Construction of a Nanosecond Fluorometric System for Applications to Biological Samples at Cell or Tissue Levels

Abstract

A fluorometric system utilizing the single photon counting technique which records the decay of the intensity and the anisotropy of fluorescence following pulse excitation is described. Both macroscopic observations on cellular suspensions and measurements on individual cells under an optical microscope are possible. The system is so designed to permit precise evaluation of complex anisotropy decays, which is essential for studies of cells or tissues. Data are processed by a method which properly analyzes these complex decays. A preliminary study on erythrocyte membrane has confirmed the high resolution and precision of the system.

A theory of fluorescence polarization decay in membranes

“…For the evolution of Wwith time, we define G(;&s, fil, t' Pa, Pe, t) as the probability that amolecule with orientation J;&a ;&'I at time t' will rotate into a new orientation $P&a, AeI by time t. Thus, W(jia, ;&e, t) = ffW(J&a'',JL t') G(pa, , t' ;a,pe, t) djda. d (7) Here we understand that both W and G contain a factor 6(P,aflecos X) which assures the fixed angle between the two moments. For the isotropic sample, stationary distribution W5 is given by WS(paLe) = ( 1/87r2) (La6 gecos X).…”

“…12 is further rewritten as follows. We again expand P2(1a -p&) as P2(;a #Ae) = P2(Sa ILAD)P2(X yAe) + AP2(pa ye) 22(Lj ye) cos (v -+ AP2( A 2 * e-)P2(p1 * pe)cos2(I -{/) (14) BIoPHYSICAL JOURNAL VOLUME 20 1977 (7) where 6 and i1' are the azimuthal angles of u4 and ye around ye. In case a the second and the third terms in Eq.…”

“…These requirements are met by a pulse fluorometer employing the single photon counting technique (1,2). An apparatus specifically designed for the present purpose has been described elsewhere (7). With currently used pulse fluorometers, a precise r(t) is obtained only through a deconvolution procedure that corrects the observed data for the time characteristics of the apparatus.…”

“…In membranes, several nanosecond experiments to date (5)(6)(7)(8) indicate a general pattern: when fluorescent molecules embedded in membranes are excited by a pulse of polarized light, the polarization of emitted fluorescence is maximal at the moment of excitation and decays to a stationary value after a certain period of time. This pattern suggests that molecules in membranes exhibit wobbling motions rather than free rotation.…”

“…The theory deals with suspensions of membranes (intact or ghost cells, liposomes, etc. ), because precise measurements can readily be made on macroscopic suspensions (7). The primary system considered is that of lipid bilayers containing fluorescent molecules which may be lipid analogues or (labeled) membrane proteins.…”