A CMOS image sensor using high-speed lock-in pixels for stimulated Raman scattering (SRS) spectroscopy is presented in this paper. The effective SRS signal from the stimulated emission of SRS mechanism is very small in contrast to the offset of a probing laser source, which is in the ratio of 10-4 to 10-5. In order to extract this signal, the common offset component is removed, and the small difference component is sampled using switched-capacitor integrator with a fully differential amplifier. The sampling is performed over many integration cycles to achieve appropriate amplification. The lock-in pixels utilizes high-speed lateral electric field charge modulator (LEFM) to demodulate the SRS signal which is modulated at high-frequency of 20MHz. A prototype chip is implemented using 0.11μm CMOS image sensor technology.
We describe rare-earth-doped nanophosphors (RE-NPs) for biological imaging using cathodoluminescence (CL) microscopy based on scanning transmission electron microscopy (STEM). We report the first demonstration of multicolor CL nanobioimaging using STEM with nanophosphors. The CL spectra of the synthesized nanophosphors (Y2O3:Eu, Y2O3:Tb) were sufficiently narrow to be distinguished. From CL images of RE-NPs on an elastic carbon-coated copper grid, the spatial resolution was beyond the diffraction limit of light. Y2O3:Tb and Y2O3:Eu RE-NPs showed a remarkable resistance against electron beam exposure even at high acceleration voltage (80 kV) and retained a CL intensity of more than 97% compared with the initial intensity for 1 min. In biological CL imaging with STEM, heavy-metal-stained cell sections containing the RE-NPs were prepared, and both the CL images of RE-NPs and cellular structures, such as mitochondria, were clearly observed from STEM images with high contrast. The cellular CL imaging using RE-NPs also had high spatial resolution even though heavy-metal-stained cells are normally regarded as highly scattering media. Moreover, since the RE-NPs exhibit photoluminescence (PL) excited by UV light, they are useful for multimodal correlative imaging using CL and PL.
A high speed Lateral Electric Field Modulator (LEFM) and lock-in pixels amplifiers for stimulated Raman
scattering (SRS)imager is presented. Since the generated signal from the SRS process is very small compared to
the offset signal, a technique suitable for extracting and amplifying the SRS signal is needed. The offset can be
canceled by tuning the phase delay between the demodulated pixel output signal and the sampling clock. The
small SRS signal in large offset is amplified by the differential integration. The proposed technique has been
investigated with an implementation of 64x8 pixels array using a pinned photodiode LEFM an lock-in pixels
amplifiers. Very small signal can be extracted from large offset signal. A ratio of the detected small SRS to
offset signal of less 10-5 is achieved.
Coherent Raman scattering provides chemical imaging by using molecular vibrational information sensitive to molecular structure. To add another information of martial symmetry, we propose using fourth order coherent Raman scattering for imaging, because the even order nonlinear phenomenon is forbidden for centro-symmetric material. We have developed a multiplex fourth order coherent Raman scattering microscopy system using a femtosecond laser. A narrowband beam of 17 cm-1 bandwidth and a broadband beam generated by a photonic crystal fiber enables to obtain a spectrum of fourth order coherent Raman scattering at once. We demonstrate the fourth order coherent Raman, hyper-Raman and second harmonics of trans-4'-(dimethylamino)-N-methyl-4- stilbazolium tosylate crystal by using the developed microscope.
We develop a coherent anti-Stokes Raman scattering (CARS) microscopy system equipped with a tunable picosecond laser for high-speed wavelength scanning. An acousto-optic tunable filter (AOTF) is integrated in the laser cavity to enable wavelength scanning by varying the radio frequency waves applied to the AOTF crystal. An end mirror attached on a piezoelectric actuator and a pair of parallel plates driven by galvanometer motors are also introduced into the cavity to compensate for changes in the cavity length during wavelength scanning to allow synchronization with another picosecond laser. We demonstrate fast spectral imaging of 3T3-L1 adipocytes every 5 cm −1 in the Raman spectral region around 2850 cm −1 with an image acquisition time of 120 ms. We also demonstrate fast switching of Raman shifts between 2100 and 2850 cm −1 , corresponding to CD 2 symmetric stretching and CH 2 symmetric stretching vibrations, respectively. The fast-switching CARS images reveal different locations of recrystallized deuterated and nondeuterated stearic acid.
