High-NA two-photon single cell imaging with remote focusing using a diffractive tunable lens

Fast, volumetric structural and functional imaging of cellular and sub-cellular dynamics inside the living brain is one of the most desired capabilities in the neurosciences, but still faces serious challenges. Specifically, while few solutions for rapid 3D scanning exist, it is generally much easier to facilitate fast in-plane scanning than it is to scan axially at high speeds. Remote focusing in which the imaging plane is shifted along the optical axis by a tunable lens while maintaining the position of the sample and objective is a promising approach to increase the axial scan speed, but existing techniques often introduce severe optical aberrations in high-NA imaging systems, eliminating the possibility of diffraction-limited single-cell imaging. Here, we demonstrate near diffraction-limited, volumetric two-photon fluorescence microscopy in which we resolve the deep sub-micron structures of single microglia cells with axial scanning performed using a novel high-NA remote focusing method. Image contrast is maintained to within 7% compared to mechanical sample stepping and the focal volume remains nearly diffraction-limited over an axial range greater than 86 µm.     

Adaptive optics enables aberration-free single-objective remote focusing for two-photon fluorescence microscopy

Two-photon fluorescence microscopy has been widely applied to three-dimensional (3D) imaging of complex samples. Remote focusing by controlling the divergence of excitation light is a common approach to scanning the focus axially. However, microscope objectives induce distortion to the wavefront of non-collimated excitation beams, leading to degraded imaging quality away from the natural focal plane. In this paper, using a liquid-crystal spatial light modulator to control the divergence of the excitation beam through a single objective, we systematically characterized the aberrations introduced by divergence control through microscope objectives of NA 0.45, 0.8, and 1.05. We used adaptive optics to correct the divergence-induced-aberrations and maintain diffraction-limited focal quality over up to 800-µm axial range. We further demonstrated aberration-free remote focusing for in vivo imaging of neurites and synapses in the mouse brain.     

Miniature structured illumination microscope for in vivo 3D imaging of brain structures with optical sectioning

We present a high-resolution miniature, light-weight fluorescence microscope with electrowetting lens and onboard CMOS for high resolution volumetric imaging and structured illumination for rejection of out-of-focus and scattered light. The miniature microscope (SIMscope3D) delivers structured light using a coherent fiber bundle to obtain optical sectioning with an axial resolution of 18 µm. Volumetric imaging of eGFP labeled cells in fixed mouse brain tissue at depths up to 260 µm is demonstrated. The functionality of SIMscope3D to provide background free 3D imaging is shown by recording time series of microglia dynamics in awake mice at depths up to 120 µm in the brain.     

Optimal lens design and use in laser-scanning microscopy

In laser-scanning microscopy often an off-the-shelf achromatic doublet is used as a scan lens which can reduce the available diffraction-limited field-of-view (FOV) by a factor of 3 and introduce chromatic aberrations that are scan angle dependent. Here we present several simple lens designs of superior quality that fully make use of high-NA low-magnification objectives, offering diffraction-limited imaging over a large FOV and wavelength range. We constructed a two-photon laser-scanning microscope with optimized custom lenses which had a near diffraction limit point-spread-function (PSF) with less than 3.6% variation over a 400 µm FOV and less than 0.5 µm lateral color between 750 and 1050 nm.OCIS codes: (180.0180) Microscopy, (180.1790) Confocal microscopy, (180.4315) Nonlinear microscopy, (220.3620) Lens system design, (220.3630) Lenses     

Design of high-performance adaptive objective lens with large optical depth scanning range for ultrabroad near infrared microscopic imaging

