Lymph node (LN) metastasis is one of the most important prognostic factors in several common malignancies such as
gastric cancer and breast cancer. The frozen section method is widely used for intraoperative pathological diagnosis.
However, there are some issues with this process. In other words, experience is essential for specimen preparation and
diagnosis, and freezing causes severe tissue damage. Microscopy with ultraviolet surface excitation (MUSE) has
potential to provide rapid diagnosis with simple technique comparing to conventional histopathology based on
hematoxylin and eosin (H&E) staining. We established a fluorescent staining protocol for Deep UV-excitation
fluorescence imaging by using terbium ion and Hoechst 33342 that has enabled clear discrimination of nucleoplasm,
nucleolus, and cytoplasm. In formalin-fixed paraffin-embedded (FFPE) thin-sliced tissue sections of metastasis-positive/-
negative LNs of gastric cancer patients, the performance of cancer detection by patch-based training with a
deep convolutional neural network (DCNN) on the fluorescence images was comparable with that of H&E images.
However, MUSE images from non-thin-sliced tissue are difficult for pathologists to label training data for a supervised
learning manner. We attempt a deep-learning pipeline model for LN metastasis detection, in which CycleGAN translates
MUSE images to FFPE thin-sliced tissue images, and diagnostic prediction is performed using deep convolutional neural
network trained on FFPE images. The modality translation using CycleGAN was able to improve the pathological
diagnosis of non-thin-sliced surface images using DCNN model trained by FFPE images.
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.
Nerve-sparing surgery is essential to avoid functional deficits of the limbs and organs. Raman scattering, a label-free, minimally invasive, and accurate modality, is one of the best candidate technologies to detect nerves for nerve-sparing surgery. However, Raman scattering imaging is too time-consuming to be employed in surgery. Here we present a rapid and accurate nerve visualization method using a multipoint Raman imaging technique that has enabled simultaneous spectra measurement from different locations (n=32) of a sample. Five sec is sufficient for measuring n=32 spectra with good S/N from a given tissue. Principal component regression discriminant analysis discriminated spectra obtained from peripheral nerves (n=863 from n=161 myelinated nerves) and connective tissue (n=828 from n=121 tendons) with sensitivity and specificity of 88.3% and 94.8%, respectively. To compensate the spatial information of a multipoint-Raman-derived tissue discrimination image that is too sparse to visualize nerve arrangement, we used morphological information obtained from a bright-field image. When merged with the sparse tissue discrimination image, a morphological image of a sample shows what portion of Raman measurement points in arbitrary structure is determined as nerve. Setting a nerve detection criterion on the portion of “nerve” points in the structure as 40% or more, myelinated nerves (n=161) and tendons (n=121) were discriminated with sensitivity and specificity of 97.5%. The presented technique utilizing a sparse multipoint Raman image and a bright-field image has enabled rapid, safe, and accurate detection of peripheral nerves.
The peripheral nervous system plays an important role in motility, sensory, and autonomic functions of the human body. Preservation of peripheral nerves in surgery, namely nerve-sparing surgery, is now promising technique to avoid functional deficits of the limbs and organs following surgery as an aspect of the improvement of quality of life of patients. Detection of peripheral nerves including myelinated and unmyelinated nerves is required for the nerve-sparing surgery; however, conventional nerve identification scheme is sometimes difficult to identify peripheral nerves due to similarity of shape and color to non-nerve tissues or its limited application to only motor peripheral nerves. To overcome these issues, we proposed a label-free detection technique of peripheral nerves by means of Raman spectroscopy. We found several fingerprints of peripheral myelinated and unmyelinated nerves by employing a modified principal component analysis of typical spectra including myelinated nerve, unmyelinated nerve, and adjacent tissues. We finally realized the sensitivity of 94.2% and the selectivity of 92.0% for peripheral nerves including myelinated and unmyelinated nerves against adjacent tissues. Although further development of an intraoperative Raman spectroscopy system is required for clinical use, our proposed approach will serve as a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.
Accurate and rapid evaluation of lymph node metastasis is required in tumor staging and the decision of treatment strategy.
General intraoperative pathological evaluation, however, takes at least a few tens of minutes or longer for metastasis
diagnosis. 5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is a solution for accurate and ultrarapid diagnosis
of malignant lesions. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA,
protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue
chromophores, such as collagen and flavins. To enhance the accuracy of the diagnosis of malignant lesions based on the
PPIX fluorescence, elimination of the autofluroescence is required. In this study, we proposed and experimentally
demonstrated background-free PPIX fluorescence estimation method by simplified and optimized multispectral imaging.
To realize background-free PPIX fluorescence estimation, we computationally optimized observation wavelength regions
in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen
and flavins. We verified the fundamental detection capability of our method by using known-chemical mixtures.
Furthermore, we applied our method to lymph node metastasis, and successfully realized background-free
histopathological evaluation of metastatic lesions of lymph node metastasis. Our results confirmed the potential of the
background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions,
and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis.
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.
