Measurement of intracellular concentration of fluorescently-labeled targets in living cells

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells

ОСОБЕННОСТИ ФОКУСИРОВКИ СВЕТА ПЛОСКОЙ ЛИНЗОЙ НА ОСНОВЕ СТРУКТУРЫ МЕТАЛЛ–ДИЭЛЕКТРИК

It is shown that lenses based on the metal – dielectric (MD) structure are energetically more efficient in ultraviolet and violet ranges of wavelengths. Phase characteristics of flat lenses in ultraviolet as well as in visible ranges for the p-polarized light have a concave shape, which is the necessary condition of focusing. For the first time, direct measurement is made of the flat optically thin two-layered MD-structure (Ag/SiO2 ). It has been found experimentally that for an incident spherical wave with radial polarization (p-polarization), the focusing regime is realized, and with azimuthal polarization (s-polarization) the channeling regime is realized.Показано, что линзы на основе структуры металл–диэлектрик (МД) энергетически наиболее эффективны в ультрафиолетовом и фиолетовом диапазонах длин волн. Фазовые характеристики плоской линзы как в ультрафиолетовом, так и в видимом диапазонах для p-поляризованного света имеют вогнутую форму, что и является необходимым условием фокусировки. Впервые проведено прямое измерение угловой зависимости фазового сдвига свето- вой волны, прошедшей через плоскую оптически тонкую двухслойную МД-структуру (Ag/SiO2 ). Экспериментально установлено, что для падающей сферической волны с радиальной поляризацией (p-поляризация) реализуется режим фокусирования, а с азимутальной поляризацией (s-поляризация) реализуется режим каналирования