We report on the theory and design of adaptive objective lens for ultra broadband near infrared light imaging with large dynamic optical depth scanning range by using an embedded tunable lens, which can find wide applications in deep tissue biomedical imaging systems, such as confocal microscope, optical coherence tomography (OCT), two-photon microscopy, etc., both in vivo and ex vivo. This design is based on, but not limited to, a home-made prototype of liquid-filled membrane lens with a clear aperture of 8mm and the thickness of 2.55mm ~3.18mm. It is beneficial to have an adaptive objective lens which allows an extended depth scanning range larger than the focal length zoom range, since this will keep the magnification of the whole system, numerical aperture (NA), field of view (FOV), and resolution more consistent. To achieve this goal, a systematic theory is presented, for the first time to our acknowledgment, by inserting the varifocal lens in between a front and a back solid lens group. The designed objective has a compact size (10mm-diameter and 15mm-length), ultrabroad working bandwidth (760nm - 920nm), a large depth scanning range (7.36mm in air) — 1.533 times of focal length zoom range (4.8mm in air), and a FOV around 1mm × 1mm. Diffraction-limited performance can be achieved within this ultrabroad bandwidth through all the scanning depth (the resolution is 2.22 μm - 2.81 μm, calculated at the wavelength of 800nm with the NA of 0.214 - 0.171). The chromatic focal shift value is within the depth of focus (field). The chromatic difference in distortion is nearly zero and the maximum distortion is less than 0.05%.OCIS codes: (220.0220) Optical design and fabrication, (220.1080) Active or adaptive optics, (080.2468) First-order optics, (080.2740) Geometric optical design, (170.3890) Medical optics instrumentation, (170.6900) Three-dimensional microscopy, (170.1790) Confocal microscopy, (170.4500) Optical coherence tomography     

Non-interferometric volumetric imaging in living human retina by confocal oblique scanning laser ophthalmoscopy

Three-dimensional (3D) imaging of the human retina is instrumental in vision science and ophthalmology. While interferometric retinal imaging is well established by optical coherence tomography (OCT), non-interferometric volumetric imaging in the human retina has been challenging up to date. Here, we report confocal oblique scanning laser ophthalmoscopy (CoSLO) to fill that void and harness non-interferometric optical contrast in 3D. CoSLO decouples the illumination and detection by utilizing oblique laser scanning and oblique imaging to achieve ∼4x better axial resolution than conventional SLO. By combining remote focusing, CoSLO permits the acquisition of depth signals in parallel and over a large field of view. Confocal gating is introduced by a linear sensor array to improve the contrast and resolution. For the first time, we reported non-interferometric 3D human retinal imaging with >20° viewing angle, and revealed detailed features in the inner, outer retina, and choroid. CoSLO shows potential to be another useful technique by offering 3D non-interferometric contrasts.     

Brightfield,fluorescence, and phase-contrast whole slide imaging via dual-LED autofocusing

Whole slide imaging (WSI) systems convert the conventional biological samples into digital images. Existing commercial WSI systems usually require an expensive high-performance motorized stage to implement the precise mechanical control, and the cost is prohibitive for most individual pathologists. In this work, we report a low-cost WSI system using the off-the-shelf components, including a computer numerical control (CNC) router, a photographic lens, a programmable LED array, a fluorescence filter cube, and a surface-mount LED. To perform real-time single-frame autofocusing, we exploited two elements of a programmable LED array to illuminate the sample from two different incident angles. The captured image would contain two copies of the sample with a certain separation determined by the defocus distance of the sample. Then the defocus distance can be recovered by identifying the translational shift of the two copies. The reported WSI system can reach a resolution of ∼0.7 µm. The time to determine the optimal focusing position for each tile is only 0.02 s, which is about an 83% improvement compared to our previous work. We quantified the focusing performance on 1890 different tissue tiles. The mean focusing error is ∼0.34 µm, which is well below the ± 0.7 µm depth of field range of our WSI system. The reported WSI system can handle both the semitransparent and the transparent sample, enabling us to demonstrate the implementation of brightfield, fluorescence, and phase-contrast WSI. An automatic digital distortion correction strategy is also developed to avoid the stitching errors. The reported prototype has an affordable cost and can make it broadly available and utilizable for individual pathologists as well as can promote the development of digital pathology.     

Simultaneous scattering compensation at multiple points in multi-photon microscopy

The two-photon fluorescence imaging depth has been significantly improved in recent years by compensating for tissue scattering with wavefront correction. However, in most approaches the wavefront corrections are valid only over a small sample region on the order of 1 to 10 µm. In samples where most scattering structures are confined to a single plane, sample conjugate correction geometries can increase the observable field to a few tens of µm. Here, we apply a recently introduced fast converging scheme for sensor-less scattering correction termed “Dynamic Adaptive Scattering compensation Holography” (DASH) in a sample conjugate configuration with a high pixel count nematic liquid crystal spatial light modulator (LC-SLM). Using a large SLM allows us to simultaneously correct for scattering at multiple field points, which can be distributed over the entire field of view provided by the objective lens. Despite the comparably slow refresh time of LC-SLMs, we achieve correction times on the order of 10 s per field point, which we show is sufficiently fast to counteract scattering at multiple sites in living mouse hippocampal tissue slices.     

