The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient’s blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.
Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.
Adaptive optics is a strategy to compensate for sample-induced aberrations in microscopy applications. Generally, it requires the presence of "guide stars" in the sample to serve as localized reference targets. We describe an implementation of conjugate adaptive optics that is amenable to widefield (i.e. non-scanning) microscopy, and can provide aberration corrections over potentially large fields of view without the use of guide stars. A unique feature of our implementation is that it is based on wavefront sensing with a single-shot partitioned-aperture sensor that provides large dynamic range compatible with extended samples. Combined information provided by this sensor and the imaging camera enable robust image de-blurring based on a rapid estimation of sample and aberrations obtained by closed-loop feedback. We present the theoretical principle of our technique and experimental demonstrations using both trans-illumination and fluorescence microscopes. Finally, we apply our technique to mouse brain imaging.
We demonstrate feasibility of a an spatially-modulated multi-photon microscopy (S-MPM) technique that can image through complex media that strongly scatters light, and we describe performance achieved with a prototype instrument. S-MPM’s imaging advantages are enabled by a high-speed, microelectromechanical spatial light modulator (MEMS SLM) subsystem with 1020 independently controllable mirror segments.
The design, fabrication, and characterization of an upconversion-luminescence enhancer based on a two-dimensional plasmonic crystal are described. Full-wave finite-difference time domain analysis was used for optimizing the geometrical parameters of the plasmonic crystal for maximum plasmon activity, as signified by minimum light reflection. The optimum design produced >20× enhancement in the average electromagnetic field intensity within a one-micron-thick dielectric film over the plasmonic crystal. The optimized plasmonic upconverter was fabricated and used to enhance the upconversion efficiency of sodium yttrium fluoride: 3% erbium, 17% ytterbium nanocrystals dispersed in a poly(methylmethcrylate) matrix. A thin film of the upconversion layer, 105 nm in thickness, was spin-coated on the surface of the plasmonic crystal, as well as on the surfaces of planar gold and bare glass, which were used as reference samples. Compared to the sample with a planar gold back reflector, the plasmonic crystal showed an enhancement of 3.3× for upconversion of 980-nm photons to 655-nm photons. The upconversion enhancement was 25.9× compared to the same coating on bare glass. An absorption model was developed to assess the viability of plasmonically enhanced upconversion for photovoltaic applications.
We demonstrate enhanced focusing of polychromatic light through strongly scattering media. The experimental results
validate a theoretical relationship among source bandwidth, sample bandwidth, and initial contrast of a far-field
speckle. For various combinations of source bandwidth and sample bandwidth, we optimize far-field focal intensity
enhancement using a MEMS spatial light modulator to modulate the source beam prior to its propagation through the
medium. We achieve focus optimization using a sequential coordinate descent algorithm and Hadamard basis
functions to control the spatial phase of the modulator. Enhancement, the ratio of optimized focal intensity to initial
speckle mean intensity, is shown to vary monotonically with initial contrast.
Enhancement of electromagnetic field by two dimensional arrays of rectangular and cylindrical nanopillars of both gold
and silver metals arranged in either square or triangular lattices was investigated. We simulated these gratings by 3D
Finite Difference Time Domain (3D-FDTD) method in visible and near infrared (NIR) wavelengths regime and
investigated field enhancement by exciting surface plasmon polaritons (SPPs) as a function of geometrical parameters of
grating. It was found that the geometrical grating parameters such as period, shape, thickness and size can be tuned for
excitation of SPPs at particular frequency of interest. The tuned grating would lead to an electric field intensity
enhancement by greater than 100× near the grating surface due to excitation of SPPs. Cylindrical gratings tuned for 750
nm at zero degree incident angle showed that the thickness of grating is the most sensitive geometrical parameter of
resonance. Furthermore, triangular lattice gratings have wider bandwidth of resonance than square lattice gratings.
Meanwhile, wavelength versus incident angle diagram showed that the enhancement was highly sensitive with angle of
incidence.
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