Two-photon photoacoustic microscopy (TP-PAM) can visualize deep structures in living tissues with high spatial resolution determined by the volume of nonlinear absorption. Generally, the out-of-focus background fluorescence limits the imaging depth in nonlinear optical microscopies. In this study, to overcome this drawback that is also expected to exist in TP-PAM, we propose TP-PAM with spatial overlap modulation using femtosecond optical pulse train. Because the modulation depth of the spatial overlap in the focal region is much greater than those in out-of-focus regions, the out-of-focus background is effectively rejected by extracting the modulated photoacoustic signals.
Photoacoustic microscopy (PAM) is a biological visualization technique that can provide high spatial resolution and high contrast images of deep structures in living tissues. However, because of the spherical aberration of the objective lens and the wavefront distortion due to the surface shape and light scattering of the specimen, obtained photoacoustic images in deep tissues are sometimes blurred or distorted. In order to solve this problem, we have developed a PAM using a transmissive liquid-crystal adaptive optics (AO) element. The transmissive and thin structure of the AO element can be easily installed in the PAM system. Using photoacoustic images of a USAF 1951 resolution test target measured through the glass substrate (thickness; 1.5-mm), the lateral resolutions in PAM were estimated with and without the AO element, when a flashlamp-pumped nanosecond pulse laser (pulse width, 5-ns; wavelength, 500-nm) and water-immersion objective lens (NA = 0.8) were employed. The lateral resolution of PAM at the depth of 1.5-mm was improved from 1.04 ± 0.04 μm to 0.53 ± 0.10 μm by optimizing AO corrections. We have also visualized small blood vessels in mouse ear in vivo by PAM with AO correction. Thus, by optimizing the AO correction according to the imaging depth, our proposed PAM improves the spatial resolution in biological tissues.
Photoacoustic microscopy (PAM) is a biological visualization technique that can provide high spatial resolution and high contrast images of deep structures in living tissues. In PAM, the lateral resolution is determined by the size of the focus spot. Generally, because the wavefront aberration, due to the difference of refractive index between samples and air (water) and the shape of samples, enlarges the focus spot, obtained deep images are blurred or distorted. In order to solve this problem, we corrected the wavefront aberration occurring in samples using a transmissive liquid-crystal adaptive optics (AO) element. Our AO element consists of three liquid-crystal layers which have different ITO (indium tin oxide) patterns and are controlled independently. Their patterns are designed to correct the wavefront aberration suitable for a 40X waterimmersion objective lens. The AO element with transmissive and thin structure is easily installed in the PAM system. Also, our AO element is inexpensive and has low power consumption. In this study, we compared photoacoustic images obtained without and with the AO element for a USAF test target, polystyrene beads diffused in glycerol and various tissue specimens. As a result, we found that the use of transmissive AO element improves the lateral resolution and signal-tonoise ratio in PAM.
Compressional wave detection is useful means for health monitoring of building, detection of abnormal vibration of moving objects, defect evaluation, and biomedical imaging such as echography and photoacoustic imaging. The frequency of the compressional wave is varied from quasi-static to a few tens of megahertz depending on applications. Since the dynamic range of general compressional wave detectors is limited, we need to choose a proper compressional wave detector depending on applications. For the compressional wave detection with wide dynamic range, two or more detectors with different detection ranges is required. However, these detectors with different detection ranges generally has different accuracy and precision, disabling the seamless detection over these detection ranges. In this study, we proposed a compressional wave detector employing optical frequency comb (OFC). The compressional wave was sensed with a part of an OFC cavity, being encoded into OFC. The spectrally encoded OFC was converted to radio-frequency by the frequency link nature of OFC. The compressional wave-encoded radio-frequency can therefore be directly measured with a high-speed photodetector. To enhance the dynamic range of the compressional wave detection, we developed a cavityfeedback-based system and a phase-sensitive detection system, both of which the accuracy and precision are coherently linked to these of the OFC. We provided a proof-of-principle demonstration of the detection of compressional wave from quasi-static to ultrasound wave by using the OFC-based compressional wave sensor. Our proposed approach will serve as a unique and powerful tool for detecting compressional wave versatile applications in the future.
Conventional one-photon photoacoustic microscopy (PAM) utilizes high-frequency components of generated photoacoustic waves to improve the depth resolution. However, to obtain optically-high resolution in PAM in the depth direction, the use of high-frequency ultrasonic waves is to be avoided. It is because that the propagation distance is shortened as the frequency of ultrasonic waves becomes high. To overcome this drawback, we have proposed and developed two-photon photoacoustic microscopy (TP-PAM). Two-photon absorption occurs only at the focus point. TPPAM does not need to use the high-frequency components of photoacoustic waves. Thus, TP-PAM can improve the penetration depth while preserving the spatial resolution. However, the image acquisition time of TP-PAM is longer than that of conventional PAM, because TP-PAM needs to scan the laser spot both in the depth and transverse directions to obtain cross-sectional images. In this paper, we have introduced a focus-tunable electrically-controlled liquid lens in TP-PAM. Instead of a mechanical stepping-motor stage, we employed electrically-controlled liquid lens so that the depth of the focus spot can be quickly changed. In our system, the imaging speed of TP-PAM using the liquid lens and one-axis stepping-motor stage was 10 times faster than that using a two-axis stepping-motor stage only. TP-PAM with focus-scanning head consisting of the liquid lens and stepping-motor stage will be a promising method to investigate the inside of living tissues.
