KEYWORDS: Signal detection, Sensors, Amplifiers, Pulse shaping, Luminescence, In vivo imaging, Two photon excitation microscopy, Oscillators, Photomultipliers, Microscopy
A broad-bandwidth oscillator and pulse shaper with a compensation phase mask applied produce sub-10 fs laser pulses is used to induce selective two-photon excitation in the 380 to 500 nm range. The output is split into two arms with different second order dispersion (SOD). The recombined beams create a train of pulses with phase-shape switching at a rate of 162 MHz. Each pulse induces selective TPEF on the sample at wavelengths determined by the amount of SOD in the beam, which tunes the selective TPEF wavelength. Fluorescence is detected by a single fast photomultiplier tube (PMT) detector; therefore, signal from the PMT detector contains fluorescence signals from two different selectivelyexcited fluorophores. The two separate signals are isolated by quadrature detection using a lock-in amplifier. Images are obtained from the two different fluorophores simultaneously at 81MHz. The wide tunability of the two-photon excitation wavelength, fast switching rate between the selective excitation, and low photodamage (due to low power of laser beam) enables potential application of this method for in vivo dynamic imaging in biological samples
We demonstrate a modular and versatile experimental setup that enables straightforward compression (and then shaping)
of ultrashort laser pulses at the imaging plane of a two-photon microscope. A commercially available pulse shaper is
used in conjunction with a commercially available broadband Ti:Sapphire oscillator to produce sub-8fs pulses at the
focus of a high-numerical-aperture objective. Automated adaptive pulse compression, based on multiphoton intrapulse
interference phase scan (MIIPS), is verified in situ by shaper assisted interferometric autocorrelation. Two-photon
excited fluorescence image of a mouse kidney slide is obtained to confirm microscopic sectioning capabilities.
The fundamental difficulty of achieving a coherently enhanced sensing method at standoff distances greater than 10
meters has been solved by single-beam coherent anti-Stokes Raman scattering and by actively measuring and
eliminating chromatic dispersion experienced by the broad-bandwidth (100 nm) laser pulses. Characteristic Raman
spectra for several chemicals in gas, liquid, and solid states, are successfully obtained from a 12 meter standoff distance.
The results obtained indicate this is a promising approach to standoff detection of chemicals, hazardous contaminations,
and explosives.
A number of nonlinear imaging modalities, such as two-photon excitation and second harmonic generation, have gained popularity during the last decade. These, and related methods, have in common the use of a femtosecond laser in the near infrared, with the short pulse duration making the nonlinear excitation highly efficient. Efforts toward the use of pulses with pulse duration at or below 10 fs, however, have been a great challenge, in part due to the fact that shorter pulses have been found to cause greater sample damage. Here we provide a brief review of the MIIPS method for correction of phase distortions introduced by high numerical aperture objectives and the introduction of simple phase functions capable of preventing three-photon induced damage, reducing autofluorescence, and providing selective probe excitation.
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