A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

A topical review of numerical and experimental studies of supercontinuum generation in photonic crystal fiber is presented over the full range of experimentally reported parameters, from the femtosecond to the continuous-wave regime. Results from numerical simulations are used to discuss the temporal and spectral characteristics of the supercontinuum, and to interpret the physics of the underlying spectral broadening processes. Particular attention is given to the case of supercontinuum generation seeded by femtosecond pulses in the anomalous group velocity dispersion regime of photonic crystal fiber, where the processes of soliton fission, stimulated Raman scattering, and dispersive wave generation are reviewed in detail. The corresponding intensity and phase stability properties of the supercontinuum spectra generated under different conditions are also discussed.

Supercontinuum generation, photonic crystal fiber

Supercontinuum Generation in Optical Fibers: Preface

Three-photon microscopy has been increasingly adopted for probing neural activities beyond the typical two-photon imaging depth. In this review, we outline the unique properties that differentiate three-photon microscopy from two-photon microscopy for in vivo imaging in biological samples, especially in the mouse brain. We present a systematic summary of the optimization of three-photon imaging parameters for neural imaging, based on their effects on calcium imaging quality and perturbation to brain tissues. Furthermore, we review the existing techniques for volumetric imaging and discuss their prospects in mesoscale three-photon imaging in deep tissue.

Three-photon neuronal imaging in deep mouse brain

Measurements of two-photon fluorescence excitation (TPE) spectra are presented for 11 common molecular fluorophores in the excitation wavelength range 690 nm < λ < 1050 nm. Results of excitation by ∼100-fs pulses of a mode-locked Ti:sapphire laser are corroborated by single-mode cw Ti:sapphire excitation data in the range 710 nm < λ < 840 nm. Absolute values of the TPE cross section for Rhodamine B and Fluorescein are obtained by comparison with one-photon-excited fluorescence, assuming equal emission quantum efficiencies. TPE action cross sections for the other nine fluorophores are also determined. No differences between one-photon- and two-photon-excited fluorescence emission spectra are found. TPE emission spectra are independent of excitation wavelength. With both pulsed and cw excitation the fluorescence emission intensities are strictly proportional to the square of the excitation intensity to within ±4% for excitation intensities sufficiently below excited-state saturation.

Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm

Deep-tissue multiphoton microscopy (MPM) enables noninvasive optical imaging into the deep regions of the tissue in animal models in vivo . High-order MPM techniques, such as three- and four-photon fluorescence microscopy at the 1700-nm window, are emerging as promising imaging techniques for deeper penetration. Currently, signal depletion at large imaging depth sets the depth limit for these imaging techniques. As a result, how to further boost signal level is the key to achieving a larger imaging depth. Contrary to the previous thought that overfilling the back aperture of the objective yields the highest multiphoton fluorescence signal, in this paper, through numerical simulation, we show that, due to the effect of exponential decay of the excitation beam, the signal generation is maximized for certain underfilling of the back aperture of the objective lens. This will provide a simple strategy for signal generation maximization in deep-tissue MPM and potentially enables deeper imaging penetration into the tissue.

Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy

Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known ...

In Vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots.

1700-nm window has been demonstrated to be a promising excitation window for deep-tissue multiphoton microscopy (MPM). Long working-distance water immersion objective lenses are typically used for deep-tissue imaging. However, absorption due to immersion water at 1700 nm is still high and leads to dramatic decrease in signals. In this paper, we demonstrate measurement of absorption spectrum of deuterium oxide (D2O) from 1200 nm to 2600 nm, covering the three low water-absorption windows potentially applicable for deep-tissue imaging (1300 nm, 1700 nm, and 2200 nm). We apply this measured result to signal enhancement in MPM at the 1700-nm window. Compared with water immersion, D2O immersion enhances signal levels in second-harmonic generation imaging, 3-photon fluorescence imaging, and third-harmonic generation imaging by 8.1, 24.8, and 24.7 times with 1662-nm excitation, in good agreement with theoretical calculation based on our absorption measurement. This suggests D2O a promising immersion medium for d...

Measurement of absorption spectrum of deuterium oxide (D2O) and its application to signal enhancement in multiphoton microscopy at the 1700-nm window

Here we demonstrate deep-brain 2-photon fluorescence microscopy in
mouse in vivo, excited at the 1700 nm window. Through signal versus
power measurement, we show that indocyanine green (ICG) is a promising
2-photon fluorescent dye excitable at the 1700 nm window. In order
to excite ICG circulating in the vasculature in the deep brain, we employ
a circular-polarization soliton self-frequency shift technique to generate
energetic femtosecond pulses at 1617 nm. Combining the labeling and
laser technologies, we achieve a record 2-photon fluorescence brain
vasculature imaging depth of 2000 μm in vivo. Both the
effective attenuation length measurement and signal-to-background ratio
measurement indicate that we have reached the theoretical depth limit in
2-photon fluorescence microscopy.

Deep-brain 2-photon fluorescence microscopy in vivo excited at the 1700-nm window

To enhance signal levels in multiphoton microscopy (MPM) at the deep-tissue excitation window (1600-1820 nm) with oil immersion, we demonstrate: First, the absorption spectra of several commonly immersion oils are characterized, which were unknown before. Second, new material with lower absorption based on mixing is proposed. Third, optimal selection of excitation wavelength within this window is proposed based on absorption spectra characterization. Second and third harmonic generation imaging of mouse tissue corroborate our selection: 1600-nm excitation leads to notable orders-of-magnitude increase in MPM signal, compared with 1700-nm excitation, enabling 200-µm imaging depth of mouse skin while 1700-nm excitation could resolve virtually no structure.

Ping Qiu

Papers

Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy

In Vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots.

Measurement of absorption spectrum of deuterium oxide (D2O) and its application to signal enhancement in multiphoton microscopy at the 1700-nm window

Deep-brain 2-photon fluorescence microscopy in vivo excited at the 1700-nm window

Order-of-magnitude multiphoton signal enhancement based on characterization of absorption spectra of immersion oils at the 1700-nm window