Photo-acoustic imaging is a promising modality for deep tissue imaging with high spatial resolution in the field of biology and medicine. High penetration depth and spatial resolution of the photo-acoustic imaging is achieved by means of the advantages of optical and ultrasound imaging, i.e. tightly focused beam confines ultrasound-generated region within micrometer scale and the ultrasound can propagate through tissues without significant energy loss. To enhance the detection sensitivity and penetration depth of the photo-acoustic imaging, highly sensitive ultrasound detector is greatly desired. In this study, we proposed a novel ultrasound detector employing optical frequency comb (OFC) cavity. Ultrasound generated by the excitation of tightly focused laser beam onto a sample was sensed with a part of an OFC cavity, being encoded into OFC. The spectrally encoded OFC was converted to radio-frequency by the frequency link nature of OFC. The ultrasound-encoded radio-frequency can therefore be directly measured with a high-speed photodetector. We constructed an OFC cavity for ultrasound sensing with a ring-cavity erbium-doped fiber laser. We provided a proof-of-principle demonstration of the detection of ultrasound that was generated by a transducer operating at 10 MHz. Our proposed approach will serve as a unique and powerful tool for detecting ultrasounds for photo-acoustic imaging in the future.
To improve the penetration depth in photoacoustic microscopy while preserving high spatial resolution, we have proposed two-photon absorption-induced photoacoustic microscopy (TP-PAM). However, in tissue imaging, unwanted one-photon photoacoustic signals impair the image constructed from the two-photon photoacoustic signals, because the cross-section of two-photon absorption is smaller than that of one-photon absorption. To overcome this drawback, it is important to enhance (or extract) only the photoacoustic signals generated by two-photon absorption. In this study, to improve the detection selectivity and efficiency of two-photon photoacoustic signals, we investigated the dependence of TP-PAM signal intensity and image quality on the detection frequency range and excitation pulse duration in detail. The comparison among photoacoustic signals generated by optical pulses with various pulse durations (femtosecond to sub-nanosecond) enabled us to find that, the shorter the pulse duration is, the higher the generation efficiency of two-photon photoacoustic signals is. We also applied the confocal configuration between optical (excitation) and acoustic (detection) foci to TP-PAM. The optimization of the pulse duration, frequency filtering and confocal configuration improves the selectivity and efficiency of the TP-PAM signal. Such improvements can reduce the photon number required to obtain TP-PAM images and thus make the imaging speed faster and avoid tissue damage.
Recently autofluorescence imaging (AFI) endoscopy, visualizing tissue fluorescence in combination with reflected light,
has been adopted as a technique for detecting neoplasms in the colon and other organs. However, autofluorescence
colonoscopy is not infallible, and improvement of the detection method can be expected to enhance the performance.
Colonic mucosa contains metabolism-related fluorophores, such as reduced nicotinamide adenine dinucleotide, which
may be useful for visualizing neoplasia in autofluorescence endoscopy. We examined sliced cross-sections of
endoscopically resected tubular adenomas under a microscope. Fluorescence images acquired at 365-nm excitation
(F365ex) and 405-nm excitation (F405ex), and reflectance images acquired at 550 nm (R550) were obtained. Fluorescence
ratio (F365ex/F405ex) images and reflectance/fluorescence ratio (R550/F405ex) images were calculated from the acquired
images. The fluorescence ratio images could distinguish adenomatous mucosa from normal mucosa more clearly than the
reflectance/fluorescence ratio images. The results showed that the autofluorescence ratio imaging is a potential technique
for increasing the diagnostic power of autofluorescence endoscopy.
Multiphoton excitation-induced photoacoustic microscopy (MEPAM) can be used to investigate the interior of dense
objects precisely and directly because the multiphoton excitation occurs only at the focal point. This method makes it
possible to avoid the strong signal from the surface of dense objects. However, in the case of tissue imaging, one-photon
photoacoustic signals affect the image constructed from MEPAM signals, owing to the smaller cross section of
multiphoton absorption compared with that of one-photon absorption. Thus, in order to apply MEPAM for precise
investigation in living tissues, it is important to enhance (or extract) only the photoacoustic signals induced by
multiphoton excitation.
In this study, we examined the use of frequency-selective detection (frequency filtering) in multiphotonphotoacoustic
imaging by evaluating the depth discrimination and penetration. Because MEPAM signals are generated in
a very small region, they include higher frequency components compared with one-photon photoacoustic signals. We
measured the images at the cross sections of blood-vessel phantoms visualized by MEPAM using the high-frequency
components. We found that the images visualized using only
high-frequency components showed better contrast
compared with those visualized using all frequency components. We conclude that the combination of frequency
filtering and MEPAM demonstrates great potential for precise observation of cross sections of blood vessels in living
tissues.
