A single-laser dual-spectrum source designed for integrated optical coherence and multiphoton microscopy is demonstrated. The source implements the laser characteristics needed to optimally perform both modalities while extending the spectral range for this imaging technique. It consists of a widely tunable, mode-locked, Ti-sapphire laser with a portion of its output spectrally broadened via continuum generation in a photonic crystal fiber. The continuum-broadened beam allows for enhanced optical sectioning with optical coherence microscopy, while the unbroadened beam from the ultrashort-pulse Ti-sapphire laser optimally excites fluorescent markers. The noise power of the continuum-broadened beam is less than 1.1 dBm/Hz higher than the Ti-sapphire laser in the range from 1 Hz to 25 MHz, and the fiber shows no sign of damage after ~100 h of use. We demonstrate the use of this source across a wide spectral range by imaging green fluorescent protein-transfected mouse fibroblast cells costained with fluorescent dyes that are maximally excited at various wavelengths. Images of unstained in vivo human skin are also presented. This source extends the feasibility of this integrated imaging modality and will facilitate new investigations in in vivo microscopy, tissue engineering, and cell biology.
We validate a molecular imaging technique called Nonlinear Interferometric Vibrational Imaging (NIVI)
by comparing vibrational spectra with those acquired from Raman microscopy. This broadband coherent
anti-Stokes Raman scattering (CARS) technique uses heterodyne detection and OCT acquisition and design
principles to interfere a CARS signal generated by a sample with a local oscillator signal generated
separately by a four-wave mixing process. These are mixed and demodulated by spectral interferometry. Its
confocal configuration allows the acquisition of 3D images based on endogenous molecular signatures.
Images from both phantom and mammary tissues have been acquired by this instrument and its spectrum is
compared with its spontaneous Raman signatures.
A four-wave mixing process of 4700 cm-1 Stokes-shift is stimulated by pumping a 20-cm commercial large-mode-area
photonic crystal fiber with amplified Ti:sapphire femtosecond pulses. The phase-matching condition is realized through
an intermodal scheme which promotes the process in the largely normal dispersion regime of the fiber. The walk-off
effect of the interacting pulses is minimized by introducing an initial chirp to the pump pulses. Conversion efficiency
over 7% from near-infrared pump input to visible anti-Stokes signal can be achieved.
We demonstrate imaging with the technique of nonlinear interferometric vibrational imaging (NIVI). Experimental
images using this instrumentation and method have been acquired from both phantom and biological tissues. In our
system, coherent anti-Stokes Raman scattering (CARS) signals are detected by spectral interferometry, which is able to
fully restore high resolution Raman spectrum on each focal spot of a sample covering multiple Raman bands using
broadband pump and Stokes laser beams. Spectral-domain detection has been demonstrated and allows for a significant
increase in image acquiring speed, in signal-to-noise, and in interferometric signal stability.
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