We have developed a high speed spectral tuning CARS microscopy system using a mode-locked Ti:Sapphire laser
with an acousto-optic tunable filter (AOTF) in the cavity. Since the wavelength of the laser is tunable with the
applied radio frequency to the AOTF, the wavelength is electrically tunable.The pulse duration of the laser is
about 10 ps, tunable range is 800 nm to 930 nm, and the tuning speed is ms order. The laser is synchronized
with another mode-locked Ti:Sapphire laser laser our own method using a balance cross-correlator and phase
lock loop technique. The synchronized lasers are used for light source of multi-focus CARS microscopy system
using a microlens array scanner, and the hyperspectral imaging of adipocyte cells is demonstrated.
We propose a technique that employs single fluorescent molecules for visualizing the distribution of strain induced in
microstructures. We sprayed single-molecule tracers on microstructures by ultrasonic atomization and traced the position
and orientation of the tracers by a single-molecule detection technique with a three-dimensional (3D) orientation
microscope, which consists of a conventional fluorescent microscope and a polarization-mode converter. By using 3D
spline interpolation, we visualized the surface geometry of a microelectromechanical (MEMS) device. We tracked the
3D position and orientation of tracers attached to a supporting beam of the MEMS mirror. The surface declination angles
calculated from the orientation of the tracers were in agreement with the tilt angle obtained from the 3D position of the
tracers.
We developed a polarization-mode controllable coherent anti-Stokes Raman scattering microscope. The polarizationmode
of excitations beams such as linear, radial, or azimuth polarization were switched with compact polarization
mode converters made of eight-segmented liquid-crystal spatial-light-modulators. The polarization-mode of the
excitation beams is electrically controllable without any mechanical operation. We demonstrated the detection
of the molecular orientation of liquid crystals with the developed microscope.
We investigated photo-induced cell damage for multi-focus CARS (coherent anti-Stokes Raman scattering) microscopy. In general, using a near-infrared pulse light source, photo-induced damage is dominantly caused via multi-photon induced phenomena, and the peak power of the excitation light is limited for the non-invasive imaging. We obtained cell viability images during single- or multi-focus (7 foci) exposure of which wavelength and pulse duration were 709 nm and 5 ps. The laser power of one focal spot was respectively set to 27.8 mW and 14.5 mW for single- and multi-focus excitation because those excitation beams induce the comparable signals for third-order nonlinear phenomena. The cell viability was observed using DAPI fluorophore that mainly stains DNA of dead cells. As a result, we found that the single-focus excitation with 27.8 mW/spot caused cell damage within 6 min. In contrast, photo-induced damage was not detected until 20 min for the multi-focus excitation with 14.5 mW/spot and 7 foci. The results suggest that the photo-induced damage is a serious problem on the single-focus excitation, and the multi-focus excitation method is preferable for CARS imaging.
We demonstrate the real-time imaging of laser-induced disruption of the cellular membrane in a living HeLa cell and its cellular response with a multifocus coherent anti-Stokes Raman scattering (CARS) microscope. A near-infrared pulsed laser beam tightly focused on the cellular membrane of a living cell induces ablation at the focal point causing a local disruption of the cellular membrane. After the membrane disruption a dark spot decreasing CARS intensity of 2840 cm−1 Raman shift at the disrupted site appears. This dark spot immediately disappears and a strong CARS signal is observed around the disrupted site. This increase of the CARS signal might be caused by resealing of the disrupted site via aggregation of the patch lipid vesicles in the cytoplasm. The accumulation of lipids around the disrupted site is also confirmed with three-dimensional CARS images of a cell before and after membrane disruption. The temporal behavior of the CARS signal at the disrupted site is observed to detect the fusion dynamics of patch vesicles
KEYWORDS: Laser ablation, CARS tomography, Microscopes, Plasma, Real time imaging, Image acquisition, Visualization, Molecules, Pulsed laser operation, Digital signal processing
We demonstrated real-time imaging of organelles in a living HeLa cell using a multi-focus excitation CARS
(coherent anti-Stokes Raman scattering) microscope. Chemical selective CARS imaging of lipids and proteins
was demonstrated by observing CH2 and CH3 vibrations. Real-time imaging of lipid rich organelles such as the
plasma membrane, mitochondria, and lipid rich vesicles was achieved by observing CH2 stretching vibrations
of lipids. The image acquisition rate of 5 frames per second was achieved without any staining. We also
demonstrated real-time CARS imaging of laser-induced disruption and reaction of organelles in a living HeLa
cell. A near-infrared pulsed laser beam tightly focused on an organelle in a living cell produces ablation at the
focal point, causing local disruption of the organelle. We visualized the spatial and temporal distributions of a
lipid rich organelles in the cytoplasm of a living HeLa cell in
laser-induced dissection. We also demonstrated
real-time CARS imaging of disruption of a plasma membrane and its repair.
