Two-photon Fluorescence Lifetime Imaging
Two-photon (2P) microscopy has become increasingly important for the examination of living tissues. Due to the fact that the probability of simultaneous absorption of two photons is proportional to the square of the excitation light intensity, fluorescence excitation in 2P microscopy is mainly restricted to a small focal volume. Thus, 2P microscopy provides confocal detection without a pinhole before the detector to reject out-of focus fluorescence light, increasing the detectable signal. In addition, less-scattered near infrared (NIR) excitation light as used in 2P microscopy permits deep tissue imaging and penetration depths up to 1 mm. The low-energy NIR excitation light and the highly localised excitation also strongly reduce global photobleaching of the fluorescent dye as well as tissue damages.
Mode-locked Ti:sapphire lasers are widely used for 2P microscopy, but they are expensive and technically complex systems which prevent many researchers from upgrading existing 1P confocal systems. An inexpensive alternative is a nontunable laser, working at a fixed wavelength (e.g. C-Fiber A 780, MenloSystems). The fixed wavelength is not necessarily a disadvantage. Compared with 1P absorption spectra, 2P absorption spectra tend to be broader, meaning that a nontunable laser can excite spectrally different dyes, even simultaneously.
Combination of 2P microscopy with fluorescence lifetime imaging microscopy (FLIM) has become a powerful tool for imaging cellular processes within living tissue and, thus, provides a technical basis for the investigation of many biomedical problems. FLIM is a time-resolved image acquisition method based on measuring the decay time of fluorescent dyes. In contrast to the fluorescence intensity, the decay time is mostly independent of variations in dye concentration, illumination intensity or photobleaching. Thus, absolute quantification of ion concentrations in biological preparations by using ion-sensitive fluorescent dyes with FLIM seems to be more reliable.
Biological application of 2P-FLIM
The frequency doubled Er-doped fiber laser system of MenloSystems can be applied for 2P-FLIM. The efficient 2P excitation at 780 nm could be shown for two widely used fluorescent dyes, the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE, when loaded into a living insect tissue.
In contrast to 1P excitation the 2P excitation of dye-loaded tissue unraveled the inhomogeneous dye distribution within the cells of the tubular salivary ducts. The nuclei were stained weakly and the cytosol displayed a lamelar staining profile, most likely due to deep infolfings of the basolateral and apical plasma membrane. Straight forward in situ calibration methods are possible to link the fluorescence decay properties with the respective ion concentrations resulting in spatially resolved intracellular pH and chloride concentration measurements in living tissue.
2P-FLIM setup
2P excitation was generated by a mode-locked femtosecond fiber laser (MenloSystems C-Fiber A 780) operating at 780 nm wavelength, 50 MHz pulse repetition rate and 90 fs pulse width. The output power of the NIR laser beam (>60 mW) could be optionally adjusted by a circular neutral density filter before entering the microscope. Time-resolved fluorescence image acquisition occurred with the time-correlated single-photon counting (TCSPC) technique by rasterscanning the tissue and recording every detected photon with its individual timing.
Author
Dr. Carsten Hille is member of the BMBF-funded InnoProfile Junior Research Group "Applied Laser Sensing" at the University of Potsdam.