Confocal Gate Research Articles

Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.

Time-domain full-field optical coherence tomography with digital confocal line scanning.

Full-field optical coherence tomography (FF-OCT) is a camera-based interferometric microscopy technique that can image deep in tissue with high spatial resolution. However, the absence of confocal gating leads to suboptimal imaging depth. Here, we implement digital confocal line scanning in time-domain FF-OCT by exploiting the row-by-row detection feature of a rolling-shutter camera. A digital micromirror device (DMD) is used in conjunction with the camera to produce synchronized line illumination. An improvement in the SNR by an order of magnitude is demonstrated on a sample of a US Air Force (USAF) target mounted behind a scattering layer.

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.

Closed-loop wavefront sensing and correction in the mouse brain with computed optical coherence microscopy.

Optical coherence microscopy (OCM) uses interferometric detection to capture the complex optical field with high sensitivity, which enables computational wavefront retrieval using back-scattered light from the sample. Compared to a conventional wavefront sensor, aberration sensing with OCM via computational adaptive optics (CAO) leverages coherence and confocal gating to obtain signals from the focus with less cross-talk from other depths or transverse locations within the field-of-view. Here, we present an investigation of the performance of CAO-based aberration sensing in simulation, bead phantoms, and ex vivo mouse brain tissue. We demonstrate that, due to the influence of the double-pass confocal OCM imaging geometry on the shape of computed pupil functions, computational sensing of high-order aberrations can suffer from signal attenuation in certain spatial-frequency bands and shape similarity with lower order counterparts. However, by sensing and correcting only low-order aberrations (astigmatism, coma, and trefoil), we still successfully corrected tissue-induced aberrations, leading to 3× increase in OCM signal intensity at a depth of ∼0.9 mm in a freshly dissected ex vivo mouse brain.

Super-resolution retinal imaging using optically reassigned scanning laser ophthalmoscopy.

Super-resolution optical microscopy techniques have enabled the discovery and visualization of numerous phenomena in physics, chemistry and biology1-3. However, the highest resolution super-resolution techniques depend on nonlinear fluorescence phenomena and are thus inaccessible to the myriad applications that require reflective imaging4,5. One promising super-resolution technique is optical reassignment6, which so far has only shown potential for fluorescence imaging at low speeds. Here, we present novel advances in optical reassignment to adapt it for any scanning microscopy, including reflective imaging, and enable an order of magnitude faster image acquisition than previous optical reassignment techniques. We utilized these advances to implement optically reassigned scanning laser ophthalmoscopy, an in vivo super-resolution human retinal imaging device not reliant on confocal gating. Using this instrument, we achieved high-resolution imaging of living human retinal cone photoreceptor cells (determined by minimum foveal eccentricity) without adaptive optics or chemical dilation of the eye7.

Standard-unit measurement of cellular viability using dynamic light scattering optical coherence microscopy.

Dynamic light scattering optical coherence microscopy (DLS-OCM) integrates DLS, which measures diffusion or flow of particles by analyzing fluctuations in light scattered by the particles, and OCM, which achieves single-cell resolution by combining coherence and confocal gating, integratively enabling cellular-resolution 3D mapping of the diffusion coefficient, and flow velocity. The diffusion coefficient mapping has a potential for the non-destructive measurement of cellular viability in the standard unit but has not been validated yet. Here, we present DLS-OCM imaging of intra-cellular motility (ICM) as a surrogate of cellular viability. For this purpose, we have simultaneously obtained and compared ICM-contrast DLS-OCM images and calcium fluorescence-contrast images of retinal ganglion cells, and then characterized the responses of the measured ICM to a change in cellular viability induced by environmental conditions such as temperature and pH. The diffusion-coefficient-represented ICM exhibits consistent changes with the manipulated cellular viability.

High speed adaptive optics ophthalmoscopy with an anamorphic point spread function.

Retinal imaging working with a line scan mechanism and a line camera has the potential to image the eye with a near-confocal performance at the high frame rate, but this regime has difficulty to collect sufficient imaging light while adequately digitize the optical resolution in adaptive optics imaging. To meet this challenge, we have developed an adaptive optics line scan ophthalmoscope with an anamorphic point spread function. The instrument uses a high-speed line camera to acquire the retinal image and act as a confocal gate. Meanwhile, it employs a digital micro-mirror device to modulate the imaging light into a line of point sources illuminating the retina. The anamorphic mechanism ensures adequate digitization of the optical resolution and increases light collecting efficiency. We demonstrate imaging of the living human retina with cellular level resolution at a frame rate of 200 frames/second (FPS) with a digitization of 512 × 512 pixels over a field of view of 1.2° × 1.2°. We have assessed cone photoreceptor structure in images acquired at 100, 200, and 800 FPS in 2 normal human subjects, and confirmed that retinal images acquired at high speed rendered macular cone mosaic with improved measurement repeatability.

