Diesel2p mesoscope with dual independent scan engines for flexible capture of dynamics in distributed neural circuitry.

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large field-of-view Two-Photon imaging (Diesel2p). Combining optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view of ~25 mm2, encompassing distances up to 7 mm, with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain.

Flexible simultaneous mesoscale two-photon imaging of neural activity at high speeds.

Understanding brain function requires monitoring local and global brain dynamics. Two-photon imaging of the brain across mesoscopic scales has presented trade-offs between imaging area and acquisition speed. We describe a flexible cellular resolution two-photon microscope capable of simultaneous video rate acquisition of four independently targetable brain regions spanning an approximate five-millimeter field of view. With this system, we demonstrate the ability to measure calcium activity across mouse sensorimotor cortex at behaviorally relevant timescales.

Fast holographic scattering compensation for deep tissue biological imaging.

Scattering in biological tissues is a major barrier for in vivo optical imaging of all but the most superficial structures. Progress toward overcoming the distortions caused by scattering in turbid media has been made by shaping the excitation wavefront to redirect power into a single point in the imaging plane. However, fast, non-invasive determination of the required wavefront compensation remains challenging. Here, we introduce a quickly converging algorithm for non-invasive scattering compensation, termed DASH, in which holographic phase stepping interferometry enables new phase information to be updated after each measurement. This leads to rapid improvement of the wavefront correction, forming a focus after just one measurement iteration and achieving an order of magnitude higher signal enhancement at this stage than the previous state-of-the-art. Using DASH, we demonstrate two-photon fluorescence imaging of microglia cells in highly turbid mouse hippocampal tissue down to a depth of 530 µm.

Method for Multiplexed Dynamic Intravital Multiphoton Imaging.

Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.

Biocompatible diameter-oscillating fiber with microlens endface.

Optical fibers have been widely applied to life science, such as laser delivering, fluorescence collection, biosensing, bioimaging, etc. To resolve the challenges of advanced multiphoton biophotonic applications utilizing ultrashort laser pulses, here we report a flexible diameter-oscillating fiber (DOF) with microlens endface fabricated by using Polydimethylsiloxane (PDMS) elastomers. The diameter of the DOF is designed to longitudinally vary for providing accurate dispersion management, which is important for near-infrared multiphoton biophotonics that usually involves ultrashort laser pulses. The variation range and period of the DOF's diameter can be flexibly adjusted by controlling the parameters during the fabrication, such that dispersion curves with different oscillation landscapes can be obtained. The dispersion oscillating around the zero-dispersion baseline gives rise to a minimized net dispersion as the ultrashort laser pulse passes through the DOF - reducing the temporal broadening effect and resulting in transform-limited pulsewidth. In addition, the endface of the DOF is fabricated with a microlens, which is especially useful for laser scanning/focusing and fluorescence excitation. It is anticipated that this new biocompatible DOF is of great interest for biophotonic applications, particularly multiphoton microscopy deep inside biological tissues.

Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae.

Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy-imaging the sample from different angles followed by 3D image reconstruction-was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas.

Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of ∼16,000 neurons at 7.5 Hz from a 9 mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements.

Learned adaptive multiphoton illumination microscopy for large-scale immune response imaging.

Multiphoton microscopy is a powerful technique for deep in vivo imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to generate contrast in scattering tissue, while minimizing photobleaching and phototoxicity. We show here how adaptive imaging can optimize illumination power at each point in a 3D volume as a function of the sample's shape, without the need for specialized fluorescent labeling. Our method relies on training a physics-based machine learning model using cells with identical fluorescent labels imaged in situ. We use this technique for in vivo imaging of immune responses in mouse lymph nodes following vaccination. We achieve visualization of physiologically realistic numbers of antigen-specific T cells (~2 orders of magnitude lower than previous studies), and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response. We provide a step-by-step tutorial for implementing this technique using exclusively open-source hardware and software.

Intravital Imaging of Vascular Permeability by Two-Photon Microscopy.

