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.

An adaptive excitation source for high-speed multiphoton microscopy.

Optical imaging is important for understanding brain function. However, established methods with high spatiotemporal resolution are limited by the potential for laser damage to living tissues. We describe an adaptive femtosecond excitation source that only illuminates the region of interest, which leads to a 30-fold reduction in the power requirement for two- or three-photon imaging of brain activity in awake mice for improved high-speed longitudinal neuroimaging.

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.

Intravital Imaging of the Murine Subventricular Zone with Three Photon Microscopy.

The mouse subventricular zone (SVZ) produces neurons throughout life. It is useful for mechanism discovery and is relevant for regeneration. However, the SVZ is deep, significantly restricting live imaging since current methods do not extend beyond a few hundred microns. We developed and adapted three-photon microscopy (3PM) for non-invasive deep brain imaging in live mice, but its utility in imaging the SVZ niche was unknown. Here, with fluorescent dyes and genetic labeling, we show successful 3PM imaging in the whole SVZ, extending to a maximum depth of 1.5 mm ventral to the dura mater. 3PM imaging distinguished multiple SVZ cell types in postnatal and juvenile mice. We also detected fine processes on neural stem cells interacting with the vasculature. Previous live imaging removed overlying cortical tissue or lowered lenses into the brain, which could cause inflammation and alter neurogenesis. We found that neither astrocytes nor microglia become activated in the SVZ, suggesting 3PM does not induce major damage in the niche. Thus, we show for the first time 3PM imaging of the SVZ in live mice. This strategy could be useful for intravital visualization of cell dynamics, molecular, and pathological perturbation and regenerative events.

Microscopic sensors using optical wireless integrated circuits.

We present a platform for parallel production of standalone, untethered electronic sensors that are truly microscopic, i.e., smaller than the resolution of the naked eye. This platform heterogeneously integrates silicon electronics and inorganic microlight emitting diodes (LEDs) into a 100-µm-scale package that is powered by and communicates with light. The devices are fabricated, packaged, and released in parallel using photolithographic techniques, resulting in ∼10,000 individual sensors per square inch. To illustrate their use, we show proof-of-concept measurements recording voltage, temperature, pressure, and conductivity in a variety of environments.

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.

Fabrication of Injectable Micro-Scale Opto-Electronically Transduced Electrodes (MOTEs) for Physiological Monitoring.

In vivo, chronic neural recording is critical to understand the nervous system, while a tetherless, miniaturized recording unit can render such recording minimally invasive. We present a tetherless, injectable micro-scale opto-electronically transduced electrode (MOTE) that is ~60µm × 30µm × 330µm, the smallest neural recording unit to date. The MOTE consists of an AlGaAs micro-scale light emitting diode (µLED) heterogeneously integrated on top of conventional 180nm complementary metal-oxide-semiconductor (CMOS) circuit. The MOTE combines the merits of optics (AlGaAs µLED for power and data uplink), and of electronics (CMOS for signal amplification and encoding). The optical powering and communication enable the extreme scaling while the electrical circuits provide a high temporal resolution (<100µs). This paper elaborates on the heterogeneous integration in MOTEs, a topic that has been touted without much demonstration on feasibility or scalability. Based on photolithography, we demonstrate how to build heterogenous systems that are scalable as well as biologically stable - the MOTEs can function in saline water for more than six months, and in a mouse brain for two months (and counting). We also present handling/insertion techniques for users (i.e. biologists) to deploy MOTEs with little or no extra training.

Multiphoton imaging provides a superior optical biopsy to that of confocal laser endomicroscopy imaging for colorectal lesions.

BACKGROUND: Confocal laser endomicroscopy (CLE) requires fluorescence agents, the use of which leads to blurred images and low diagnostic accuracy owing to fluorescein leakage. We aimed to explore whether multiphoton imaging (MPI) could serve as a better method of optical biopsy. METHODS: First, a pilot study was performed to set up the optical diagnostic criteria of MPI for benign or malignant colorectal lesions in 30 patients. Then, a blinded study was conducted to compare the sensitivity, specificity, and accuracy of MPI versus CLE imaging in 79 patients. RESULTS : In the pilot study, MPI revealed regular tissue architecture and cell morphology in the normal tissue, and irregular tubular structures, and cellular and nuclear pleomorphism in the abnormal tissue. In the blinded study, compared with CLE imaging, MPI significantly improved the diagnostic sensitivity, specificity, and accuracy of the optical biopsy (89.74 % vs. 61.54 %, P = 0.008; 92.5 % vs. 67.5 %, P = 0.009; and 91.14 % vs. 64.56 %, P = 0.001, respectively). CONCLUSIONS : MPI can provide a superior optical biopsy to that of CLE imaging for colorectal lesions.

