Browsing by Author "Miyazaki, Jun"

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Biological imaging with nonlinear photothermal microscopy using a compact supercontinuum fiber laser source
(2015-04-20)
He, Jinping
;
Miyazaki, Jun
;
Wang, Nan
;
Tsurui, Hiromichi
;
Kobayashi, Takayoshi
Nonlinear photothermal microscopy is applied in the imaging of biological tissues stained with chlorophyll and hematoxylin. Experimental results show that this type of organic molecules, which absorb light but transform dominant part of the absorbed energy into heat, may be ideal probes for photothermal imaging without photochemical toxicity. Picosecond pump and probe pulses, with central wavelengths of 488 and 632 nm, respectively, are spectrally filtered from a compact supercontinuum fiber laser source. Based on the light source, a compact and sensitive super-resolution imaging system is constructed. Further more, the imaging system is much less affected by thermal blurring than photothermal microscopes with continuous-wave light sources. The spatial resolution of nonlinear photothermal microscopy is similar to 188 nm. It is similar to 23% higher than commonly utilized linear photothermal microscopy experimentally and similar to 43% than conventional optical microscopy theoretically. The nonlinear photothermal imaging technology can be used in the evaluation of biological tissues with high-resolution and contrast. (C) 2015 Optical Society of America
Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy
(2016-05-01)
Miyazaki, Jun
;
Iida, Tadatsune
;
Tanaka, Shinji
;
Hayashi-Takagi, Akiko
;
Kasai, Haruo
A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 mu s. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. (C) 2016 Optical Society of America
Label-free imaging of biological tissues with nonlinear photothermal microscopy
(2015)
He, Jinping
;
Miyazaki, Jun
;
Wang, Nan
;
Kobayashi, Takayoshi
;
電子物理學系
Nonlinear photothermal microscopy has been applied in imaging of mouse melanoma and spinal cord without any staining. The resolution is higher compared with linear mechanism.
Label-free imaging of melanoma with nonlinear photothermal microscopy
(2015-04-01)
He, Jinping
;
Miyazaki, Jun
;
Wang, Nan
;
Tsurui, Hiromichi
;
Kobayashi, Takayoshi
Nonlinear photothermal microscopy, in which the intensity of the pump heating beam is modulated at f and the photothermal signal is extracted from the probe beam with a lock-in amplifier referred to 2f, is applied to the imaging of mouse melanoma without any staining. The pump and probe pulses, with central wavelengths of 488 and 632 nm, and a pulse duration of similar to 100 ps, are filtered from a compact commercial supercontinuum fiber laser source. An auto-balanced detector is applied to accumulate the signal and remove the laser noise of the probe. The spatial resolution of the nonlinear photothermal imaging is enhanced by similar to 18% in both theoretical calculations and experiments, compared with a linear photothermal mechanism, and the resolution enhancement is theoretically similar to 42% compared with conventional optical microscopy. This imaging technique shows possibilities for the clinical evaluation of melanoma with a high contrast and spatial resolution. (C) 2015 Optical Society of America
Optimal detection angle in sub-diffraction resolution photothermal microscopy: application for high sensitivity imaging of biological tissues
(2014-08-11)
Miyazaki, Jun
;
Tsurui, Hiromichi
;
Kawasumi, Koshi
;
Kobayashi, Takayoshi
;
電子物理學系
We evaluated the optimal detection angle for maximizing the signal to noise ratio (SNR) in sub-diffraction resolution photothermal microscopy. The angular dependent photothermal signal was calculated based on scattering theory using the temporally modulated Yukawa potential, and its detection angle and modulation frequency dependencies were analyzed. We verified the theoretical findings by imaging gold nanoparticles using laser diode based photothermal microscopy with balanced detection scheme. High-sensitivity (SNR similar to 40) photothermal biological imaging of a mouse brain was also demonstrated.