We developed a high speed CARS (coherent anti-Stokes Raman scattering) spectral-imaging system using an
acousto optic tunable filter and multi-focus excitation system. We compared two methods of CARS emission
filtering and CARS excitation filtering. In both case, two laser pulses with narrow band (picosecond laser) and
broad band (femotosecond laser)were used for the light source. For CARS emission filtering, the generated CARS
was filtered by an AOTF, and for excitation filtering the broad band femtosecond laser pulse were filtered by an
AOTF before excitation. The experimental results indicated that the CARS emission filtering was suitable for
CARS microscopy.
We developed a multifocus excitation coherent anti-Stokes Raman scattering microscope using a microlens array
scanner for realtime molecular imaging. Two picoseond mode-locked lasers tightly synchronized were splited to
a few tens of foci with the microlens array, the foci excited the sample parallely and the generated CARS from
each spot was detected with an image sensor at once. By the multifocus excitation, exposure time was prolonged
proportionally to the number of the foci because of parallel excitation and detection. The video-rate (frame
rate of 30 fps) imaging of polystyrene beads in water was demonstrated, and the Brownian motion of beads
were clearly obtained. The three-dimensional reconstructed imaging of living HeLa cells (frame rate of 5 fps, 85
images) was also demonstrated.
KEYWORDS: Microscopy, Sensors, Signal detection, Raman spectroscopy, Signal to noise ratio, Microlens array, CARS tomography, Imaging spectroscopy, Laser systems engineering, Digital signal processing
We realized realtime molecular imaging with low excitation laser intensity using a multi-focus excitation CARS (coherent anti-Stokes Raman scattering) microscope. We demonstrated realtime CARS images of polystyrene beads and lipid vesicles. Time series CARS images of the polystyrene beads in water was obtained with the frame rate of 30 fps. The three dimensional lipids vesicle which consists of 50 slices was observed within 7 s (100 ms/image).
We demonstrated high-speed imaging of the distribution of DPPC (dipalmitoylphosphatidylcholine), d62-DPPC (deuterated DPPC), and DOPC (dioleoylphosphatidylcholine) lipids in a lipid vesicle with a multi-focus excitation CARS (coherent anti-Stokes Raman scattering) microscope using a microlens array scanner. By the multi-focus excitation, the dwell time is increased in proportion to the number of focal spots compared with a single beam scanning, and high-speed and high-quality CARS imaging is possible without increasing the peak power of each spot. We demonstrated the selectively visualization of DPPC and d62-DPPC lipid vesicles, in which the vesicles contain a type of lipid, by observing at 2840 cm-1 and 2090 cm-1. We also visualized the DOPC and DPPC lipids distribution in a lipid mixture vesicle observed at 1440 cm-1 and 1655 cm-1. The image acquisition time of 10 s/image at each Raman shift was realized. The signal ratio of 1440 cm-1 and 1655 cm-1 was locally intense on the lipid vesicle. It must be because the gel phase domain of DPPC lipids was exists in the DOPC lipids which were liquid-crystalline phase at room temperature.
We developed a compact polarization-converter using two liquid-crystal spatial-light-modulators with eight electrodes. The converter converted a linearly polarized beam to two orthogonal linearly polarized beams and a radially polarized beam, and the direction of the electric filed at the focal point were controlled three-dimensionally. We constructed a second-harmonic-generation microscope using the polarization-converter to observe three-dimensional molecular orientation and demonstrated the detectability of molecular orientation.