The regulation of vascular permeability is critical in inflammation. It controls the distribution of water and plasma contents such as immunoglobulins in peripheral tissues. To regulate allergic diseases, it is important to study vascular biology especially in inflammation. Since the vascular permeability changes in minutes upon the exposure to proinflammatory mediators, intravital imaging system is a powerful technique to capture such dynamic responses. We here describe how to evaluate vascular permeability in vivo using multiphoton microscopy. We use various sizes of fluorescence-labeled dextran to visualize how leaky the blood vessels are in the steady state and in inflammation. Using this assay system, we can illustrate the dynamic kinetics of vascular permeability in vivo in real-time. This assay system provides a novel convenient way to study vascular biology that is beneficial in the assessment of various animal models of allergic disease.

Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness.

Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.

A novel setup for simultaneous two-photon functional imaging and precise spectral and spatial visual stimulation in Drosophila.

Motion vision has been extensively characterised in Drosophila melanogaster, but substantially less is known about how flies process colour, or how spectral information affects other visual modalities. To accurately dissect the components of the early visual system responsible for processing colour, we developed a versatile visual stimulation setup to probe combined spatial, temporal and spectral response properties. Using flies expressing neural activity indicators, we tracked visual responses in the medulla, the second visual neuropil, to a projected colour stimulus. The introduction of custom bandpass optical filters enables simultaneous two-photon imaging and visual stimulation over a large range of wavelengths without compromising the temporal stimulation rate. With monochromator-produced light, any spectral bandwidth and centre wavelength from 390 to 730 nm can be selected to produce a narrow spectral hue. A specialised screen material scatters each band of light across the visible spectrum equally at all locations of the screen, thus enabling presentation of spatially structured stimuli. We show layer-specific shifts of spectral response properties in the medulla correlating with projection regions of photoreceptor terminals.

In vivo two-photon microscopic observation and ablation in deeper brain regions realized by modifications of excitation beam diameter and immersion liquid.

In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.

Aberration-free 3D imaging via DMD-based two-photon microscopy and sensorless adaptive optics.

In this Letter, we present a new, to our knowledge, aberration-free 3D imaging technique based on digital micromirror device (DMD)-based two-photon microscopy and sensorless adaptive optics (AO), where 3D random-access scanning and modal wavefront correction are realized using a single DMD chip at 22.7 kHz. Specifically, the DMD is simultaneously used as a deformable mirror to modulate a distorted wavefront and a fast scanner to maneuver the laser focus in a 3D space by designed binary holograms. As such, aberration-free 3D imaging is realized by superposing the wavefront correction and 3D scanning holograms. Compared with conventional AO devices and methods, the DMD system can apply optimal wavefront correction information to different imaging regions or even individual pixels without compromising the scanning speed and device resolution. In the experiments, we first focus the laser through a diffuser and apply sensorless AO to retrieve a corrected focus. After that, the DMD performs 3D scanning on a Drosophila brain labeled with green fluorescent protein. The two-photon imaging results, where optimal wavefront correction information is applied to 3×3 separate regions, demonstrate significantly improved resolution and image quality. The new DMD-based imaging solution presents a compact, low-cost, and effective solution for aberration-free two-photon deep tissue imaging, which may find important applications in the field of biophotonics.

Scanless two-photon excitation with temporal focusing.

Temporal focusing, with its ability to focus light in time, enables scanless illumination of large surface areas at the sample with micrometer axial confinement and robust propagation through scattering tissue. In conventional two-photon microscopy, widely used for the investigation of intact tissue in live animals, images are formed by point scanning of a spatially focused pulsed laser beam, resulting in limited temporal resolution of the excitation. Replacing point scanning with temporally focused widefield illumination removes this limitation and represents an important milestone in two-photon microscopy. Temporal focusing uses a diffusive or dispersive optical element placed in a plane conjugate to the objective focal plane to generate position-dependent temporal pulse broadening that enables axially confined multiphoton absorption, without the need for tight spatial focusing. Many techniques have benefitted from temporal focusing, including scanless imaging, super-resolution imaging, photolithography, uncaging of caged neurotransmitters and control of neuronal activity via optogenetics.