The peripheral nervous system plays an important role in motility, sensory, and autonomic functions of the human body. Preservation of peripheral nerves in surgery is essential for improving quality of life of patients. To preserve peripheral nerves, detection of ne peripheral nerves that cannot be identi ed by human eye or under white light imaging is necessary. In this study, we sought to provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues against adjacent tissues that employs spontaneous Raman microspectroscopy. A line-illumination confocal Raman microscope was used for the experiment. A laser operating at the wavelength of 532 nm was used as an excitation laser light. We obtained Raman spectra of peripheral nerve, brous connective tissue, skeletal muscle, blood vessel, and adipose tissue of Wistar rats, and extracted speci c spectral features of peripheral nerves and adjacent tissues. By applying multivariate image analysis, peripheral nerves were clearly detected against adjacent tissues without any preprocessing neither xation nor staining. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.
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.
Tumor invasion to the peritoneum is a poor prognostic factor in cancer patients. Accurate diagnosis of disseminated
peritoneal tumors is essential to accurate cancer staging. To date, peritoneal washing cytology during laparotomy has
been used for diagnosis of peritoneal dissemination of gastrointestinal cancer, but its sensitivity has not been satisfactory.
Thus, a more direct approach is indispensable to detect peritoneal dissemination in vivo. Fluorescein diacrylate (FDAcr)
is an esterase-sensitive fluorescent probe derived from fluorescein. In cancer cells, fluorescent fluorescein generated by
exogenous application of FDAcr selectively deposits owing to its stronger hydrolytic enzyme activity and its lower
leakage rate. We examined whether FDAcr can specifically detect disseminated peritoneal tumors in athymic nude
mouse models. Intraperitoneally administered FDAcr revealed disseminated peritoneal microscopic tumors not readily
recognized on white-light imaging. These results suggest that FDAcr is a useful probe for detecting disseminated
peritoneal tumors.
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.
Noninvasive and straightforward methods to inactivate selected proteins in the living cell with high spatiotemporal
resolution are eagerly sought for elucidation of protein function in the post-genome-mapping era. Chromophore-assisted
laser inactivation (CALI) facilitates inactivation of proteins by photochemically generated reactive oxygen species
(ROS), but CALI using single-photon excitation thus far has presented several drawbacks, including its complex
procedure, low efficiencies of inactivation with a certain chromophore, and photodamage effects. We here show that by
application of multiphoton excitation to CALI using near-infrared femtosecond laser, enhanced green fluorescent protein
(EGFP) can work as an effective chromophore for inactivation of a protein's function without nonspecific photodamage
in the living cell.
We develop a compact scanning head for use in laser confocal fluorescence microscopy for in situ fluorescence imaging of organs. The head, cylindrical in shape, has 3.5 mm diameter and 30 mm length, and is thus small enough to operate in a living rat heart. The lateral and axial resolutions, defined as full widths at half maximum (FWHM) of a point spread function (PSF), measures 1.0 and 5.0 µm, respectively, for 488-nm excitation and 1.0 and 5.4 µm, respectively, for 543-nm excitation. The chromatic aberration between 488- and 543-nm laser beams is well suppressed. We perform Ca2+ imaging in cardiomyocytes through the right ventricular chamber of a perfused rat heart in line-scan mode with 2.9-ms time resolution. We also carried out two-color imaging of a fixed mouse heart and liver with subcellular resolution. The compact head of the microscope equipped with a line-scan imaging mode and two-color imaging mode is useful for in situ imaging in living organs with subcellular resolution and can advantageously be applied to in vivo research.
We have demonstrated that intracellular Ca2+ waves in a living HeLa cell can be induced by femtosecond near-infrared laser pulses. In this paper, we present the results of investigation on the process of the Ca2+ wave generation using pharmacological methods to determine generation mechanisms. A mode-locked Ti:Sapphire laser (780 nm, 80 fs, 82 MHz) was used as a wave-triggering light source. The laser beam was focused into HeLa cells by using a water immersion objective lens (NA 0.9). Ca2+ waves were visualized by using a fluorescent Ca2+ indicator (Fluo-4) and monitored by a fluorescence microscope. Three mechanisms for the Ca2+ wave generation were considered; (1) Ca2+ flow into cells by destruction of the cell membrane, (2) mechanical stress by shock waves associated with the laser absorption, and (3) the leaking of Ca2+ through the destruction of intracellular Ca2+ stores. To investigate the mechanisms, we have performed experiments to determine the dependence of the probability of Ca2+ wave generation with two kinds of extracellular solutions; (a) a Ca2+ free extracellular solution (by use of EGTA), and (b) a solution containing U-73122 to inhibit the response to shockwave-based mechanical effects. From these experimental results, we can conclude the main mechanism of Ca2+ wave generation by laser irradiation is due to the leaking of Ca2+ through the destruction of intracellular Ca2+ stores.
A real-time confocal multiphoton fluorescence microscope was developed to observe Ca2+ dynamics in living rat- cardiac muscle cells. The real-time imaging was achieved by multifocus excitation of a specimen with a rotating microlens-array disk. A pinhole-array disk for confocal detection was introduced in the microscope to improve the spatial resolution and the contrast of fluorescence images. Ca2+ wave and Ca2+ transient in cultured rat- cardiac cells were successfully observed with the developed microscope.
The use of a confocal pinhole-array in a real-time two-photon fluorescence microscope with a microlens array is proposed to increase the three-dimensional resolution and the depth- penetration property. Enhancement of the lateral and axial resolution in the proposed microscope is observed in our experiments. Comparisons of images taken with and without the pinhole-array to verify the sharp depth-imaging performance are shown.
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