We have developed a second-harmonic-generation (SGH) microscope to observe the three-dimensional molecular orientation with three-dimensional high spatial resolution using a polarization mode converter. The mode converter consists of a parallel-aligned nematic-liquid-crystal spatial-light-modulator (PAL-SLM) and quarter-waveplates, and converts a incident linearly polarized beam to orthogonal linearly polarized beams or radially polarized beam. We combined the mode converter with SHG microscope to obtain the local information of the three-dimensional molecular orientation. We demonstrated the detection of three-dimensional molecular orientation of collagen fiber in human Achilles' tendon. For high precision three-dimensional molecular orientation measurement, we propose a technique to calibrate the dependence of SHG detection efficiencies on molecular orientation using a liposome.
Optical microscopy that can visualize the molecular vibration with a nanometric spatial resolution has been realized by a combination of near-field optics and coherent anti-Stokes Raman scattering (CARS) spectroscopy. A metallic probe with a sharp tip is used to strongly enhance optical near-field in the local vicinity of the tip owing to the excitation of local surface plasmon polariton. CARS signals of molecules in the local area can be strongly induced by the plasmonic field. We have visualized DNA molecules and single-walled carbon nanotubes (SWNTs) with a spatial resolution far beyond the diffraction limit by the tip-enhanced near-field CARS microscopy.
A metallic nano-probe has locally induced coherent anti-Stokes Raman scattering (CARS) of adenine molecules in a nanometric DNA network structure. The excitation fields and CARS polarization are enhanced by the tip apex of the nano-probe through the excitation of local surface plasmons. Owing to the third-order nonlinearity, the excitation of the CARS polarization is extremely confined to the end of the tip apex, resulting in the spatial resolution far beyond the diffraction limit of light. Our CARS microscope using a silver-coated probe visualized the DNA network structure at a specific vibrational frequency (~1337 cm-1) of adenine molecules with a spatial resolution of ~15 nm and sufficient sensitivity.
A light microscope capable to show images of molecules in nanometer scale has been a dream of scientists, which, however, is difficult due to the strict limitation of spatial resolution due to the wave nature of light. While there have been attempts to overcome the diffraction limit by using nonlinear response of materials, near-field optical microscopy could provide better detecting accuracy. In this paper, we present molecular distribution nano-imaging colored by Raman-scattering spectral shifting, which is probed with a metallic tip. The metallic probe tip has been used to enhance the optical field only in the vicinity of probe tip. The effect is similar to the one seen in the detection of molecules on the metal-island film, known as surface-enhanced Raman spectroscopy (SERS), while in this case a single metallic tip works for the field enhancement in nanometer scale.
II-V-based magnetic semiconductors are gathering great interest because of their potential utility as new functional materials that are expected to lead to the introduction of spin degree-of-freedom for semiconductor devices. New magnetic semiconductor heterostructure, InMnAsSb/InSb, was proposed and grown by low temperature molecular beam epitaxy (MBE). Mid-infrared (2-6 μm) light-induced ferromagnetism was observed at temperatures lower than 40-50K and this light-induced magnetization remained even after the stop of light irradiation. Observed characteristics are applicable to the mid-infrared sensor/memory devices. New magnetic semiconductor GaCrN layers were grown by electron-cyclotron-resonance (ECR) plasma-assisted MBE and the ferromagnetic characteristics were observed at 7-400K. Clear hysteresis and clear saturation were observed in the magnetization versus magnetic field (M-H) curves at all measuring temperatures. We also observed the photoluminescence (PL) emission from GaCrN layers. Applications to the novel devices controlling charges (electrons and holes), spins and photons are expected.
We propose a new laser scanning microscope using coherent anti-Stokes Raman spectroscopy. As the proposed method is a kind of Raman spectroscopy, molecular structural information are obtained without any staining. The imaging property is theoretically estimated by using the 3D optical transfer function. It is shown that the proposed microscope has 3D resolution in any case of measuring for the weak or the high contrast object with or without a pinhole before a detector. Spatial resolution of micrometer order along the optical axis is demonstrated.
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