Three-dimensional light-field microendoscopy with a GRIN lens array

Optical endoscopy has emerged as an indispensable clinical tool for modern minimally invasive surgery. Most systems primarily capture a 2D projection of the 3D surgical field. Currently available 3D endoscopes can restore stereoscopic vision directly by projecting laterally shifted views of the operating field to each eye through 3D glasses. These tools provide surgeons with informative 3D visualizations, but they do not enable quantitative volumetric rendering of tissue. Therefore, advanced tools are desired to quantify tissue tomography for high precision microsurgery or medical robotics. Light-field imaging suggests itself as a promising solution to the challenge. The approach can capture both the spatial and angular information of optical signals, permitting the computational synthesis of the 3D volume of an object. In this work, we present GRIN lens array microendoscopy (GLAM), a single-shot, full-color, and quantitative 3D microendoscopy system. GLAM contains integrated fiber optics for illumination and a GRIN lens array to capture the reflected light field. The system exhibits a 3D resolution of ∼100 µm over an imaging depth of ∼22 mm and field of view up to 1 cm². GLAM maintains a small form factor consistent with the clinically desirable design, making the system readily translatable to a clinical prototype.     

Dual-color multiple-particle tracking at 50-nm localization and over 100-μm range in 3D with temporal focusing two-photon microscopy

Nanoscale particle tracking in three dimensions is crucial to directly observe dynamics of molecules and nanoparticles in living cells. Here we present a three-dimensional particle tracking method based on temporally focused two-photon excitation. Multiple particles are imaged at 30 frames/s in volume up to 180 × 180 × 100 µm³. The spatial localization precision can reach 50 nm. We demonstrate its capability of tracking fast swimming microbes at speed of ~200 µm/s. Two-photon dual-color tracking is achieved by simultaneously exciting two kinds of fluorescent beads at 800 nm to demonstrate its potential in molecular interaction studies. Our method provides a simple wide-field fluorescence imaging approach for deep multiple-particle tracking.OCIS codes: (170.2520) Fluorescence microscopy, (170.7160) Ultrafast technology, (180.4315) Nonlinear microscopy     

Optical axial scanning in confocal microscopy using an electrically tunable lens

This paper presents the use and characterization of an electrically focus tunable lens to perform axial scanning in a confocal microscope. Lateral and axial resolution are characterized over a >250 µm axial scan range. Confocal microscopy using optical axial scanning is demonstrated in epithelial tissue and compared to traditional stage scanning. By enabling rapid axial scanning, minimizing motion artifacts, and reducing mechanical complexity, this technique has potential to enhance in vivo three-dimensional imaging in confocal endomicroscopy.OCIS codes: (170.0110) Imaging systems, (170.1790) Confocal microscopy, (170.3880) Medical and biological imaging, (170.3890) Medical optics instrumentation, (170.6900) Three-dimensional microscopy     

Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens

Functional two-photon Ca(2+)-imaging is a versatile tool to study the dynamics of neuronal populations in brain slices and living animals. However, population imaging is typically restricted to a single two-dimensional image plane. By introducing an electrically tunable lens into the excitation path of a two-photon microscope we were able to realize fast axial focus shifts within 15 ms. The maximum axial scan range was 0.7 mm employing a 40x NA0.8 water immersion objective, plenty for typically required ranges of 0.2-0.3 mm. By combining the axial scanning method with 2D acousto-optic frame scanning and random-access scanning, we measured neuronal population activity of about 40 neurons across two imaging planes separated by 40 μm and achieved scan rates up to 20-30 Hz. The method presented is easily applicable and allows upgrading of existing two-photon microscopes for fast 3D scanning.     

