Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.

White light phosphors have many potential applications such as solid‐state lighting, full color displays, light source for plant growth, and crop improvement. However, most of these phosphors are rare‐earth‐based materials which are costly and would be facing the challenge of resource issue due to the extremely low abundance of these elements on earth. A new white color composite consisted of a graphitic‐phase nitrogen carbon (g‐C3N4) treated with nitric acid and copper‐cysteamine Cu3Cl(SR)2 is reported herein. Under a single wavelength excitation at 365 nm, these two materials show a strong blue and red luminescence, respectively. It is interesting to find that the white light emission with a quantum yield of 20% can be obtained by mixing these two self‐activated luminescent materials at the weight ratio of 1:1.67. Using a 365 nm near‐ultraviolet chip for excitation, the composite produces a white light‐emitting diode that exhibits an excellent color rendering index of 94.3. These white‐emitting materials are environment friendly, easy to synthesize, and cost‐effective. More importantly, this will potentially eliminate the challenge of rare earth resources. Furthermore, a single chip is used for excitation instead of a multichip, which can greatly reduce the cost of the devices.

Fluorescence is an established technology for studying molecular processes and molecular interactions. More recently fluorescence became a leading method for detection, sensing, medical diagnostics, biotechnology, imaging, DNA analysis, and gene expression. Consequently, precise and accurate measurements in various conditions have become more critical for proper result interpretations. Previously, in Part 1, we discussed inner filter effect type I, which is a consequence of the instrumental geometrical sensitivity factor and absorption of the excitation. In this part, we analyze inner filter effect type II and discuss the practical consequences for fluorescence measurements in samples of high optical density (absorbance/scattering). We consider both the standard square and front-face experimental configurations, discuss experimental approaches to limit/mitigate the effect and discuss methods for correcting and interpreting experimental results.

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