Real-time in vivo optical biopsy using confocal laser endomicroscopy to evaluate distal margin in situ and determine surgical procedure in low rectal cancer.

BACKGROUND: In low rectal cancer, a negative distal margin (DM) is necessary for R0 radical resection, and therefore, the choice of surgical procedure is dependent on whether the planned transection rectum has residual cancer or not. Currently, surgeons choose surgical procedures according to intraoperative in vitro DM frozen sections. This study aimed to investigate the feasibility of real-time in vivo optical biopsy using confocal laser endomicroscopy (CLE) to evaluate DM in situ and determine the surgical procedure in low rectal cancer. METHODS: Optical biopsy using CLE was performed when the rectum was dissected at the levator ani plane and rectum transection was ready. For negative DM, the surgical procedure of low anterior resection (LAR) was chosen. For positive DM, the surgical procedure of abdominoperineal resection (APR) was chosen. The specimen at the site of the planned transection rectum underwent intraoperative frozen section and routine pathological procedures. RESULTS: Eighteen patients underwent real-time in vivo optical biopsy using CLE in surgery. Eleven patients' CLE images of DM showed a regular, round crypt, and round luminal opening covered by a simple layer of columnar epithelial cells and goblet cells. LAR was then performed. Pathology revealed that the 11 DMs were negative, and the median length of the DMs was 2.0 cm. The remaining seven patients' CLE images of the planned transection rectum showed the loss of crypt architecture and irregular epithelial layer with loss of goblet cells. APR was then performed. Pathology confirmed cancer invasion, and the median distance from tumor to dentate line was 1.0 cm. The sensitivity, specificity, and accuracy of CLE optical biopsy of DM were 85.71%, 100%, and 94.44%, respectively. CONCLUSIONS: It is feasible to perform real-time in vivo optical biopsy using CLE to evaluate DM in situ and determine the surgical procedure in low rectal cancer.

Multiple Regions of Interest on Multiparametric Magnetic Resonance Imaging are Not Associated with Increased Detection of Clinically Significant Prostate Cancer on Fusion Biopsy.

PURPOSE: We sought to determine the association between multiple regions of interest on prebiopsy magnetic resonance imaging and the detection of clinically significant prostate cancer in men undergoing magnetic resonance imaging-ultrasound fusion biopsy. MATERIALS AND METHODS: We performed a retrospective, single institution analysis of men who underwent fusion biopsy. Men with prior positive biopsies, magnetic resonance imaging performed elsewhere and/or magnetic resonance imaging prior to release of the PI-RADS™ (Prostate Imaging Reporting and Data System) version 2 were excluded from study, resulting in 381 participants. Modeled independent variables included patient age, number of regions of interest with a PI-RADS categorization of 3 or greater, body mass index, prostate specific antigen, prostate volume and PI-RADS categorization. Multivariable logistic regression was performed to determine factors associated with finding clinically significant prostate cancer (Gleason 7 or greater) on biopsy. RESULTS: Median age was 67.2 years (IQR (61.6-73.0) and median prostate specific antigen was 6.6 ng/ml (5.0-10.0). Adjusted analysis demonstrated that age (OR 1.10, 95% CI 1.06-1.15, p ≤0.001), body mass index (OR 1.08, 95% CI 1.01-1.16, p = 0.038) and prostate specific antigen (OR 1.06, 95% CI 1.01-1.10, p = 0.015) were associated with detection of clinically significant prostate cancer. PI-RADS categories 4 (OR 4.62, 95% CI 2.23-9.33) and 5 (OR 6.75, 95% CI 2.72-16.71, each p <0.001) were associated with greater odds of clinically significant prostate cancer. Multiple regions of interest were not associated with the detection of clinically significant prostate cancer (OR 1.05, 95% CI 0.60-1.84, p = 0.857). CONCLUSIONS: Multiple regions of interest do not portend a greater likelihood of finding clinically significant prostate cancer. Physicians should recognize that multiple regions of interest should not influence the decision to perform fusion biopsy. Our findings may ease patient anxiety concerning these findings.

Investigation of the long wavelength limit of soliton self-frequency shift in a silica fiber.