Optimal detection angle in sub-diffraction resolution photothermal microscopy: application for high sensitivity imaging of biological tissues
(2014-08-11)
Miyazaki, Jun
;
Tsurui, Hiromichi
;
Kawasumi, Koshi
;
Kobayashi, Takayoshi
;
電子物理學系
We evaluated the optimal detection angle for maximizing the signal to noise ratio (SNR) in sub-diffraction resolution photothermal microscopy. The angular dependent photothermal signal was calculated based on scattering theory using the temporally modulated Yukawa potential, and its detection angle and modulation frequency dependencies were analyzed. We verified the theoretical findings by imaging gold nanoparticles using laser diode based photothermal microscopy with balanced detection scheme. High-sensitivity (SNR similar to 40) photothermal biological imaging of a mouse brain was also demonstrated. (C) 2014 Optical Society of America
"Optimal detection angle in sub-diffraction resolution photothermal microscopy: application for high sensitivity imaging of biological tissues": reply
(2015-03-09)
Miyazaki, Jun
;
Tsurui, Hiromichi
;
Kawasumi, Koshi
;
Kobayashi, Takayoshi
;
電子物理學系
The differences between our model and existing models are rationalized in terms of the experimental conditions. The theory in [Opt. Express 22(16), 18833-18842 (2014)] is applicable when the temperature increase is moderate (similar to 1 K) and the spatial extend of refractive index being modulated is comparable to or smaller than the wavelength, which are in accordance with our experiment. (C) 2014 Optical Society of America
Photothermal imaging of skeletal muscle mitochondria
(2017-06-01)
Tomimatsu, Toru
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Miyazaki, Jun
;
Kano, Yutaka
;
Kobayashi, Takayoshi
;
電子物理學系
The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers. (C) 2017 Optical Society of America
Photothermal Microscopy for High Sensitivity and High Resolution Absorption Contrast Imaging of Biological Tissues
(2017-06-01)
Miyazaki, Jun
;
Kobayahsi, Takayoshi
;
電子物理學系
;
Department of Electrophysics
Photothermal microscopy is useful to visualize the distribution of non-fluorescence chromoproteins in biological specimens. Here, we developed a high sensitivity and high resolution photothermal microscopy with low-cost and compact laser diodes as light sources. A new detection scheme for improving signal to noise ratio more than 4-fold is presented. It is demonstrated that spatial resolution in photothermal microscopy is up to nearly twice as high as that in the conventional widefield microscopy. Furthermore, we demonstrated the ability for distinguishing or identifying biological molecules with simultaneous muti-wavelength imaging. Simultaneous photothermal and fluorescence imaging of mouse brain tissue was conducted to visualize both neurons expressing yellow fluorescent protein and endogenous non-fluorescent chromophores.
Reduction of distortion in photothermal microscopy and its application to the high-resolution three-dimensional imaging of nonfluorescent tissues
(2015-09-01)
Miyazaki, Jun
;
Tsurui, Hiromichi
;
Kobayashi, Takayoshi
;
電子物理學系
;
Department of Electrophysics
A scheme for reducing image distortion in photothermal microscopy is presented. In photothermal microscopy, the signal shape exhibits twin peaks corresponding to the focusing or defocusing of the probe beam when a sample is scanned in the axial direction. This causes a distortion when imaging a structured sample in the axial plane. Here, we demonstrate that image distortion caused by the twin peaks is effectively suppressed by providing a small offset between two the focal planes of the pump and the probe beams. Experimental results demonstrate improvement in resolution, especially in the axial direction, over conventional optical microscopy-even with the focal offset. When a dry objective lens with a numerical aperture of 0.95 is used, the full width at half the maximum of the axial point spread function is 0.6 mu m, which is 50% (62%) smaller than the focal spot sizes of the pump (probe) beam. Herein, we present high-resolution three-dimensional imaging of thick biological tissues based on the present scheme. (C) 2015 Optical Society of America
Resolution enhancement of pump-probe microscope with an inverse-annular filter
(2018-04-01)
Kobayashi, Takayoshi
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Kawasumi, Koshi
;
Miyazaki, Jun
;
Nakata, Kazuaki
;
電子物理學系
