Fluorescence Absorbance Inner-Filter Decomposition: The Role of Emission Shape on Estimates of Free Ca²⁺ Using Rhod-2

Abstract

A method for decomposing complex emission spectra by correcting for known inner-filter effects is described. This approach builds on previous work using a linear combination of model emission spectra and combines the known absorption characteristics of the system to fit the composite emission spectrum. Rhod-2, which has a small Stokes shift and significant self-absorption, was used as the model system. By adding the absorption characteristics of Rhod-2 to the model, the degree of fit was significantly improved, thus minimizing residuals, and accurately predicted the spectral shape changes with increasing concentration, [Rhod-2]. More complex studies were conducted with Rhod-2 in isolated cardiac mitochondria with multiple emission and absorption elements. By including known absorbances to the spectral decomposition, the overall precision increased almost four fold. Moreover, this approach eliminated the significant [Rhod-2] dependence on the apparent K(50) and therefore improved the accuracy of free [Ca(2+)] calculations. These data demonstrate that secondary inner-filter correction can significantly improve spectral decomposition of complex emission spectra, which are used in a variety of biological applications.

Selective mitochondrial staining with small fluorescent probes: importance, design, synthesis, challenges and trends for new markers

“…In order to probe the mitochondrial calcium flux, a charged calcium indicator family was developed (Rhod-2, rhod-5N, X-rhod-1, X-rhod-5F, X-rhod-FF and X-rhod-5N), reporting on its accumulation in mitochondria. [72][73][74] There are two approaches to probe mitochondrial calcium flux: (i) By using calcium indicator alone. Once mitochondria accumulate calcium these organelles become visible due to the fluorescent signal emitted by the calcium indicator.…”

Accounting for Secondary Inner Filter Effect in Fluorescence Spectra from Solid Samples

“…Secondary inner filter effect is observed when there is a strong overlap between absorption and emission spectra, leading to the possibility of reabsorption of emitted light by another molecule in close vicinity. In this case, self-absorption of the blue end of emission results in distortion of the emission spectrum [11][12][13][14] . In extreme situations, a progressive red shift (bathochromic shift) in emission maximum is observed with increase in concentration 7 .…”

On the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correction

“…Many practical fluorescence applications involve samples that present high optical densities due to a required high concentration of an active compound or due to its biological or chemical background. This includes optically dense samples like blood or tissue but also may involve studies of systems like proteins that physiologically require significant high micro/ millimolar concentrations [7][8][9][10][11]. Similarly, some investigations require increasing the concentration of one compound, like the concentration of an absorbing quencher in a quenching experiment (e.g.…”