The 1700-nm window has emerged as a promising excitation window for multiphoton microscopy (MPM). On one hand, the combined low tissue absorption and scattering make this window well suited for deep-tissue MPM; on the other hand, the long excitation wavelength makes higher-order MPM in biological tissues feasible, e.g., recently 4-photon fluorescence MPM in mouse brain has been demonstrated. Objective lens is a key optical component in the entire MPM setup. Multiphoton signal levels are largely dependent on the transmittance of objective lens. Here we demonstrate experimental results of transmittance measurement of two water immersion objective lenses commonly used for MPM at the 1700-nm window, covering both the excitation and the signal window. Our target application is MPM of even higher order excited at this window, i.e., 4th harmonic generation (FHG) imaging and 5-photon fluorescence generation. Our results show that, although the customized objective lens offers higher transmittance at the excitation window, it suffers from dramatically degraded transmittance at the signal window, compared with the non-customized objective lens. These results will offer guidelines for selection of proper objective lens for higher-order MPM at the 1700-nm window.
Larger imaging depth is the quest of almost all the imaging modalities, including multiphoton microscopy (MPM). Recently, it has been domonstrated that excitation at the 1700-nm helps extending imaging depth in MPM, optical coherence tomography, as well as photoacoustic imaging compared with excitation at other wavelengths. In MPM, immersion objective lenses with high numerical aperture (NA) are typically used to achieve better signal resolution, higer signal collection efficiency, and stronger signal generation. Although physically short (~mm), this extra optical path length traversed by the excitation light inevitably introduces absorption of the excitation light, and as a result leads to a decrease in the signal generation. Here we demonstrate experimental characterization of absorption spectrum of various immersion media at the 1700-nm window, including water (H2O), deuterium oxide (D2O), and several brands of immersion oil. Our results identify either the best immersion medium for a specific wavelength, or the best wavelength for a specific immersion medium at the 1700-nm window. Furthermore, through quantitative MPM experiments comparing different immersion media, we show that the MPM signal levels can be enhanced by more than ten fold simply by selecting the proper immersion medium, in good agreement with theoretical expectation based on the absorption measurement. Our results will offer guidelines for signal optimization in MPM at the 1700-nm window.
Multiphoton microscopy (MPM) has benefitted dramatically from the development of fiber-based technology. Among these technologies, soliton self-frequency shift (SSFS) in photonic crystal (PC) rods uniquely combines high soliton energy (~100 nJ) and broad wavelength tuning range (>100 nm). Recently, high-energy, 1700-nm femtosecond pulses generated by SSFS in a PC rod enabled 3-photon microscopy of mouse hippocampus in vivo. Below damage threshold, acquisition speed is proportional to the repetition rate of the laser. Here we demonstrate a technique for increasing the soliton repetition rate in a PC rod. Through polarization division and polarization multiplexing, we can double the repetition rate of the optical solitons generated in a PC rod. The orthogonally polarized solitons have virtually the same spectrum, pulse width and energy. As an application, we demonstrate that compared with single-polarization soliton, imaging signals are increased by two fold in MPM using our technology, corresponding to the repetition rate doubling of the optical solitons. We expect this technology will help increase the imaging speed or signal levels in MPM. Besides, this technology may overcome the signal generation limit due to the maximum available soliton energy from solid-core PC rods.
Tunable optical solitons generated by soliton self-frequency shift (SSFS) have become valuable tools for multiphoton microscopy (MPM). Recent progress in MPM using 1700 nm excitation enabled visualizing subcortical structures in mouse brain in vivo for the first time. Such an excitation source can be readily obtained by SSFS in a large effective-mode-area photonic crystal rod with a 1550-nm fiber femtosecond laser. A longpass filter was typically used to isolate the soliton from the residual in order to avoid excessive energy deposit on the sample, which ultimately leads to optical damage. However, since the soliton was not cleanly separated from the residual, the criterion for choosing the optimal filtering wavelength is lacking. Here, we propose maximizing the ratio between the multiphoton signal and the n’th power of the excitation pulse energy as a criterion for optimal spectral filtering in SSFS when the soliton shows dramatic overlapping with the residual. This optimization is based on the most efficient signal generation and entirely depends on physical quantities that can be easily measured experimentally. Its application to MPM may reduce tissue damage, while maintaining high signal levels for efficient deep penetration.
In actively modulated laser systems such as the time-lens source, quadratic temporal phase modulation was naturally considered as the “ideal” modulation. However, it is unknown that whether a quadratic temporal phase modulation (equivalently, a linear chirp) translates into a quadratic spectral phase modulation, which can be easily eliminated using compressors. Here we study this phase transfer both analytically and numerically. Our results indicate that non-quadratic spectral phase aberration arises even if the temporal phase modulation is quadratic. Consequently, there is observable difference between the compressed pulses with a flat spectral phase and with quadratic spectral phase compensation only. However, as quadratic temporal phase modulation increases, non-quadratic spectral phase modulation decreases, and pulse compression with quadratic spectral phase tends to that with a flat spectral phase.
Soliton self-frequency shift (SSFS) is a versatile technique for generating ultrashort optical pulses with customized wavelengths suitable for nonlinear optical microscopy. However, spectral overlapping of the soliton with the residual sometimes causes extra power deposition onto the biological samples, which may further induce optical damage. Consequently, how to choose the optimal optical filter is of vital importance for increasing signal level and minimizing damage. Here we propose maximizing the ratio between multi-photon signal and the nth power of the excitation pulse energy as a criterion for optimal spectral filtering in SSFS. This optimization is based on most efficient signal generation and entirely depends on physical quantities that can be easily measured experimentally.
Tunable optical solitons due to intra-pulse stimulated Raman scattering are well-known for their ultrashort pulse width,
superb pulse quality and broadband tunability. Consequently they are suitable for a variety of applications, especially
multi-photon microscopy (MPM). Recent progress in MPM demands two- or multi-color excitation to match the
absorption peaks of multiple fluorophores. Here we propose a new scheme to generate two-color solitons with each
wavelength individually tunable. The wavelength of the most energetic soliton can be easily tuned through energy
tuning, while stretching the input pulse width narrows the wavelength separation between the most and the second most
energetic solitons. This tunable two-color source may find application in various modalities of MPM.
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