Spectroscopic properties of fluorescein in living lymphocytes

Abstract

Detailed studies have been performed on various spectroscopic properties such as time dependence and excitation wavelength dependence of the fluorescence anisotropy for fluorescein molecules introduced into rat thymus lymphocytes. Experimental results have been found to be well interpreted in terms of the coexistence of two types of dye molecules, i.e., free and bound molecules. The fluorescence spectrum of only the bound molecules has been obtained from the difference in the time-resolved spectra of fluorescence with two polarization directions. The time gate has been set at a sufficiently late time after the excitation, so that the polarization memories of the free molecules are lost. The spectrum thus determined agrees very well with that calculated from the spectral data in the stationary condition. From the above results, we come to the conclusion that the main factors which determine the fluorescence anisotropy inside the cell are the fraction and the anisotropy of the bound dye molecules. Finally, we discuss how these factors are related to biological quantities.Key terms: Neoplasms, spectrometry, fluorescence, lasers, fluorescence polarization Recently, several experiments have been reported in which human cancers are detected by the measurement of fluorescence depolarization in lymphocytes. This method is based on the fact that the degree of polarization of fluorescence from fluorescein introduced into living cells changes according to antigenic stimulation of lymphocytes. Cercek and Cercek (3) examined this property in peripheral lymphocytes from various cancer patients and found that the ratio of the change in the degree of polarization by the stimulation with nonspecific mitogen PHA (phytohemagglutinin) to that with socalled cancer basic proteins, gives a good measure for the purpose of the diagnosis of human cancers. This finding has been confirmed by many researchers Although this method provides an easy way of a medical checkup for human cancers, there exist clear limitations to the wide applicability of this method at the present time. First, a very complicated pretreatment method for this assay lowers the reproducibility of the experiment. Second, the detection of early-stage cancers is limited by its relatively low sensitivity. However, these difficulties may possibly be solved if the mechanism of this phenomenon is clarified from the physical and biological viewpoints.Various mechanisms to explain this fluorescence depolarization in living cells have been proposed so far, (6,8,13,(17)(18)(19)21,23). such as free rotational Brownian motion of the dye molecules in the cytoplasm (2,241, restricted motion of the dye molecules bound to proteins (20), and the mixture of these two (16,251. However, no model has been accepted generally yet, and also, the quantitative evaluation of the degree of polarization has not been made. To determine the physical mechanism of this fluorescence depoIarization, we have measured the time dependence of the fluorescence anisotropy of flourescein introduced into rat th...

Fluorescence quenching of fluorescein by Merocyanine 540 in liposomes

“…Besides, the spectral characteristic of fluorescent probes depends on the variety of the depths that probe molecules are located at and the surrounding environment [7]. FL and MC540 molecules are dye compounds that are exploited in different fields [8][9][10]. For example, MC540 is used in the photodynamic treatment of various human cancer cells as photosensitizer [11][12][13] whereas FL is used for labeling membrane and proteins [14].…”

Polarization of scatter and fluorescence signals in flow cytometry

“…Later (3), they used the method to verify the occurrence of energy transfer between labeled cell‐surface receptors. Other investigators have used similar methods to measure the fluorescence polarization of intracellular fluorescein (4–7) and of various DNA‐ and membrane‐bound dyes (8–15), because polarization provides information on the cellular context of these molecules. Measurements of fluorescence polarization of peripheral blood cells have been reported to be useful for the study and diagnosis of cancer (16) and Alzheimer's disease (17).…”

Fluorescence lifetime imaging microscopy reveals quenching of fluorescein within corneal epithelium

“…Multiple fluorophores bound in proximity on the same protein often show quenching (Lakowicz, 2007). So it is not surprising fluorescein bound to albumin demonstrates quenching, both static and dynamic (Barbero et al, 2009; Kinoshita et al,1987). Fluorescein entry into human lymphocytes has been proffered to exist in both bound and free states (Meisingset and Steen, 1981).…”

“…Fluorescein entry into human lymphocytes has been proffered to exist in both bound and free states (Meisingset and Steen, 1981). The bound fraction predominates, involves many intracellular proteins and shows increased anisotropy (Kinoshita et al, 1987; Meisingset and Steen, 1981). These data are consonant with the findings presented here for corneal epithelial cells.…”