Abstract
Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.
Highlights
Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers
Two-photon excitation fluorescence (TPEF) microscopy [1] is a powerful technique for sensitive, high-resolution and deep tissue imaging [2]
In biological imaging the tissue penetration depth is limited to ~100μm-1mm [2], so the study of deeper regions and internal organs can only be conducted by employing fiber endoscopes to overcome the penetration limit set by scattering in tissue
Summary
Two-photon excitation fluorescence (TPEF) microscopy [1] is a powerful technique for sensitive, high-resolution and deep tissue imaging [2]. Current laser systems for TPEF imaging employ ultra-short pulse lasers (~100fs-10ps) in order to achieve the high intensities required for non-linear excitation This is because the average power is limited by the sample damage threshold and most systems run at a fixed, very high pulse repetition rate (~80MHz). We present a TPEF system that is fiber-based and uses 0.4-10ns pulses from a fiber master oscillator power amplifier (MOPA) This laser has freely programmable pulse repetition rates, which allows realization of duty cycles comparable to ultra-fast laser systems (10 4-10 5). For a 1ns pulse with 100W peak power we estimate a transient temperature increase of roughly 1°C in the focus of an NA = 1 objective, if we assume 1064nm laser wavelength and use the linear absorption coefficient of water. Such arbitrarily adjustable pulse patterns or sequences can be realized with the presented fiber MOPA
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