We explore the long wavelength limit of soliton self-frequency shift in silica-based fibers experimentally and using numerical simulation. We found that the longest wavelength soliton generated by soliton self-frequency shift is approximately 2500 nm because the soliton loses its energy rapidly at wavelength beyond 2400 nm due to material absorption by silica and water. We demonstrate 1580-2520 nm wavelength-tunable, high-pulse energy soliton generation using soliton self-frequency shift in a large-mode-area silica fiber pumped by a compact fiber source. Soliton pulses with pulse width of ~100 fs and pulse energy up to 73 nJ were obtained. Second harmonic generation of the solitons enables a wavelength-tunable femtosecond source from 950 nm to 1260 nm, with pulse energy up to 21 nJ. Using such energetic pulses, we demonstrate in vivo two-photon excited fluorescence imaging of vasculature and neurons in a mouse brain at wavelength beyond the tuning range of a mode-locked Ti:Sapphire lasers.

Multi-color background-free coherent anti-Stokes Raman scattering microscopy using a time-lens source.

We demonstrate a multi-color background-free coherent anti-Stokes Raman scattering (CARS) imaging system, using a robust, all-fiber, low-cost, multi-wavelength time-lens source. The time-lens source generates picosecond pulse trains at three different wavelengths. The first is 1064.3 nm, the second is tunable between 1052 nm and 1055 nm, and the third is tunable between 1040 nm and 1050 nm. When the time-lens source is synchronized with a mode-locked Ti:Sa laser, two of the three wavelengths are used to detect different Raman frequencies for two-color on-resonance imaging, whereas the third wavelength is used to obtain the off-resonance image for nonresonant background subtraction. Mixed poly(methyl methacrylate) (PMMA) and polystyrene (PS) beads are used to demonstrate two-color background-free CARS imaging. The synchronized multi-wavelength time-lens source enables pixel-to-pixel wavelength-switching. We demonstrate simultaneous two-color CARS imaging of CH2 and CH3 stretching vibration modes with real-time background subtraction in ex vivo mouse tissue.

Characterization and adaptive compression of a multi-soliton laser source.

Ultrashort pulse generation in the 1600 nm wavelength region is required for deep-tissue biomedical imaging. We report on the characterization and adaptive compression of a multi-soliton output spanning >300 nm from a large-mode area photonic-crystal fiber rod for two separate laser setups. Sub-30 fs pulses are generated by first compressing of each soliton individually, and then followed by coherently combining all of the pulses in the train, which are separated by hundreds of femtoseconds. Simulations of the source, together with amplitude and phase coherence measurements are provided.

Two-photon Shack-Hartmann wavefront sensor.

We introduce a simple wavefront sensing scheme for aberration measurement of pulsed laser beams in near-infrared wavelengths (<2200 nm), where detectors are not always available or are very expensive. The method is based on two-photon absorption in a silicon detector array for longer wavelengths detection. We demonstrate the simplicity of such implementations with a commercially available Shack-Hartmann wavefront sensor and discuss the detection sensitivity of this method.

Nonresonant background suppression for coherent anti-Stokes Raman scattering microscopy using a multi-wavelength time-lens source.

We demonstrate a robust, all-fiber, two-wavelength time-lens source for background-free coherent anti-Stokes Raman scattering imaging. The time-lens source generates two picosecond pulse trains simultaneously: one at 1064 nm and the other tunable between 1040 nm and 1075 nm (~400 mW for each wavelength). When synchronized to a mode-locked Ti:Sapphire laser, the two wavelengths are used to obtain on- and off-resonance coherent anti-Stokes Raman scattering images. Real-time subtraction of the nonresonant background in the coherent anti-Stokes Raman scattering image is achieved by the synchronization of the pixel clock and the time-lens source. Background-free coherent anti-Stokes Raman scattering imaging of sebaceous glands in ex vivo mouse tissue is demonstrated.

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.

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.

Measurement of third order coherence by in situ autocorrelation for determining three-photon cross-sections.

We show theoretically that the third order coherence at zero delay can be obtained by measuring the second and third order autocorrelation traces of a pulsed laser. Our theory enables the measurement of a fluorophore's three-photon cross-section without prior knowledge of the temporal profile of the excitation pulse by using the same fluorescent medium for both the measurement of the third order coherence at zero delay as well as the cross-section. Such an in situ measurement needs no assumptions about the pulse shape nor group delay dispersion of the optical system. To verify the theory experimentally, we measure the three-photon action cross-section of Alexa Fluor 350 and show that the measured value of the three-photon cross-section remains approximately constant despite varied amounts of chirp on the excitation pulses.

Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo.

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 µm into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 µm of the murine spinal cord, in vivo.