Comelli et al, 2004(Comelli et al, , 2005, remote sensing (Esposito, 2012;Ge et al, 2012;Lin et al, 2012), lipid order problems in physical chemistry (Togashi et al, 2005), the spatial distribution of dopants in perovskite oxides (Rodenbücher et al, 2013) and temperature sensing (Bennet et al, 2011;Benninger et al, 2006;Graham et al, 2010;Mendels et al, 2008;Okabe et al, 2012) have also been reported. FLIM has been carried out from the UV (Li et al, 2004;Schüttpelz et al, 2006) to the near infrared (Becker and Shcheslavskiy, 2013) and it is not surprising that fluorescence lifetime-based imaging is widely used in the biomedical sciences, and that this trend shows no signs of abating.…”