En-face time-domain optical coherence tomography with dynamic focus for high-resolution imaging.

Optical coherence tomography (OCT) is capable of imaging microstructures within translucid samples. A time-domain version of the OCT technology is employed here due to its compatibility with the dynamic focus (DF) procedure. DF means moving the confocal gate in synchronism with the depth scanning via the coherence gate. A DF-OCT setup was implemented for imaging samples at 1300 nm. Its confocal gate of 180 ?? ? m allows the achievement of good and similar transversal resolution along its much larger axial range. Images of a phantom, human skin, teeth, and larynx with and without DF are demonstrated.

Enhancement of optical coherence microscopy in turbid media by an optical parametric amplifier.

We report the enhancement in imaging performance of a spectral-domain optical coherence microscope (OCM) in turbid media by incorporating an optical parametric amplifier (OPA). The OPA provides a high level of optical gain to the sample arm, thereby improving the signal-to-noise ratio of the OCM by a factor of up to 15 dB. A unique nonlinear confocal gate is automatically formed in the OPA, which enables selective amplification of singly scattered (ballistic) photons against the multiply-scattered light background. Simultaneous enhancement in both imaging depth and spatial resolution in imaging microstructures in highly light-scattering media are demonstrated with the combined OPA-OCM setup. Typical OCM inteferograms (left) and images (right) without and with OPA.

Investigation of basal cell carcinoma using dynamic focus optical coherence tomography

Optical coherence tomography (OCT) is becoming a popular modality for skin tumor diagnosis and assessment of tumor size and margin status. We conducted a number of imaging experiments on periocular basal cell carcinoma (BCC) specimens using an OCT configuration. This configuration employs a dynamic focus (DF) procedure where the coherence gate moves synchronously with the peak of the confocal gate, which ensures better signal strength and preservation of transversal resolution from all depths. A DF-OCT configuration is used to illustrate morphological differences between the BCC and its surrounding healthy skin in OCT images. The OCT images are correlated with the corresponding histology images. To the best of our knowledge, this is the first paper to look at DF-OCT imaging in examining periocular BCC.

Quantitative evaluation of scattering in optical coherence tomography skin images using the extended Huygens–Fresnel theorem

An optical properties extraction algorithm is developed based on enhanced Huygens-Fresnel light propagation theorem, to extract the scattering coefficient of a specific region in an optical coherence tomography (OCT) image. The aim is to quantitatively analyze the OCT images. The algorithm is evaluated using a set of phantoms with different concentrations of scatterers, designed based on Mie theory. The algorithm is then used to analyze basal cell carcinoma and healthy eyelid tissues, demonstrating distinguishable differences in the scattering coefficient between these tissues. In this study, we have taken advantage of the simplification introduced by the utilization of a dynamic focus OCT system. This eliminates the need to deconvolve the reflectivity profile with the confocal gate profile, as the sensitivity of the OCT system is constant throughout the axial range.

Megahertz streak-mode Fourier domain optical coherence tomography

Here we present an ultrahigh-speed Fourier-domain optical coherence tomography (OCT) that records the OCT spectrum in streak mode with a high-speed area scan camera, which allows higher OCT imaging speed than can be achieved with a line-scan camera. Unlike parallel OCT techniques that also use area scan cameras, the conventional single-mode fiber-based point-scanning mechanism is retained to provide a confocal gate that rejects multiply scattered photons from the sample. When using a 1000 Hz resonant scanner as the streak scanner, 1,016,000 A-scans have been obtained in 1 s. This method's effectiveness has been demonstrated by recording in vivo OCT-image sequences of embryonic chick hearts at 1000 frames/s. In addition, 2-megahertz OCT data have been obtained with another high speed camera.

High-resolution optical coherence microscopy for high-speed, in vivo cellular imaging.

Optical coherence microscopy (OCM) is demonstrated with a high-speed, broadband, reflective-grating phase modulator and a femtosecond Ti:Al2O3 laser. The novel system design permits high-resolution OCM imaging in a new operating regime in which a short coherence gate is used to relax the requirement for high-numerical-aperture confocal axial sectioning. In vivo cellular imaging is demonstrated in the Xenopus laevis tadpole and in human skin with a 3-microm coherence gate and a 30-microm confocal gate. The ability to achieve cellular imaging with a lower numerical aperture should facilitate the development of miniaturized probes for in vivo imaging applications.