Optical pump-probe microscopy can provide images by detecting changes in probe light intensity induced by stimulated emission, photoinduced absorbance change, or photothermal-induced refractive index change in either transmission or reflection mode. Photothermal microscopy, which is one type of optical pump-probe microscopy, has intrinsically super resolution capability due to the bilinear dependence of signal intensity of pump and probe. We introduce new techniques for further resolution enhancement and fast imaging in photothermal microscope. First, we introduce a new pupil filter, an inverse-annular pupil filter in a pump-probe photothermal microscope, which provides resolution enhancement in three dimensions. The resolutions are proved to be improved in lateral and axial directions by imaging experiment using 20-nm gold nanoparticles. The improvement in X (perpendicular to the common pump and probe polarization direction), Y (parallel to the polarization direction), and Z (axial direction) are by 15 +/- 6, 8 +/- 8, and 21 +/- 2% from the resolution without a pupil filter. The resolution enhancement is even better than the calculation using vector field, which predicts the corresponding enhancement of 11, 8, and 6%. The discussion is made to explain the unexpected results. We also demonstrate the photothermal imaging of thick biological samples (cells from rabbit intestine and kidney) stained with hematoxylin and eosin dye with the inverse-annular filter. Second, a fast, high-sensitivity photothermal microscope is developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope using a Galvano mirror. We confirm a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrates simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 mu s. The fluorescence image visualizes neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures most probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. Third, we have made further resolution improvement of high-sensitivity laser scanning photothermal microscopy by applying non-linear detection. By this, the new method has super resolution with 61 and 42% enhancement from the diffraction limit values of the probe and pump wavelengths, respectively, by a second-order non-linear scheme and a high-frame rate in a laser scanning microscope. The maximum resolution is determined to be 160 nm in the second-order non-linear detection mode and 270 nm in the linear detection mode by the PT signal of GNPs. The pixel rate and frame rate for 300 x 300 pixel image are 50 mu s and 4.5 s, respectively. The pixel and frame rate are shorter than the rates, those are 1 ms and 100 s, using the piezo-driven stage system.
Resolution improvement in laser diode-based pump-probe microscopy with an annular pupil filter
(2014-07-15)
Miyazaki, Jun
;
Kawasumi, Koshi
;
Kobayashi, Takayoshi
;
電子物理學系
;
Department of Electrophysics
We experimentally demonstrate the use of annular beams to improve lateral resolution in laser-diode-based pump-probe microscopy. We found that the width of the point-spreading function in the case of the annular pump-probe beams is 162 nm, which is 30% smaller than that of the circular beams (232 nm). Furthermore, side lobes were efficiently suppressed at the focal plane since the pump-probe signal is proportional to the product of the two beam intensities. This scheme is demonstrated for the photothermal signal of nonfluorescent gold nanoparticles and the stimulated emission signal of fluorescence beads. (C) 2014 Optical Society of America
Sensitivity enhancement of photothermal microscopy with radially segmented balanced detection
(2015-02-15)
Miyazaki, Jun
;
Tsurui, Hiromichi
;
Kawasumi, Koshi
;
Kobayashi, Takayoshi
;
電子物理學系
A novel detection method is proposed for highly sensitive photothermal microscopic imaging. This method is based on the characteristics of an angular-dependent photothermal signal; it improves signal intensity by up to two times and rejects the intensity noise of the probe beam. The subdiffraction resolution photothermal imaging of mouse skin melanoma is demonstrated using a laser diode-based photothermal microscopy system to evaluate this method. We confirm that the signal intensity is enhanced 1.7 times compared with the conventional detection method. Moreover, the intensity noise of the laser diode used for the probe beam is effectively reduced by approximately 31 dB, even for a sample with non-uniformity of the refractive index and stationary absorption. This method is implemented by means of a commonly used balanced detector and is thus potentially useful for high-speed imaging. (C) 2015 Optical Society of America
Simultaneous dual-wavelength imaging of nonfluorescent tissues with 3D subdiffraction photothermal microscopy
(2015-02-09)
Miyazaki, Jun
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Tsurui, Hiromichi
;
Kawasumi, Koshi
;
Kobayashi, Takayoshi
;
電子物理學系
