First Clinical Applications on the Horizon for FLIM From microscopy of ocular diseases to imaging in neurosurgery to microscopes and nanoscopes, endoscopes tomography in patients with skin cancer and brain tumors, fluorescence and tomographs, but can be also acquired an a macroscopic level," said Karsten lifetime imaging microscopy is the versatile partner that can be teamed Koenig, professor at Saarland University with a host of other imaging techniques to produce striking results. in Saarbrücken, Germany, and CEO of the laser medicine company JenLab GmbH in Jena, Germany. BY MARIE FREEBODY, CONTRIBUTING EDITOR By detecting either intrinsic or extrin­ sic fluorophores in a specific nano- or luorescence lifetime imaging mi­ lt is favored for binding studies and micro-environment, FLIM can be used to croscopy (FLIM) emerged in the late molecule characterizations, occupying a probe intermolecular interactions within F1980s with the development of pulsed secure position in both life and materials a 10-nm distance, such as bindings, by lasers. But it was not until the 1990s when, science for more than a decade. Förster resonance energy transfer (FRET) thanks to the introduction of two-photon "The term FLIM is defined as fluo­ and anisotropy measurements. FLIM is femtosecond laser scanning microscopes, rescence lifetime imaging [microscopy] also applied in animal studies, artwork, FLIM gained significant popularity. where the fluorescence I i fetime is microfluidics andin forensic science. Today, FLIM has become an important depicted with spatial resolution. FLIM "FLIM images are often false-color add-on tool for fluorescence microscopy. i mag es are typica lly generated with coded where the colors reflect values. FLIM images can be taken in the fre­ quency mode (measurement of phase shift and demodulation with a continuous or pulsed laser), as well as in the time mode using time-gated cameras, streak cameras and time-correlated single photon count­ ing (TCSPC) units," Koenig said. In fact, it is TCSPC-based FLIM that is currently being used to explore one of the most challenging and exciting applica­ tions: in vivo clinical imaging of patients. In this approach, one-photon excita- tion comes courtesy of picosecond laser diodes in the visible spectral range while two-photon excitation comes from the application of near-infrared femtosecond Ti:sapphire lasers. Thanks to the focus being on measur­ ing the fluorescent lifetime of a fluoro­ phore (the time a fluorophore stays in the excited state), rather than its intensity, FLIM is independent of a whole hast of factors that otherwise afflict traditional fluorescence microscopy techniques. "lt is independent of experiment pa­ rameters, such as illumination intensity, light path length, detection efficiency, scatter and photobleaching," said Klaus Weisshart, product manager at Carl Zeiss Microscopy GmbH in Jena, Ger­ many. "lt is thus able to provide contrast 800 ps Fluor. Lifetime 2500 ps between different fractions of the same fluorescent species in different molecular High FRET Efficiency Low environment[s], or between fluorescence Human skin , stained with pH-sensitive dye. Fluorescence liletime represents pH. Courtesy of Wolfgang species with identical or strongly overlap­ Becker, Becker & Hickl, Germany. ping emission spectra." 1 44 PHOTON1cs) MEDIA BioPhotonics • April 2016 -, matically increased over the last decade. sure the efficacy of anti-aging drugs to FLIM in the time domain is getting faster fig ht against oxidative stress in skin and and therefore proving a more popular to trace components in situ in deep tissue. method compared with fast frequency Cancer research is also a hot topic; domain FLIM. previous studies have shown that the "Large tissue areas or a !arge number fluorescence lifetime changes the more of cells can now be imaged in the same a cell becomes dedifferentiated in the experiment, and under exactly identical course of cancer progression. Therefore, The pco.flim comero system . Courlesy of PCO AG, experimental conditions," said Wolfgang FLIM m ight be involved in diagnostics. Kelheim. Becker, co-founder of Becker & Hickl GmbH in Germany. "FLIM has been com­ Instrument advances As weil as being independent of in­ bined with STED to obtain superresolu­ Improvements in detectors are among ternal instrument settings, such as laser tion data of cellular structures. The ability the most important technological ad­ intensity or detection efficiency, FLIM to record !arge amounts of data has led to va nces. For example, the development of allows many types of fluorescent markers techniques that record temporal variations sCMOS (scientific CMOS) image sensors to be used to assess different processes of molecular parameters, at mill isecond with a