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.
In surgery, peripheral nerves should be preserved as much as possible to suppress the dysfunction and improve the quality of life after surgery. However, it is difficult to distinguish colorless, transparent, and thin nerves from other tissues. We had developed a coherent anti-Stokes Raman scattering (CARS) rigid endoscope to visualize nerves in a label-free manner. CARS allows for imaging without staining based on the information of molecular vibrations. In the conference, we show near real-time nerve visualization using CARS endoscopy and deep learning. We demonstrate that the image taken at 1.6 s/image satisfies the segmentation quality required for medical images.
Rare-earth-doped nanoparticles are one of the emerging probes for bioimaging due to their visible-to-near-infrared (NIR) upconversion emission via sequential single-photon absorption at NIR wavelengths. The NIR-excited upconversion property and high photostability make this probe appealing for deep tissue imaging. So far, upconversion nanoparticles include ytterbium ions (Yb3 + ) codoped with other rare earth ions, such as erbium (Er3 + ) and thulium (Tm3 + ). In these types of upconversion nanoparticles, through energy transfer from Yb3 + excited with continuous wave light at a wavelength of 980 nm, upconversion emission of the other rare earth dopants is induced. We have found that the use of the excitation of Er3 + in the 1550-nm wavelength region allows us to perform deep tissue imaging with reduced degradation of spatial resolution. In this excitation–emission process, three and four photons of 1550-nm light are sequentially absorbed, and Er3 + emits photons in the 550- and 660-nm wavelength regions. We demonstrate that, compared with the case using 980-nm wavelength excitation, the use of 1550-nm light enables us to moderate degradation of spatial resolution in deep tissue imaging due to the lower light scattering coefficient compared with 980-nm light. We also demonstrate that live cell imaging is feasible with this 1550 nm excitation.
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.
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 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
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