[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. Dynamic root tip growth as well as the growing speed was also observed. Applying IAA to the Arabidopsis thaliana, the authors found that the growing speed of root tip was enhanced by about 3.8 times.

[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. Clear images of single stretched DNA were obtained, while photocleavage of DNA-YOYO-1 complex was found to be naturally avoided under TIRF imaging conditions. Photobleaching of the complexes was investigated in real-time, and was greatly reduced by synchronizing the excitation of light (laser) and the exposure of detector (ICCD). The method optimized the experimental conditions for long-lasting real-time observation and imaging of single stretched DNA molecules, so as to lay a foundation for visually studying the kinetic processes of interactions between DNA and proteins.

[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. The results not only showed the different regularities of intracellular pH changes between apoptosis induced by GSNO and spontaneous apoptosis, but also provided evidence for the application of the three-channel real-time fluorescence imaging method to bio-medical studies.

[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. Combined with the merits of two-photon excitation fluorescence imaging, such as better penetration, less photon-damage, and higher signal-to-noise ratio, this approach was supposed to be a novel multi-parameter investigating tool for mouse preimplantation embryos development study.

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. We have successfully recorded the initiating time points (mostly at 2 h) of the viable-apoptotic switch in GSNO-initiated apoptosis, as well as shown the real-time differences between living and apoptotic thymocytes. These findings suggest that NO is also a switch molecule for the conversion from viable to apoptotic cell. Thymocytes cotreated by N-G-monomethyl-L-arginine acetate salt (L-NMMA) provide further evidence for this suggestion, as well as for the suggestion that L-NMMA prolongs the early stage of thymocyte apoptosis rather than strongly blocks it.