Multi-wavelength microscopic imaging is essential to visualize a variety of nanoscale cellular components with high specificity and high spatial resolution. However, previous techniques are based on fluorescence, and thus cannot visualize nonfluorescent species, which are much less suffered from photodamage or photobleaching and hence are intrinsically useful in wider range of optical microscopy. Here, we show that simultaneous multi-wavelength imaging of nonfluorescent species can be achieved with the use of a photothermal microscope. Dual-wavelength subdiffraction imaging of biological tissues stained with hematoxylin and eosin is demonstrated. Three-dimensional label-free imaging of mouse melanoma tissue section is also presented to demonstrate the effectiveness of the enhanced spatial resolution. Our technique can be implemented using cost-effective and compact laser diodes and is applicable for various types of both fluorescent and nonfluorescent tissues. (C) 2015 Optical Society of America
Sub-10 fs spectroscopy of K-TCNQ crystal for observation of intramolecular vibration modulation in melting of the Peierls dimer
(2014-08-14)
Nakata, Kazuaki
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Tokunaga, Eiji
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Du, Juan
;
Xue, Bing
;
Miyazaki, Jun
K-TCNQ crystals show photoinduced phase transition (PIPT) between a Mott insulator and a spin Peierls (SP) phase, having T-c = 395 K (= and the crystal has T-c = 395 K of the PIPT). We report the intra-molecular vibrations in the excited K-TCNQ crystal after impulsive excitation using a sub 9.4 fs pulse laser in the reflection spectrum. The frequencies of the vibrations are close to the Raman frequencies of molecules. The frequencies increase with melting of the SP phase, and then decrease with rearrangement of the dimerization. In addition, the frequencies of the vibrations are time-resolved to be modulated by the intermolecular optical phonon with 510 fs period corresponding to 65 cm(-1). This modulation is associated with the periodical change of coupling strengths of intermolecular charge transfer and/or the dipolar coupling between the counterpart molecules in the neighboring chains.
Sub-diffraction resolution pump-probe microscopy with shot-noise limited sensitivity using laser diodes
(2014-04-21)
Miyazaki, Jun
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Tsurui, Hiromichi
;
Hayashi-Takagi, Akiko
;
Kasai, Haruo
;
Kobayashi, Takayoshi
We demonstrate the use of intensity-modulated laser diodes to implement pump-probe microscopy and achieved sub-diffraction resolution imaging with shot-noise limited sensitivity with a scheme of balanced detection. This technique has several applications for various types of induced transmission change, including excited-state absorption, ground state absorption bleaching and stimulated emission. By using our technique, biological imaging of mouse T cells and the axons of neurons in the cerebral cortex was demonstrated. (C) 2014 Optical Society of America
Sub-diffraction-limit imaging using mode multiplexing
(2015-05-15)
Wang, Nan
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Miyazaki, Jun
;
He, Jinping
;
Seto, Keisuke
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Kobayashi, Takayoshi
Pixel-by-pixel processed fluorescence difference microscopy is experimentally demonstrated by multiplexing excitation laser beams with Gaussian and donut spot shapes and then demultiplexing the fluorescent signals using lock-in amplifiers. With this scheme, a fixed sample of fluorescent spheres and a slice of mouse brain tissue are imaged with resolutions that exceed the diffraction limit. Compared to previously reported subtraction imaging techniques, this pixel-by-pixel scan can be applied to improve the resolution of a moving sample without introducing subtraction errors. The synchronized signal detection feature makes this method extendible to various applications. (C) 2014 Elsevier B.V. All rights reserved.
Sub-diffraction-limit imaging using mode multiplexing
(2015-01-01)
Wang, Nan
;
He, Jinping
;
Miyazaki, Jun
;
Tsurui, Hiromichi
;
Kobayashi, Takayoshi
Simultaneous two-color subtraction microscopy using mode multiplexing is realized experimentally. The samples are irradiated with single laser diode at wavelength of 445 nm. Then the beam split laser spots generate separate solid and donut spatial modes and are multiplexed with modulators for simultaneous excitation. The produced fluorescence signals are back collected and further divided into two color bands with dichroic mirrors. Then they are detected with two photomultipliers and demultiplexed in four lock-in amplifiers. Four fluorescence images are recorded in every scan and resolution enhanced images are obtained in two color channels after applying the subtraction strategy. With this method, imaging results of microspheres stained with organic dyes and mesenteric lymph nodes of a mouse labeled with quantum dots (Q525/650) are realized. Improvement of 20% similar to 30% in resolving power of the two color channels compared with confocal microscopy is achieved in with corresponding subtraction factor of about 0.3.