High-speed multifocus phase imaging in thick tissue

Phase microscopy is widely used to image unstained biological samples. However, most phase imaging techniques require transmission geometries, making them unsuited for thick sample applications. Moreover, when applied to volumetric imaging, phase imaging generally requires large numbers of measurements, often making it too slow to capture live biological processes with fast 3D index-of-refraction variations. By combining oblique back-illumination microscopy and a z-splitter prism, we perform phase imaging that is both epi-mode and multifocus, enabling high-speed 3D phase imaging in thick, scattering tissues with a single camera. We demonstrate here 3D qualitative phase imaging of blood flow in chick embryos over a field of view of 546 × 546 × 137 µm³ at speeds up to 47 Hz.     

Human retinal imaging using visible-light optical coherence tomography guided by scanning laser ophthalmoscopy

We achieved human retinal imaging using visible-light optical coherence tomography (vis-OCT) guided by an integrated scanning laser ophthalmoscopy (SLO). We adapted a spectral domain OCT configuration and used a supercontinuum laser as the illumating source. The center wavelength was 564 nm and the bandwidth was 115 nm, which provided a 0.97 µm axial resolution measured in air. We characterized the sensitivity to be 86 dB with 226 µW incidence power on the pupil. We also integrated an SLO that shared the same optical path of the vis-OCT sample arm for alignment purposes. We demonstrated the retinal imaging from both systems centered at the fovea and optic nerve head with 20° × 20° and 10° × 10° field of view. We observed similar anatomical structures in vis-OCT and NIR-OCT. The contrast appeared different from vis-OCT to NIR-OCT, including slightly weaker signal from intra-retinal layers, and increased visibility and contrast of anatomical layers in the outer retina.OCIS codes: (110.4190) Multiple imaging, (170.0110) Imaging systems, (170.4470) Ophthalmology, (170.4500) Optical coherence tomography     

Portable boom-type ultrahigh-resolution OCT with an integrated imaging probe for supine position retinal imaging

To expand the clinical applications and improve the ease of use of ultrahigh-resolution optical coherence tomography (UHR-OCT), we developed a portable boom-type ophthalmic UHR-OCT operating in supine position that can be used for pediatric subjects, bedridden patients and perioperative conditions. By integrating the OCT sample arm probe with real-time iris display and automatic focusing electric lens for easy alignment, coupling the probe on a self-locking multi-directional manipulator to reduce motion artifacts and operator fatigue, and installing the OCT module on a moveable cart for system mobility, our customized portable boom-type UHR-OCT enables non-contact, high-resolution and high-stability retinal examinations to be performed on subjects in supine position. The spectral-domain UHR-OCT operates at a wavelength of 845 nm with 130 nm FWHM (full width at half maximum) bandwidth, achieving an axial resolution of ≈2.3µm in tissue with an A-line acquisition rate up to 128 kHz. A high-definition two-dimensional (2D) raster protocol was used for high-quality cross-sectional imaging while a cube volume three-dimensional (3D) scan was used for three-dimensional imaging and en-face reconstruction, resolving major layer structures of the retina. The feasibility of the system was demonstrated by performing supine position 2D/3D retinal imaging on healthy human subjects, sedated infants, and non-sedated awake neonates.     

Design and optimization of line-field optical coherence tomography at visible wavebands

Parallel line-field Fourier-domain optical coherence tomography (LF-FDOCT) has emerged to enable relatively higher speeds than the conventional FDOCT system. In the LF-FDOCT, one B-scan is captured at a time instead of scanning the beam to acquire hundreds of A-scans. On the other hand, spectroscopic OCT using the visible waveband provides absorption information over multiple wavelengths at each voxel. This information of spectral absorption enables quantitative measurement of blood oxygenation, voxel by voxel. Here, we presented the design and optimization of a LF-FDOCT system at the visible waveband (520–620 nm), especially using a generic Camera Link area sensor (2048 × 1088 pixels). To optimize the axial resolution and depth of imaging volume, we simulated various parameters and found that two Nyquist optima can exist, the origin and implication of which has been discussed. As a result, our system acquired 1088 A-scans in parallel at the camera’s frame rate of 281 frame per second, achieving an equivalent rate of over 300,000 A-scan/s, while minimizing sacrifice in the point spread function (2.8 × 3.1 × 3.2 µm³, x × y × z) and the field of view (750 × 750 × 750 µm³). As an example of application, we presented high-speed imaging of blood oxygenation in the rodent brain cortex.     

