Measurement of three-photon excitation cross-sections of fluorescein from 1154†nm to 1500†nm.

Measurements of three-photon action cross-sections for fluorescein (dissolved in water, pH ∼11.5) are presented in the excitation wavelength range from 1154 to 1500 nm in ∼50 nm steps. The excitation source is a femtosecond wavelength tunable non-collinear optical parametric amplifier, which has been spectrally filtered with 50 nm full width at half maximum band pass filters. Cube-law power dependance is confirmed at the measurement wavelengths. The three-photon excitation spectrum is found to differ from both the one- and two-photon excitation spectra. The three-photon action cross-section at 1154 nm is more than an order of magnitude larger than those at 1450 and 1500 nm (approximately three times the wavelength of the one-photon excitation peak), which possibly indicates the presence of resonance enhancement.

Three-Photon Adaptive Optics for Mouse Brain Imaging.

Three-photon microscopy (3PM) was shown to allow deeper imaging than two-photon microscopy (2PM) in scattering biological tissues, such as the mouse brain, since the longer excitation wavelength reduces tissue scattering and the higher-order non-linear excitation suppresses out-of-focus background fluorescence. Imaging depth and resolution can further be improved by aberration correction using adaptive optics (AO) techniques where a spatial light modulator (SLM) is used to correct wavefront aberrations. Here, we present and analyze a 3PM AO system for in vivo mouse brain imaging. We use a femtosecond source at 1300 nm to generate three-photon (3P) fluorescence in yellow fluorescent protein (YFP) labeled mouse brain and a microelectromechanical (MEMS) SLM to apply different Zernike phase patterns. The 3P fluorescence signal is used as feedback to calculate the amount of phase correction without direct phase measurement. We show signal improvement in the cortex and the hippocampus at greater than 1 mm depth and demonstrate close to diffraction-limited imaging in the cortical layers of the brain, including imaging of dendritic spines. In addition, we characterize the effective volume for AO correction within brain tissues, and discuss the limitations of AO correction in 3PM of mouse brain.

Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes.

Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 µm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600-900 µm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in the deeper regions of the LN in vivo.

Deep three-photon imaging of the brain in intact adult zebrafish.

Behaviors emerge from activity throughout the brain, but noninvasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become noninvasively accessible from embryo to sexually mature adult in a vertebrate model.

Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain.

1300 nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and perturbation to the sample. We calculated and experimentally verified the excitation pulse energy to achieve the minimum photon count required for the detection of calcium transients in GCaMP6s-expressing neurons for 920 nm two-photon and 1320 nm three-photon excitation. By considering the combined effects of in-focus signal attenuation and out-of-focus background generation, we quantified the cross-over depth beyond which three-photon microscopy outpeforms two-photon microscopy in recording fidelity. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. Increased immunoreactivity was observed with 150 mW excitation power at 1 and 1.2 mm imaging depths. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

GCaMP6 ΔF/F dependence on the excitation wavelength in 3-photon and 2-photon microscopy of mouse brain activity.

A fundamental challenge in calcium imaging is to minimize the excitation laser power while still maintaining a sufficient signal-to-noise ratio to distinguish individual transients in the fluorescence traces. It is important to characterize relative fluorescence (i.e., ΔF/F) dependence on the excitation wavelength in vivo where the environment cannot be controlled effectively during imaging. Leveraging time division multiplexing of two excitation beams to achieve nearly simultaneous 2-photon and 3-photon imaging of the calcium transients, we measured systematically the ΔF/F dependence on the excitation wavelength in 2-photon and 3-photon in vivo imaging of neuronal activity in mouse brain labeled with GCaMP6s. The technique can be applied to in vivo measurements of other fluorescence sensors.

Three-photon imaging of mouse brain structure and function through the intact skull.

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-µm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain.

High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ∼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence.

We demonstrate adaptive optics system based on nonlinear feedback from 3- and 4-photon fluorescence. The system is based on femtosecond pulses created by soliton self-frequency shift of a 1550-nm fiber-based femtosecond laser together with micro-electro-mechanical system (MEMS) phase spatial light modulator (SLM). We perturb the 1020-segment SLM using an orthogonal Walsh sequence basis set with a modified version of three-point phase shifting interferometry. We show the improvement after aberrations correction in 3-photon signal from fluorescent beads. In addition, we compare the improvement obtained in the same adaptive optical system for 2-, 3- and 4-photon fluorescence using dye pool. We show that signal improvement resulting from aberration correction grows exponentially as a function of the order of nonlinearity.

Multiphoton gradient index endoscopy for evaluation of diseased human prostatic tissue ex vivo.

Multiphoton microscopy can instantly visualize cellular details in unstained tissues. Multiphoton probes with clinical potential have been developed. This study evaluates the suitability of multiphoton gradient index (GRIN) endoscopy as a diagnostic tool for prostatic tissue. A portable and compact multiphoton endoscope based on a 1-mm diameter, 8-cm length GRIN lens system probe was used. Fresh ex vivo samples were obtained from 14 radical prostatectomy patients and benign and malignant areas were imaged and correlated with subsequent H&E sections. Multiphoton GRIN endoscopy images of unfixed and unprocessed prostate tissue at a subcellular resolution are presented. We note several differences and identifying features of benign versus low-grade versus high-grade tumors and are able to identify periprostatic tissues such as adipocytes, periprostatic nerves, and blood vessels. Multiphoton GRIN endoscopy can be used to identify both benign and malignant lesions in ex vivo human prostate tissue and may be a valuable diagnostic tool for real-time visualization of suspicious areas of the prostate.