Two-photon microscope using a fiber-based approach for supercontinuum generation and light delivery to a small-footprint optical head.

In this Letter, we report a low-cost, portable, two-photon excitation fluorescence microscopy imager that uses a fiber-based approach for both femtosecond supercontinuum (SC) generation and light delivery to the optical head. The SC generation is based on a tapered polarization-maintaining photonic crystal fiber that uses pre-chirped femtosecond narrowband pulses to generate a coherent SC spectrum with a bandwidth of approximately 300 nm. Using this approach, high-power, near-transform-limited, wavelength-selectable SC pulses are generated and directly delivered to the imaging optical head. Preliminary testing of this imager on brain slices is presented, demonstrating a high signal-to-noise ratio and sub-cellular imaging capabilities to a depth of approximately 200 µm. These results demonstrate the suitability of the technology for ex vivo and potentially in vivo cellular-level biomedical imaging applications.

Precapillary sphincters maintain perfusion in the cerebral cortex.

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow.

Measuring two-photon microscopy ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting.

Two-photon microscopy (2PM) has revolutionized biomedical imaging by allowing thin optical sectioning in relatively thick biological specimens. Because dispersive microscope components in 2PM, such as objective lens, can alter temporal laser pulse width (typically being broader at the sample plane), for accurate measurements of two-photon absorption properties, it is important to characterize pulse duration at the sample plane. We present a simple modification to a two-photon microscope light path that allows for second-harmonic-generation-based interferometric autocorrelation measurements to characterize ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting (TCSPC). We show that TCSPC can be used as a simple and versatile method to estimate the zero time delay step value between two adjacent ultrafast laser pulses for these measurements. To demonstrate the utility of this modification, we measured the Coherent Chameleon-Ultra II Ti:sapphire laser pulse width at the sample plane using a 10 × air, 40 × air, or 63 × water-immersion objective lens. At 950-nm two-photon excitation, the measured pulse width was 154 ± 32, 165 ± 13, and 218 ± 27 fs (n = 6, mean ± standard deviation), respectively.

Tissue Clearing and Deep Imaging of the Kidney Using Confocal and Two-Photon Microscopy.

Microscopic and macroscopic evaluation of biological tissues in three dimensions is becoming increasingly popular. This trend is coincident with the emergence of numerous tissue clearing strategies, and advancements in confocal and two-photon microscopy, enabling the study of intact organs and systems down to cellular and sub-cellular resolution. In this chapter, we describe a wholemount immunofluorescence technique for labeling structures in renal tissue. This technique combined with solvent-based tissue clearing and confocal imaging, with or without two-photon excitation, provides greater structural information than traditional sectioning and staining alone. Given the addition of paraffin embedding to our method, this hybrid protocol offers a powerful approach to combine confocal or two-photon findings with histological and further immunofluorescent analysis within the same tissue.

Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy.

A protocol is presented to build a custom low-cost yet high-performance femtosecond (fs) fiber laser. This all-normal-dispersion (ANDi) ytterbium-doped fiber laser is built completely using commercially available parts, including $8,000 in fiber optic and pump laser components, plus $4,800 in standard optical components and extra-cavity accessories. Researchers new to fiber optic device fabrication may also consider investing in basic fiber splicing and laser pulse characterization equipment (~$63,000). Important for optimal laser operation, methods to verify true versus apparent (partial or noise-like) mode-locked performance are presented. This system achieves 70 fs pulse duration with a center wavelength of approximately 1,070 nm and a pulse repetition rate of 31 MHz. This fiber laser exhibits the peak performance that may be obtained for an easily assembled fiber laser system, which makes this design ideal for research laboratories aiming to develop compact and portable fs laser technologies that enable new implementations of clinical multiphoton microscopy and fs surgery.