Hyper-numerical aperture (NA = 2.8) microscope using λ = 1.56 μm femtosecond source for multi-photon imaging

A new microscope is discussed, where the scanning illumination has a numerical aperture of 2.8 with λ = 1.56 µm femtosecond fiber laser. Samples are placed or grown on a silicon substrate. Multi-photon emission is imaged in transmission on a cooled CCD. Two-photon and three-photon effects are observed from the silicon/water interface and gold nanoparticles. Images of cells, reference spheres and gold nanoparticles illustrate imaging properties of the microscope. Spectral characteristics of individual particles are achieved with a blazed transmission grating. Emission properties of differently sized gold nanoparticles are studied in detail, which indicate that their emission is a two-photon effect due continuum generation. Interestingly, spectral shape and emission power are similar for 20nm, 40nm and 60nm diameter gold nanoparticles for the cases studied.OCIS codes: (180.4243) Near-field microscopy, (180.4315) Nonlinear microscopy     

Femtosecond diode-based time lens laser for multiphoton microscopy

We demonstrate a near-infrared, femtosecond, diode laser-based source with kW peak power for two-photon microscopy. At a wavelength of 976 nm, the system produces sub-ps pulses operating at a repetition rate of 10 MHz with kilowatt class peak powers suitable for deep tissue two-photon microscopy. The system, integrated with a laser-scanning microscope, images to a depth of 900 µm in a fixed sample of PLP-eGFP labeled mouse brain tissue. This represents a significant development that will lead to more efficient, compact, and accessible laser sources for biomedical imaging.     

Ultrahigh speed endoscopic optical coherence tomography for gastroenterology

We describe an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor imaging catheter. The system had a 600 kHz axial scan rate and 8 µm axial resolution in tissue. Imaging was performed with a 3.2 mm diameter imaging catheter at 400 frames per second with a 12 µm spot size. Three-dimensional OCT (3D-OCT) imaging was performed in patients with a cross section of pathologies undergoing upper and lower endoscopy. The use of distally actuated imaging catheters enabled OCT imaging with more flexibility, such as volumetric imaging in the small intestine and the assessment of hiatal hernia using retroflex imaging. The high rotational scanning stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en face OCT and cross-sectional imaging, as well as OCT angiography (OCTA) for 3D visualization of subsurface microvasculature. The ability to perform both structural and functional 3D OCT imaging in the GI tract with microscopic accuracy should enable a wide range of studies and enhance the sensitivity and specificity of OCT for detecting pathology.OCIS codes: (110.2350) Fiber optics imaging, (120.3890) Medical optics instrumentation, (120.5800) Scanners, (110.6880) Three-dimensional image acquisition, (140.7260) Vertical cavity surface emitting lasers, (170.2150) Endoscopic imaging, (170.2680) Gastrointestinal, (170.3880) Medical and biological imaging, (170.4500) Optical coherence tomography     

Development of a versatile two-photon endoscope for biological imaging

We describe a versatile, catheter-type two-photon probe, designed for in vivo and ex vivo imaging of the aqueous outflow pathway in the eye. The device consists of a silica double cladding fiber used for laser delivery and fluorescence collection, a spiral fiber scanner driven by a miniature piezoelectric tube, and an assembly of three micro-size doublet achromatic lenses used for focusing the laser and collecting the two-photon excitation signal. All the components have a maximum diameter of 2 mm and are enclosed in a length of 12-gauge stainless steel hypodermic tubing having an outer diameter of 2.8 mm. The lateral and axial resolutions of the probe are measured to be 1.5 μm and 9.2 μm, respectively. Different lens configurations and fibers are evaluated by comparing their spatial resolutions and fluorescence signal collection efficiencies. Doublet achromatic lenses and a double cladding fiber with a high inner cladding numerical aperture are found to produce a high signal collection efficiency, which is essential for imaging live tissues. Simple methods for reducing image distortions are demonstrated. Images of human trabecular meshwork tissue are successfully obtained with this miniature two-photon microscope.