The proposed system has a measured detection limit up to 5 × 10−8 M (S/N = 3) while detecting a standard fluorescence of 2′,7′-dichlorofluoresein, which is capable of detecting fluorescence samples in general applications.
Fluorescence sampling of cellular function is widely used in all aspects of biology, allowing the visualization of cellular and sub-cellular biological processes with spatial resolutions in the range from nanometers up to centimeters.
Indirect measures of NCqs are estimated by fluorescence sampling.
Wing discs and adult wings images were acquired using a Zeiss LSM510 Confocal Microscope (fluorescence samples) and a Zeiss Axiovert200 (bright-field) microscope respectively.
Fluorescence samples were prepared as described [48] and visualized by a Zeiss Axioplan 2 microscope with AxioVision digital imaging software (Zeiss).
For determining ANS or ThT fluorescence, samples were briefly mixed, and aliquots were diluted into 50 µM ANS or 5 µM ThT in 5 mM KH2PO4, 100 mM NaCl, pH 7.5 at final concentrations of 5 µM.
To control for baseline dye fluorescence, samples from each experiment were left unstimulated but stained according to the above procedure.
Fluorescence samples were quantified by a calibration curve with resorufin or 7-hydroxy-coumarin standards, using 535 or 380 nm (excitation wavelength) and 585 or 460 nm (emission wavelength), respectively.
RGS was calculated as ratio between the relative fluorescence of sample and standard.
For fluorescence staining, samples were incubated with FITC-conjugated rat anti-GFP antibodies for 2 h at 37 °C.
