Methotrexate impaired in-vivo matured mouse oocyte quality and the possible mechanisms.

Background: Methotrexate (MTX) is an antifolate agent which is widely used in clinic for treating malignancies, rheumatoid arthritis and ectopic pregnancy. As reported, MTX has side effects on gastrointestinal system, nervous system and reproductive system, while its potential damages on oocyte quality are still unclear. It is known that oocyte quality is essential for healthy conception and the forthcoming embryo development.

[Intensity loss of two-photon excitation fluorescence microscopy images of mouse oocyte chromosomes].

As an optical microscope with high resolution, two-photon excitation (TPE) fluorescence microscope is widely used in noninvasive 3D optical imaging of biological samples. Compared with confocal laser scanning microscope, TPE fluorescence microscope provides a deeper detecting depth. In spite of that, the image quality of sample always declines as the detecting depth increases when a noninvasive 3D optical imaging of thicker samples is performed.

[A method for real-time observation of intracellular free Ca2+ concentration in single bovine retinal neurons].

In order to observe the neurotrophin's influence on intracellular free calcium concentration, the test neuron was labeled by fluorescence indicator Fluo-3, and imaged by self-built real-time fluorescence microscopy system. The authors observed the changes in intracellular free calcium concentration ([Ca2+]i) in the bovine retinal neurons under the influence of four kinds of neurotrophins such as brain derived neurotrophic factor BDNF. On account of the fact that fluorescence indicator's intensity decays over time, it is necessary to apply a "decay removal correction" to the fluorescence intensity in order to show the fluorescence intensity that solely represents [Ca2+]i.

[Visualization of the age-related changes in expressions of DNA methyltransferases in mouse oocytes using two-photon imaging system].

Quantum dot (QD) is widely used as fluorescent labeling dye for its strong anti-fluorescence quenching, high quantum yield, wide absorption spectra, and narrow emission spectra. In the present paper, QD 585-labeled DNA methyltransferases (Dnmts) and Hoechst 33342-labeled chromosomes were imaged simultaneously using two-photon imaging system. The authors' results showed that aging mouse oocytes may be not suitable for in-vitro maturation: both the localizations and expression levels of Dnmts in in-vitro matured oocytes of aging mice were changed, and these changes may be related to the abnormal DNA methylation modification.

[Observation of double-stained African green monkey kidney COS-7 cells using total internal reflection double-channel fluorescence microscopy].

Using a Dual-View wavelength splitter, double-stained African green monkey kidney COS-7 cells, transfected with pEGFP-Myosin 15a and costained with Rhodamine-filopodia were observed based on an ICCD(intensified charge couple device) fluorescence micro-imaging systems. Total internal reflection fluorescence microscopy was used to observe the overexpression of Myosin 15a to the tips of the elongation filopodia. An approach to collecting fluorescence in two channels and avoiding spectra crosstalk was employed to observe Myosin 15a and filopodia distribution in African green monkey kidney COS-7 cells.

[Observation on cytoskeleton and root tip growth of Arabidopsis thaliana by two-photon laser scanning microscopy].

Cytoskeleton plays an important role in plant cells and their movements, developments, and so on. With MS culture medium, the present paper cultured GFP-fused Arabidopsis thaliana in asepsis condition, which completed its whole lifecycle in the lab, starting from germination, going through plant growth, blossom, and ending at fructification. During this process, the authors used two-photon laser scanning microscopy (TPLSM) which was suitable for 4D observation on large, thick, live samples to observe cytoskeleton in seed, root tip, vessel and root hair of the Arabidopsis thaliana.

Age-related changes in the localization of DNA methyltransferases during meiotic maturation in mouse oocytes.

The effects of maternal aging on the localization of DNA methyltransferases were evaluated during mouse oocyte maturation using fluorescence staining. And we conclude that maternal aging affects the cytoplasmic-to-nuclear trafficking of DNA methyltransferases in mouse oocytes during the time from germinal vesicle breakdown to metaphase I.

[Study on real-time imaging of single stretched DNA molecules by total internal reflection fluorescence microscopy].

Total internal reflection fluorescence microscopy (TIRF) is a powerful tool for single molecule study, since only a thin layer of about 200 nanometers is excited by the evanescent wave, resulting in high sensitivity of detection and high signal-to-noise ratio of images. Molecular combing is a convenient and efficient way to stretch DNA molecules with the help of the binding force between DNA molecule and solid surface, as well as the lateral force introduced by ambient fluid flow. In the present paper, real-time fluorescence imaging of single DNA molecules was carried out with these two techniques.