Miniature varifocal objective lens for endomicroscopy.

A miniature catadioptric lens for endoscopic imaging based on the principle of wavelength division multiplexing is presented. We demonstrate change of the magnification and the field of view (FOV) of the lens without any mechanical adjustment of the optical elements. The lens provides magnifications of ~-1.5× at 406-750 nm and ~-0.2× at 800 nm. The lens is used to demonstrate large-FOV (1.3 mm) reflectance imaging and high-resolution (0.57 µm) multiphoton fluorescence imaging of unstained mouse tissues.

Dual modality endomicroscope with optical zoom capability.

We present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance imaging with high-resolution (~0.5 µm) multiphoton intrinsic fluorescence imaging. We acquired in vivo and ex vivo images of unstained normal and tumor-laden tissues by using the large-FOV mode to navigate to the site of interest and then switching to the high-resolution modality to resolve cellular details.

Three-photon excited fluorescence imaging of unstained tissue using a GRIN lens endoscope.

We present a compact and portable three-photon gradient index (GRIN) lens endoscope system suitable for imaging of unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The lateral and axial resolution in water is 1.0 µm and 9.5 µm, respectively. The ~200 µm diameter field of view is imaged at 2 frames/s using a fiber-based excitation source at 1040 nm. Ex vivo imaging is demonstrated with unstained mouse lung at 5.9 mW average power. These results demonstrate the feasibility of three-photon GRIN lens endoscopy for optical biopsy.

In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems.

We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s. The lateral and axial resolution in water is 0.85 µm and 7.4 µm respectively. In vivo images of unstained tissues in live, anesthetized rats using the portable device are presented. These results show great promise for GRIN endoscopy to be used clinically.

In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope.

We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 µm by 115 µm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ~75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.

Multifocal multiphoton endoscope.

We report a miniaturized resonant/non-resonant multi-fiber raster scanner that is paired with a gradient-index lens assembly to achieve a compact and flexible multifocal multiphoton endoscope capable of longitudinal parallel image acquisition. Multiphoton images are obtained simultaneously at three axial depths, separated by ≥4.8 µm, by incorporating three axially offset double clad optical fibers into the miniaturized scanner. The fabricated endoscope has an outer diameter of 3 mm, a rigid length of 4 cm, and acquires images at 4 frames/s per focal plane, with lateral and axial resolutions for two-photon imaging of 0.8 and 10 µm, respectively.

Use of a lensed fiber for a large-field-of-view, high-resolution, fiber-scanning microendoscope.

We report the application of a lensed fiber to a miniaturized fiber raster scanner in order to reduce the fiber's output beam size, thereby allowing for a compact and flexible endoscope capable of a large field of view (FOV) and high spatial resolution. For a proof of principle, the fabricated lensed fiber scanner is paired with a miniaturized gradient-index assembly to achieve a one-photon lateral resolution of 1.1 µm with a FOV that has a diameter of 440 µm.

Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue.

We present a compact and flexible endoscope (3-mm outer diameter, 4-cm rigid length) that utilizes a miniaturized resonant/nonresonant fiber raster scanner and a multielement gradient-index lens assembly for two-photon excited intrinsic fluorescence and second-harmonic generation imaging of biological tissues. The miniaturized raster scanner is fabricated by mounting a commercial double-clad optical fiber (DCF) onto two piezo bimorphs that are aligned such that their bending axes are perpendicular to each other. Fast lateral scanning of the laser illumination at 4.1 frames/s (512 lines per frame) is achieved by simultaneously driving the DCF cantilever at its resonant frequency in one dimension and nonresonantly in the orthogonal axis. The implementation of a DCF into the scanner enables simultaneous delivery of the femtosecond pulsed 800-nm excitation source and epi-collection of the signal. Our device is able to achieve a field-of-view (FOV(xy)) of 110 µm by 110 µm with a highly uniform pixel dwell time. The lateral and axial resolutions for two-photon imaging are 0.8 and 10 µm, respectively. The endoscope's imaging capabilities were demonstrated by imaging ex vivo mouse tissue through the collection of intrinsic fluorescence and second-harmonic signal without the need for staining. The results presented here indicate that our device can be applied in the future to perform minimally invasive in vivo optical biopsies for medical diagnostics.

Starting dynamics of dissipative-soliton fiber laser.

We study the starting dynamics of an all-normal-dispersion Yb-doped fiber laser experimentally and compare them to an existing stochastic model of starting from quantum noise. The laser reaches mode locking 10 to 100 times faster than a soliton laser with similar parameters. According to the model, the fast starting can be attributed to the large pulse energy in the normal-dispersion laser. We also report direct observations of starting from relaxation oscillations and discuss that process in light of the theory.

Divided-pulse amplification of ultrashort pulses.

We propose and demonstrate a new approach, to the best of our knowledge, for avoiding nonlinear effects in the amplification of ultrashort optical pulses. The initial pulse is divided longitudinally into a sequence of lower-energy pulses that are otherwise identical to the original, except for the polarization. The low-intensity pulses are amplified and then recombined to create a final intense pulse. This divided-pulse amplification complements techniques based on dispersion management.