Imaging Cells By CLSM And TPLSM And Study Of Cytomixis In Plant Cells

Along the past recent years, confocal Laser scanning microcopy (CLSM) and two-photon excitation (TPE) microscopy has moved from the realms of technical curiosity to be a standard application in many advanced cell biology laboratories. Like any new technology that has to gain its own space, TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. Yet, the published reports and unpublished results point out that TPE can eventually accommodate most available protocols and probes, most of the times with evident advantages. Further, the potential for plant sciences is obvious, as plant cells possess many absorbing molecules and structures and are routinely more opaque than tissues of other organisms.However, so far, there were no reports on the probes that exhibit simultaneously the good two photon fluorescent properties and bio-imaging ability of biomolecular, such as nucleus. For thick and highly scattering specimens, two-photon excitation (TPE) microscopy can not get clear-cut images using conventional fluorescent probes, the confocal Laser scanning microcopy imaging enabled the recording of images from biofilm down to a depth of 40μm, while two photon excitation images could be recorded at depth greater than 60-70μm. These research results show that the conventional fluorescent probes are unsuitable for thick samples due to their small two-photon action cross sections and needing high incident power. So absence of suitable two-photon fluorescent probes with high two-photon action cross sections, TPE microscopy is coming slow to be a generalized technology,In this paper, firstly, we points out the obvious advantages of TPE over any other imaging method based on fluorescence, clearly improving signal-to-noise ratio and thick-tissue penetration and showing added potential for vital imaging. TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. And then we have imaged some biomolecular of cells by confocal Laser scanning microcopy (CLSM) and two-photon excitation (TPE) microscopy. Secondly, we study the character of two-photon fluorescent probes, then synthesize and character several novel two-photon fluorescent DNA probes, BMVEC,9E-BHVC and 2,7-BHVC. And then we study application of these two-photon fluorescent DNA probes to nuclear imaging in living plant cells and turbid tissues. Thirdly,3-D and dynamic observation of transgenic plant cells with confocal laser scanning microscope (CLSM) and two-photon excitation (TPE) microscopy showed the real dynamic process of the separation of chromosome during the anaphase of mitosis and migration of chromosome from one cell to neighboring cell. These results indicated that the living observation system of cytomixis study has been established successfully.