charge-swing per pixel a!Jows for simultaneously. resolution." a fast demodulation up to 40 MHz per When it comes to data analysis, any When it comes to clinical applications, pixel. background emission due to autofluores­ FLIM can be used to study the molecular "This enabled us to develop a CMOS cence from tissue can be easily removed environment within cellular compart­ camera with 1008 X 1008 pixels with by using the fluorescence lifetime infor­ ments or changes thereof including ion wh ich we can measure pixelwise phase mation. This results in higher detection concentration, pH, oxygen and membrane angles, modulation index and intensity efficiency and more accurate marker polarity. It also is a powerful method to convert this with a proper reference localization. to study interaction or conformation of measurement into fluorescence lifetime "In biological systems like cells, the proteins by using FRET techniques, or to distributions," said Gerhard Holst, who fluorophore concentration often cannot probe the positions of molecules within a heads up the science and research depart­ be accurately determined and compared macromolecular complex. ment at scientific camera specialist PCO amongst different cells," sa id Sandra An important class of applications AG, based in Kelheim, Germany. "The Orthaus-Müller, technical sales and takes advantage of FLIM's ability to FLIM measurement was possible before, support specialist for microscopy at derive metabol ic information from the but not with the same frame rate at 1 MP PicoQuant GmbH in Berlin. "Thus, FLIM fluorescence of endogenous fluorophores, of 22 frames/s." allows [users] to compare results from such as nicotinamide adenine dinucleo­ Holst believes that key to success is different cells without numerous control/ tide (NADH) and flavin adenine dinucle­ making FLIM easier to apply by non­ calibration measurements and to quantify otide (FAD). Measuring NADH in its free ex perts. "If the market is interested and the data (most important for detection of and bound form provides information the companies see economic success, it'll protein interactions)." on ox idative stress, which is of inter- be likely that they continue to improve the But for all of its advantages, there are est to the cosmetic and pharmaceutical special CMOS image sensors," he said. cha!Jenges that imaging specialists are industries. "Further, it would be nice to have a better working hard to overcome. One of the In fact, JenLab's Dermainspect and sensitivity (higher quantum efficiency) main limitations is the elaborate equip­ MPTflex is currently being used to mea- and maybe faster modulation capabilities." ment that has to be used; there has to be supplementary electronics: a fast Excitalion Filter Modulatable Light Source photodetector that is able to count single •-·-----------• •············-----------• --Gate Signal photons and additional analysis software. 1 1 • • 1 : ·4 - · : : - ~ : 1 What is more, TCSPC is hard to apply Luminescent ~ : ....__ 8 :+: . [\~ : IIIIIIII Modulalion Signal Sample Al('" : _____________. !: : _______________________~\J !: a with high imaging speed, and quantifying ·---------~ lifetime using an intensity measuring sys­ ----------~ tem such as a camera is tricky, resulting ,.,_.,.,,,!'"··, : pco.fllm ------------- in data that is complicated to interpret. i"1 111! 1 :• ~----------1 1 11 .0 1·-- 1 - t . -i,- FLIM microscopes, tomographs and : ------!~ :' ·.., -- lf :+ ~ L...... ! ·--- --- . 0• ....,..... 1 endoscopes remain expensive and re la­ 1 1 :~~ ftTI ~ .. iij 1 1 tively bulky, limiting their adoption to a 8 ! [___________________________ o ·. i·1: ·~~,M _ specialized market. But with the avail­ ~------------- 1 .1 ability of ultracompact, Jow-cost femto­ ·----------·. second laser sources within the next few lmaging Oplics years, Koenig predicts that FLIM tools will become increasingly available and clinical applications will begin to emerge. While medical FLIM is still considered Struclurol overview of a suggested setup for luminescence lifetime imoging using o PCO FLIM camera early stage, image resolution has dra- syslem . Courtesy of PCO AG, Kelheim. FLIM ods, such as spectral, dynamic or even 1 topological information from atomic force microscopy (AFM). f Such combined measurement methods open promising prospects by accessing different information types from the same sample space in a single experiment. "For example, acquiring topological information from AFM and molecular behavior as detected by FLIM was previ­ ously limited to correlative experiments, requiring !arge amounts of statistics especially for heterogeneous biological samples," said PicoQuant's Orthaus­ Müller. "With a combined FLIM-AFM set-up, as can be realized by interfacing the MicroTime