The microscope technology with wider view field, deeper penetration depth, higher spatial resolution and higher imaging speed are required to investigate the intercellular dynamics or interactions of molecules and organs in cells or a tissue in more detail. The two-photon microscope with a near infrared (NIR) femtosecond laser is one of the technique to improve the penetration depth and spatial resolution. However, the video-rate or high-speed imaging with wide view field is difficult to perform with the conventional two-photon microscope. Because point-to-point scanning method is used in conventional one, so it’s difficult to achieve video-rate imaging. In this study, we developed a two-photon microscope with spinning disk beam scanner and femtosecond NIR fiber laser with around 10 W average power for the microscope system to achieve above requirements. The laser is consisted of an oscillator based on mode-locked Yb fiber laser, a two-stage pre-amplifier, a main amplifier based on a Yb-doped photonic crystal fiber (PCF), and a pulse compressor with a pair of gratings. The laser generates a beam with maximally 10 W average power, 300 fs pulse width and 72 MHz repetition rate. And the beam incident to a spinning beam scanner (Yokogawa Electric) optimized for two-photon imaging. By using this system, we achieved to obtain the 3D images with over 1mm-penetration depth and video-rate image with 350 x 350 um view field from the root of Arabidopsis thaliana.
The esophageal cancer has a tendency to transfer to another part of the body and the surgical operation itself sometimes gives high risk in vital function because many delicate organs exist near the esophagus. So the esophageal cancer is a disease with a high mortality. So, in order to lead a higher survival rate five years after the cancer’s treatment, the investigation of the diagnosis methods or techniques of the cancer in an early stage and support the therapy are required. In this study, we performed the ex vivo experiments to obtain the Raman spectra from normal and early-stage tumor (stage-0) human esophageal sample by using Raman spectroscopy. The Raman spectra are collected by the homemade Raman spectrometer with the wavelength of 785 nm and Raman probe with 600-um-diameter. The principal component analysis (PCA) is performed after collection of spectra to recognize which materials changed in normal part and cancerous pert. After that, the linear discriminant analysis (LDA) is performed to predict the tissue type. The result of PCA indicates that the tumor tissue is associated with a decrease in tryptophan concentration. Furthermore, we can predict the tissue type with 80% accuracy by LDA which model is made by tryptophan bands.
Our Raman probe that is called as ball-lens hollow fiber Raman probe (BHRP) had been proved
possessing capability to detect the biochemical alteration within biological tissue. Whether BHRP
has high capability and sensitivity in diagnosing the biochemical changing of tissue or not, mouse's
normal rectal and anorectal prolapse (AP) were decided to be used as a model for this non invasive
method. This AP is azoxymethane and DSS-induced mouse’s anorectal prolapse. Main outcome of
BHRP will be potential for non-invasive method in tumor diagnosing. BHRP spectra obtained were
a high quality and allowed analysis of their differences between normal rectal (control group) and
AP. After spectral acquisition and comparison with corresponding images of hematoxylin/eosinstained
section observation used to make the histopathologic diagnosing, BHRP detected some
differences within the region of moiety of DNA, protein (i.e. collagen) and lipid, then following with
the alteration of symmetric P=O stretching vibration compared with the normal rectal tissue. BHRP
discriminate normal tissue and AP in the real-time.
The esophageal cancer is a disease with a high mortality. In order to lead a higher survival rate five years after the
cancer’s treatment, we inevitably need a method to diagnose the cancer in an early stage and support the therapy. Raman spectroscopy is one of the most powerful techniques for the purpose. In the present study, we apply Raman spectroscopy to obtain ex vivo spectra of normal and early tumor human esophageal sample. The result of principal component analysis indicates that the tumor tissue is associated with a decrease in tryptophan concentration. Furthermore, we can predict the tissue type with 80% accuracy by linear discriminant analysis which model is made by tryptophan bands.
The CARS spectroscopy system using the dual-wavelength oscillation electronically wavelength tuned laser as a
pumping light source were constructed to realizes more sensitive full range non-chromosomal spectroscopy. Simpler
configuration of CARS optical system was realized by using the laser. To realize the CARS system, the methods to
synchronize the pulses with two different wavelengths generated from the dual-wavelength oscillation electronically
wavelength tuned laser was demonstrated by using sum frequency generation.
Ball-lens hollow fiber Raman Probe (BHRP) and FTIR spectroscopy were main tools in this study. Thus, both of
equipments detected the alteration of antisymmetric and symmetric P=O stretching vibration within our mice colorectal
tumor models. Some differences of spectra due to randomly the edge of each BHRP and FTIR attached the surface of
tumor during measurements. Meanwhile, the application of FTIR potentially differentiates the grade levels of non-clinic
samples colorectal tumor models at four different grades (normal, grade 1, grade 2 and grade 3). Detailed investigations
were assignable to wave numbers that publicized to represent biochemical alteration. The whole of investigated spectra
in the fingerprint region revealed some different peaks and shoulders, most of which were assignable to wave numbers
that exposed to represent biochemical alteration within the tissue. Differences in peak heights and peak ratio indicated
differences in biochemical composition of cancer from different grade level. However, all collected colorectal tumor
model at different peak was distinguishable, where antisymmetric and symmetric P=O stretching vibration was imaged
and mapped clearly by both equipments. Therefore, BHRP were comfortable for in vivo studies. Meanwhile FTIR
spectral analysis in combination with calibration curve might be used to distinguish cancer grade within colorectal tumor
model tissue for ex vivo study.