While multiphoton microscopy (MPM) has been performed with a wide range of excitation wavelengths, fluorescence emission has been limited to the visible spectrum. We introduce a paradigm for MPM of near-infrared (NIR) fluorescent molecular probes via nonlinear excitation at 1550 nm. This all-NIR system expands the range of available MPM fluorophores, virtually eliminates background autofluorescence, and allows for use of fiber-based, turnkey ultrafast lasers developed for telecommunications.
We describe multimodal nonlinear microscopy using a compact, turnkey femtosecond fiber laser at 1.5 μm. The system
allows for multiplexed detection of near infrared and visible contrast agents through two-and three-photon excitation
fluorescence microscopy as well as structural imaging viaauto-confocal microscopy (ACM). This platform expands the
available emission spectrum for multiphoton microscopy, enables simultaneous structural and functional imaging, and
offers advantages in penetration depth, contrast, and simplicity as compared to conventional MPM near 800 nm
excitation.
Spectral-Domain Optical Coherence Tomography (SDOCT) allows for in-vivo video-rate investigation of biomedical
tissue depth structure with the purpose of non-invasive optical diagnostics. In ophthalmic applications, it has been
suggested that Optical Coherence Tomography (OCT) can be used for diagnosis of glaucoma by measuring the thickness
of the Retinal Nerve Fiber Layer (RNLF). We present here an automated method for determining the RNFL thickness
map from a 3-D dataset. Boundary detection has been studied since the early days of computer vision and image
processing, and different approaches have been proposed. The procedure described here is based on edge detection using
a deformable spline (snake) algorithm. As the snake seeks to minimize its overall energy, its shape will converge on the
image contour, the boundaries of the nerve fiber layer. In general, the snake is not allowed to travel too much, and
therefore, proper initialization is required. The snake parameters, elasticity, rigidity, viscosity, and external force weight
are set to allow the snake to follow the boundary for a large number of retinal topographies. The RNFL thickness map is
combined with an integrated reflectance map of the retina and retinal cross-sectional images (OCT movie), to provide
the ophthalmologist with a familiar image for interpreting the OCT data. The video-rate capabilities of our SDOCT
system allow for mapping the true retinal topography since the motion artifacts are significantly reduced as compared to
slower time-domain systems.
Spectral-Domain Optical Coherence Tomography (SDOCT) allows for in-vivo video-rate investigation of biomedical tissue depth structure intended for non-invasive optical diagnostics. It has been suggested that OCT can be used for di-agnosis of glaucoma by measuring the thickness of the Retinal Nerve Fiber Layer (RNLF). We present an automated method for determining the RNFL thickness from a 3-D dataset based on edge detection using a deformable spline algo-rithm. The RNFL thickness map is combined with an integrated reflectance map and retinal cross-sectional images to provide the ophthalmologist with a familiar image for interpreting the OCT data. The video-rate capabilities of our SDOCT system allow for mapping the true retinal topography since motion artifacts are significantly reduced as com-pared to slower time-domain systems. Combined with Doppler Velocimetry, SDOCT also provides information on retinal blood flow dynamics. We analyzed the pulsatile nature of the bidirectional flow dynamics in an artery-vein pair for a healthy volunteer at different locations and for different blood vessel diameters. The Doppler phase shift is determined as the phase difference at the same point of adjacent depth profiles, and is integrated over the area delimited by two circles corresponding to the blood vessels location. Its temporal evolution clearly shows the blood flow pulsatile nature, the cardiac cycle, in both artery and vein. The artery is identified as having a stronger variation of the integrated phase shift. We observe that artery pulsation is always easily detectable, while vein pulsation seems to depend on the veins diameter.
Spectral-Domain Polarization-Sensitive Optical Coherence Tomography (SD-PS-OCT) is a technique developed to measure the thickness and birefringence of the nerve fiber layer in vivo as a tool for the early diagnosis of glaucoma. A clinical SD-PS-OCT system was developed and scans were made around the optic nerve head (ONH) using ten concentric circles of increasing diameter. One healthy volunteer was imaged. Retinal nerve fiber layer thickness and birefringence information was extracted from the data. Polarization-sensitive OCT images were acquired at video rate (29 frames per second (fps), 1000 A-lines / frame) and at 7 fps (1000 A-lines / frame). The last setting improved the signal to noise ratio by approximately 6 dB. Birefringence measurements on the healthy volunteer gave similar results as earlier reported values that were obtained with a time-domain setup. The measurement time was reduced from more than a minute to less than a second.
