Ever since Gösch et al. 1 applied fluorescence correlation spectrometry (FCS) to a microchannel flow system, this technique has attracted attention in various fields, including nano-and life-sciences, such as the flow system of fluorescent particles, 2 quantum dots, 3-5 DNA-fluorescent probe assemblies, 6,7 protein molecules, and living cell systems. [8][9][10][11][12] Further, a new technical approach has been attempted in FCS for increasing the information obtained by the cross-correlation or fluorescence resonance energy transfer (FRET) techniques. [13][14][15] A confocal optical configuration has been adopted for observing most of FCS (see the review by Cognet et al.).11 Recently, a numerical treatment or nonstandard method has been proposed by Culbertson et al. 16 However, the object dynamics vary with the flow and free Brownian motion; in some cases, calibration of the fluorescent particle size might be required.In the present note, in particular, the limitation in determining the empirical relationship between the pinhole diameter and the fluorescent particles size is investigated: we have shown that nanosize measurement is possible without any optical magnification. As a practical application, the microalgal size was measured with the present system. Experimental ReagentsAll the fluorescent particle standards were obtained from Polyscience Inc. (Fluoresbrite, Microsphere, POL series). The particles used were latex particles, whose surfaces were modified by fluorophores: the particles were stably suspended in aqueous media. We confirmed that the standard shows the maximum emission at 410 nm for an excitation wavelength of 325 nm. FCS measurement systemThe optical configuration was the same as that used for the ordinary fluorometry. Standard particles in an ordinary synthetic quartz cuvette used in fluorometry were driven by an ordinary cubic stirrer (GL Science, Type SRS 111A; with a stirring rod of Type TC-10) at a rate of 200 rpm. The excitation was performed by a He-Cd laser at 325 nm (Kinmon Koha Co. Ltd., Model IK-3251RF, 15 mW). The emission from the cuvette was directly detected by a photon counter (Hamamatsu, Model C8855) through a pinhole by employing 350-nm highpass and 410-nm (or 650-nm) band-pass filters (purchased from Edmund Optics). The diameter of the laser beam was about 0.7 mm. Therefore, observation volume can be calculated as about 0.4 × 10 -6 A ml (A = diameter (μm) of the pinhole used). The signal was processed by a real-time correlator (ALV-multiple tau digital real correlator 5000/60XO, ALV-GmbH, Germany) and transferred to a Windows XP computer to which the photon counting signal was also simultaneously transferred for display. Pinholes were purchased from Sigma Koki Co. In the present optical configuration, no tools were used for the magnification of the fluorescent images, i.e., a pinhole was set just in front of a photon counting detector near the sample cuvette. Cultivation of microalgaeSynechocystis sp. PCC 6803 and Chlorella kessleri C-531, a single-cell freshwater algae, ...
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