**Section 4**

**Fluorescence Spectroscopy** 

180 Macro to Nano Spectroscopy

[6] Qian Hao, Zhu Ya-fei, XU Jia-rui Spectroscopy and Spectral Analysis, 2003, 23 (4): 708-

[7] Chen Jia-xing, Joseph A. Gardella Jr. Appl. Spectroscopy, 1998, 52(3): 361.

713

**11** 

*USA* 

Alexander A. Banishev\*

**Laser Fluorescence Spectroscopy:** 

**Application in Determining the Individual** 

*Department of Physics and Astronomy, University of California, Riverside, CA,* 

This work was initiated by the problem of investigating the photophysical properties of complex protein molecules and performing the diagnostics of such molecules in water environment. At the present time, fluorescence spectroscopy (fluorimetry) is widely used to study complex organic compounds (COC) (Lakowicz, 1999). Together with spectrophotometry these methods form the basis for fast and nondestructive diagnostics of COC in the natural environment, i.e. they present the diagnostic methods *in vivo* and *in situ*. However, the conventional (linear) fluorescence spectroscopy methods can not provide complete information on fluorescent objects under study because of insufficient selectivity

(fluorescence bands of most COC are broad and structureless at room temperature).

spectroscopic methods (for example, molecular concentration (Banishev et al., 2009)).

(or, better, synthesize) several spectroscopic methods.

The capabilities of fluorescence spectroscopy can be enhanced by using the methods of laser fluorimetry, in particular nonlinear laser fluorimetry (Fadeev et al., 1999). This method allows one to get information on the molecular level and determine the photophysical parameters of molecules (absorption cross section, lifetime in excitation state, intersystem crossing and energy transfer rates, etc.). Furthermore, the parameters can be measured *in vivo* and *in situ* in the absence of *a priori* information, which is necessary for conventional

The diagnostics of protein complexes is an intricate problem if a molecule contains more than one absorption/fluorescent center (Permyakov, 1992). The problem becomes much more complex if, in addition, the protein specimen (ensemble of molecules) is a mixture of several chemically nonidentical types of molecules (subensembles) which cannot be separated, i.e. their partial concentrations are unknown. The second situation is typical for the special kind of proteins, namely, fluorescent proteins (FPs) (Piatkevich et al., 2010). The solutions of FPs are usually mixtures of several types of molecules, which are chemically different and have their own set of photophysical properties (Verkhusha et al., 2004). In this case for unambiguous interpretation of experimental data it is necessary to make simultaneous measurements of a large number of parameters, i.e. to simultaneously apply

**1. Introduction** 

\* Corresponding Author

**Photophysical Parameters of Proteins** 