Microfluidic devices are becoming increasingly popular for many applications, enabling biological and chemical reactions to be performed with nano- and picoliter sample volumes. Accurate measurement and monitoring of fluid flow behavior in the small channels of microfluidic systems is important for evaluating the performance of existing devices, and in the modeling and design of new microfluidic networks. We present here the results of experiments using spectral-domain optical Doppler tomography (SD-ODT) to measure fluid flow in single-layer microfluidic devices. The principles behind flow imaging with SD-ODT are reviewed, a method for velocity calibration is described, and cross-sectional and en-face images of fluid velocity in microfluidic channels are presented.
Microfluidic devices are becoming increasingly popular for many applications, enabling biological and chemical reactions to be performed with nano- and picoliter sample volumes. Accurate measurement and monitoring of fluid flow behavior in the small channels of microfluidic systems is important for evaluating the performance of existing devices, and in the modeling and design of new microfluidic networks. We present here the results of experiments using spectral-domain optical Doppler tomography (SD-ODT) to measure fluid flow in single-layer microfluidic devices. The principles behind flow imaging with SD-ODT are reviewed, a method for velocity calibration is described, and cross-sectional and en-face images of fluid velocity in microfluidic channels are presented.
The Reflection Grating Spectrometer (RGS) on Constellation-X is designed to supply astronomers with high spectral resolution in the soft x-ray band from 0.25 to 2 keV. High resolution, large collecting area and low mass at grazing incidence require very flat and thin grating substrates, or thin-foil optics.
Thin foils typically have a diameter-to-thickness ratio of 200 or higher and as a result very low stiffness. This poses a number of technological challenges in the areas of shaping, handling, positioning, and mounting of such optics. The most minute forces (gravity sag, friction, thermal mismatch with optic mount, etc.) can lead to intolerable deformations and limit figure metrology repeatability. We present results of our efforts in the manipulation and metrology of suitable grating substrates, utilizing a novel low-stress foil holder with friction-reducing flexures.
A large number of reflection gratings is needed to achieve the required collecting area. We have employed nanoimprint lithography (NIL) - which uses imprint films as thin as 100 nm or less - for the high-fidelity and low-stress replication from 100 mm diameter saw-tooth grating masters.
The Reflection Grating Spectrometer (RGS) on Constellation-X will require thousands of large gratings with very exacting tolerances. Two types of grating geometries have been proposed. In-plane gratings have low ruling densities (~500 l/mm) and very tight flatness and assembly tolerances. Off-plane gratings require much higher ruling densities (~5000 l/mm), but have somewhat relaxed flatness and assembly tolerances and offer the potential of higher resolution and efficiency. The trade-offs between these designs are complex and are currently being studied. To help address critical issues of manufacturability we are developing a number of novel technologies for shaping, assembling, and patterning large-area reflection gratings that are amenable to low-cost manufacturing. In particular, we report results of improved methods for patterning the sawtooth grating lines that are required for efficient blazing, including the use of anisotropic etching of specially-cut silicon wafers to pattern atomically smooth grating facets. We also report on the results of using nanoimprint lithography as a potential means for replicating sawtooth grating masters. Our Nanoruler scanning beam interference lithography tool allows us to pattern large area gratings up to 300 mm in diameter. We also report on developments in grating assembly technology utilizing lithographically patterned and micromachined silicon metrology structures ("microcombs") that have achieved submicron assembly repeatability.
We are developing a Scanning Beam Interference Lithography (SBIL) system. SBIL represents a new paradigm in semiconductor metrology, capable of patterning large-area linear gratings and grids with nanometer overall phase accuracy. Realizing our accuracy goal is a major challenge because the interference fringes have to be locked to a moving substrate with nanometer spatial phase errors while the period of the fringes has to be stabilized to approximately one part per million. In this paper, we present a review of the SBIL design, and report recent progress towards prototyping the first-ever SBIL tool.
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