Tongsheng Chen

Fluorescence correlation spectroscopy considering the effect of photobleaching

In fluorescence correlation spectroscopy (FCS), the fluorescence intensity decay curve is used to calculate the correlation time, which characterizes the diffusion feature of the molecules. However, photobleaching, an unavoidable phenomenon that happened during fluorescence excitation, would result in fluorescence decay, and thus cause error in the calculation of the correlation time and the diffusion coefficient. To compensate this effect, a probability factor is introduced. This factor describes the probability at which a molecule is photobleached by the excitation laser during fluorescence excitation. By selecting the right value of the photobleaching factor, the effect of photobleaching on the FCS measurement can be removed.

Photobleaching of fluorophores at lower concentration under two-photon excitation microscopy

The intensity-dependence of photobleaching rate with two-photon excitation (TPE) at biological imaging level has been found to be high-order (>3) relationship. This paper studied experimentally the dependence for rhodamine B (RhB) at two different concentrations under unsaturated absorption conditions. The log-log plot of photobleaching rate versus incident power at concentration of 10-2M increased with a slope of ~3.7, but that at concentration of 10-4M increased with a slope of ~2.7.

Capillary electrophoresis based on continuous-wave multiphoton excitation fluorescence

High performance capillary electrophoresis(HPCE) has been widely applied to the field of chemistry and life science for its advantages of high separation efficiency, short analysis times and low sample volume requirements. But how to improve the detection capability of capillary electrophoresis was ever a key problem. Multi-photon excitation (MPE) fluorescence detection could solve this problem, however this method needed expensive femtosecond mod-locked laser, which restricted the improvement of capillary electrophoresis. In this paper, we implemented a set of MPE-CE system based on a continuous wave (CW), successfully exploited the CW multi-photon excitation to achieve detection of capillary electrophoresis. In our current MPE-CE system we used Ti:sapphire laser to prove MPE-CE could work in CW mode. From this point, we would substitute the laser with laser diode (LD) in future. This is really a new but also practical idea in MPE-CE system based on CW.

Interaction method of photobleaching produced by the interaction between excitation photons and fluorophore molecules in excited states

We proposed and analyzed the D-P (Dye-to-Photon) photobleaching mechanism, the interaction between the excitation photon(s) and the fluorophore dye molecules in excited states. At single molecule level, the D-P photobleaching probability with One- and Two-photon excitation (OPE and TPE) should always depend linearly on the probability of fluorophore molecule in excited states. However, at biological imaging level, the dependence of the D-P photobleaching probability on the probability of fluorophore molecules in excited states may depend on the excitation way: the photobleaching states, while the photobleaching rate depends on the high-order power of the probability of fluorophore molecule in the excited states, especially in the case of high excitation intensity.

High-order photobleaching in two-photon excitation fluorescence microscopy inside live cell

The two-photon excitation (TPE) microscopy has become an important tool of noninvasive imaging due to the better penetration and relative harmlessness of the longer wavelength. However, the high photon flux in two-photon excitation can potentially lead to higher-order photobleaching within the focal volume. This paper measured the relationship between the photobleaching rate and the excitation power for Chemical dyes and green fluorescence proteins (GFPs) in vivo at biological imaging level. As expected, the photobleaching rate increased near-linearly with the excitation power for one-photon excitation. However, the two-photon photobleaching rate increased with high-order power (≥3.5) of excitation power, indicating the presence of high-order photon interaction in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study may be limited by increased photobleaching.

5-HT spatial distribution imaging with multiphoton excitation of 5-HT correlative visible fluorescence in live cells

The autofluorescence of 5-Hydroxytryptamine (5-HT) loaded rat mucosal mast cells (RBL-2H3 cells) is imaged with multiphoton excitation laser scanning microscope (MPELSM). 5-HT correlative visible fluorescence (Fco-vis) excited with 740-nm multiphoton excitation is observed in live cells for the first time, and the generating mechanism of 5-HT Fco-vis is studied. The spatial distribution of 5-HT in live cells is imaged at high spatial resolution in our experiment, which provides a new way to study the correlation between 5-HT spatial distribution and content, and the cellular functional state in live tissue or cells.

High-sensitivity detection of capillary electrophoresis with multiphoton excitation

Multiphoton excited fluorescence detection is a powerful tool for probing chemistry and structure deep within biological tissue, for performing sensitive measurement on deep-UV (ex 200-300 nm) specimen. We present an approach for rapid analysis of Rhodamine B using multiphoton excited fluorescence detection coupled to capillary electrophoresis separations. In this highly versatile approach, Rhodamine B is excited through the nearly simultaneous absorption of two low-energy photons, fluorescence can be detected efficiently with low background. In these studies, Rhodamine B is fractionated in several minutes, with mass detection limits as low as 10 amol (2nM). This approach is demonstrated to be a powerful tool for analyzing the complex biological samples of minute quantities.

Novel photobleaching model in living cells

A novel photobleaching model was proposed, in which a D-P photobleaching mechanism was developed. By combining with the conventional D-D and D-O photobleaching mechanisms, the dependence of photobleaching rate on the excitation power is well illustrated. The validity of this model was demonstrated with GFP photobleaching experiments in cases of one-photon excitation (1PE) and two-photon excitation (2PE) respectively. Previously inexplicable experimental results published in literature were also illustrated with this new model.

Data analysis in green-fluorescent-protein-based fluorescence resonance energy transfer

Green fluorescent protein based fluorescence resonance energy transfer is widely used in studies of inter-and intra-molecular dynamic interactions in alive cells. But it is a trade off between quantitative measurement and real time measurement because of the emission cross talk. In stead of using more than one excitation wavelength and several sets of filters, we proposed a more efficient correction scheme which needs only a single excitation and one filter set. This method makes use of the known emission spectra of the fluorophores and can be run in real time. The validity and advantages were demonstrated by experiments with chamaeleons.