Estimating the distribution of myocardial fibrosis after myocardial infarct is important for appropriate therapeutic
planning. Here, we applied a Raman confocal microscope equipped with slit scanner for molecular tissue imaging of rat
infarcted hearts. Raman spectra of the cytoplasm of cardiomyocytes included the resonance Raman bands at 751, 1130
and 1582 cm-1 arising mainly from reduced b- and c- type cytochromes. Raman spectra of fibrotic tissues at the borderzone
of old myocardial infarct were highly consistent with that of collagen type I. Based on these findings, we
successfully obtained Raman tissue images of a cardiomyocyte and surrounding collagen at the cellular level.
Recently, we have developed multiphoton excitation-induced photoacoustic imaging for thick tissues employing a
1064-nm nanosecond pulsed laser. The combination of multiphoton excitation and photoacoustic imaging improves the depth
resolution. To apply the multiphoton-photoacoustic imaging for precise investigation in living tissues, it is important to
enhance only the photoacoustic signals induced by multiphoton excitation, because the generation of multiphotonphotoacoustic
signals is less efficient than that of one-photon photoacoustic signals. In this study, we investigated the
relation between the signal intensity and the thermophysical properties of various solutions of fluorescent dyes in
multiphoton-photoacoustic imaging. We found that the signal intensity is proportional to the coefficient of thermal
expansion divided by the specific heat of the solvent. Thus thermophysical properties are also important, together with
absorption properties, in enhancing the multiphoton-photoacoustic signal. Based on our findings, we propose the use of
gold nanoparticles surrounded by fluorescent dyes as contrast agents. Rhodamine B, which is employed in fluorescent
dyes, selectively evokes the two-photon absorption. In addition, because gold nanoparticles have a small specific heat,
the multiphoton-photoacoustic signal generated is strong due to effective photon-to-heat conversion. We conclude that
this combination allows deeper observation in living tissues by multiphoton-photoacoustic imaging.
In recent years, various types of molecular imaging technologies have been developed, but many of them require probes
and may have some influence on the distribution of the target molecules. In contrast, Raman microscopic analysis is
effective for molecular identification of materials, and molecular imaging methods employing Raman scattering light can
be applied to living organisms without use of any exogenous probes. Unfortunately, Raman microscopic imaging is
rarely used in the biomedical field due to the weakness of Raman signals. When the conventional Raman microscopes
are used, the acquisition of an image of a cell usually takes several hours. Recently, a slit-scanning confocal Raman
microscope has been developed. It can acquire images of living cells and tissues with faster scanning speed. In this study,
we used the slit-scanning confocal Raman microscope (RAMAN-11) to image the distribution of a drug in living cells.
We could acquire images of the distribution of an anticancer reagent in living cells within several minutes. Since the
wavelength of Raman scattering light is determined by the frequency of molecular vibration, the in situ mapping of the
intracellular drugs without use of a probe is possible, suggesting that laser Raman imaging is a useful method for a
variety of pharmacokinetic studies.
Autofluorescence endoscopy is a promising modality for diagnosis of colonic tumors. This article discusses the
origin of autofluorescence of the normal colon. Excised normal colons were analyzed by using fluorescence
stereomicroscopy and a fluorescence-lifetime microscopy system. Fluorescence images showed that the mucosa had
stronger autofluorescence than the submucosa. The results of fluorescence-lifetime measurement showed that
nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) might be responsible for the
autofluorescence of the colonic epithelia. Our results suggest that the mucosal autofluorescence generates by NADH and
FAD was an important source of the green autofluorescence.
Commercial imaging systems, such as computed tomography and magnetic resonance imaging, are frequently used
powerful tools for observing structures deep within the human body. However, they cannot precisely visualized several-tens
micrometer-sized structures for lack of spatial resolution. In this presentation, we propose photoacoustic imaging
using multiphoton absorption technique to generate ultrasonic waves as a means of improving depth resolution. Since the
multiphoton absorption occurs at only the focus point and the employed infrared pulses deeply penetrate living tissues, it
enables us to extract characteristic features of structures embedded in the living tissue. When nanosecond pulses from a
1064-nm Nd:YAG laser were focused on Rhodamine B/chloroform solution (absorption peak: 540 nm), the peak
intensity of the generated photoacoustic signal was proportional to the square of the input pulse energy. This result
shows that the photoacoustic signals can be induced by the two-photon absorption of infrared nanosecond pulse laser and
also can be detected by a commercial low-frequency MHz transducer. Furthermore, in order to evaluate the depth
resolution of multiphoton-photoacoustic imaging, we investigated the dependence of photoacoustic signal on depth
position using a 1-mm-thick phantom in a water bath. We found that the depth resolution of two-photon photoacoustic
imaging (1064 nm) is greater than that of one-photon photoacoustic imaging (532 nm). We conclude that evolving
multiphoton-photoacoustic imaging technology renders feasible the investigation of biomedical phenomena at the deep
layer in living tissue.
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