[DNA nanosensor fluorescence imaging microscopy].

Quantum dots have many excellent optical properties such as high quantum yield, long fluorescence lifetime, wide excitation spectrum and narrow emission spectrum, tunable emission wavelength and so on, thus have become a newpopular type of fluorescence probes in these years. Quantum-dot-based DNA nanosensor comprising streptavidin-conjugated quantum dots, capture probes with biotin and reporter probes with Cy5 was designed to detect DNA or RNA segments. Capture probes and reporter probes were connected by the target DNA or RNA segments so that quantum dots and Cy5s could be together and FRET (fluorescence resonance energy transfer) could be detected.

A quantification model for apoptosis in mouse embryos in the early stage of fetation.

Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development.

[Study of the relationship between apoptosis and intracellular pH in single living cells using a three-channel real-time fluorescence imaging method].

Using a Dual-View wavelength splitter, a custom-made filter block and only a single intensified charge-coupled device (ICCD), a three-channel real-time fluorescence imaging method for single living cell research was established based on an ICCD fast fluorescence micro-imaging system. The relevant calibrating method for images was also developed to eliminate the influence introduced by spectral crosstalk. Double-labeled with two fluorescent indicators, Annexin V-FITC and SNARF-1, mouse thymocyte was treated with S-nitrosoglutathione (GSNO) and the relationship between their apoptosis and intracellular pH changes was studied in real-time at the single living cell level.

[ICCD-based fast fluorescence micro-imaging technique and preliminary application in living cells].

A fast fluorescence micro-imaging system using mainly intensified charge couple device (ICCD), argon-ion laser, and xenon lamp was set up, and its preliminary application in living cells was presented. Real-time observation and imaging of fast concentration and distribution changing of intracellular Ca2+ labeled by Fluo-3, a fluorescent Ca2+ indicator, in the proliferation process of rat cerebral micro-vessels endothelial cells (rCMECs) were carried out, and curves of artificial intensity versus imaging sequence of four typical points were obtained. It is shown that the ICCD-based fast fluorescence micro-imaging system is a powerful tool for recording the real-time fast processes in living cells.

[Application of two-photon excitation fluorescence imaging to real-time investigation of mouse preimplantation embryo].

The blue shift of a two-photon excitation absorption peak allows the application of a single wavelength to the simultaneous excitation of several fluorochromes with disparate emission characteristics. An output at 730 nm of a mode-locked femtosecond Ti-sapphire laser was used to excite four different commonly used fluorochromes, namely Hoechst 33342, Fluo-4, PI and Indo-1, and characteristic fluorescence images were obtained using (455 +/- 15) nm, (540 +/- 15)nm, (580 +/- 16) nm and (500 +/- 15) nm filters respectively. An approach to exciting with a single wavelength, staining with two different fluorochromes, and collecting fluorescence in two separate channels was employed to study mouse preimplantation embryos by 3D and 4D real-time imaging.

Real-time imaging of viable-apoptotic switch in GSNO-induced mouse thymocyte apoptosis.

Many scientists are focusing on the apoptotic-necrotic or the necrotic-apoptotic switch and its mechanism, but little attention has been paid to the viable-apoptotic switch. Most of the techniques and methods used for detecting apoptosis are performed on fixed samples, yielding static information of specific time points. We have studied the viable-apoptotic switch in S-nitrosoglutathione (GSNO)-induced mouse thymocyte apoptosis in real-time by means of a novel technique, intensified charge coupled device (ICCD)-based real-time fluorescence micro-imaging, coupled with Annexin V-FITC labeling for phosphatidylserine (PS) translocation in cell membrane.

[Study on multi-photon excited fluorescence combined with capillary electrophoresis].

This paper describes a new method, multiphoton excitation fluorescence detection combined with capillary electrophoresis separation. The excitation source was a self mode-locked femtosecond titanium-sapphire laser (Spectra-physics Inc.), producing a stream of pulses with a pulse duration of about 100 fs at 82 MHz repetition rate.

[Measurement of methane 13C/12C isotope ratio of slurry gas from oil wells by laser frequency-modulation absorption spectroscopy].

In this paper, we determined the ratio of C13/C12 of methane in slurry gas from oil wells by the method of laser frequency modulation absorption spectroscopy that has many advantages such as high precision, high spectroscopic resolution and rapidly and simply dealing with samples. This technique provided us with a subsidiary method for oil-gas source exploration. The method of laser frequency modulation absorption spectroscopy is based on the method of laser frequency absorption spectroscopy and is more accurate.