Recovering fluorophore concentration profiles from confocal images near lateral refractive index step changes

Abstract

Optical aberrations due to refractive index mismatches occur in various types of microscopy due to refractive differences between the sample and the immersion fluid or within the sample. We study the effects of lateral refractive index differences by fluorescence confocal laser scanning microscopy due to glass or polydimethylsiloxane cuboids and glass cylinders immersed in aqueous fluorescent solution, thereby mimicking realistic imaging situations in the proximity of these materials. The reduction in fluorescence intensity near the embedded objects was found to depend on the geometry and the refractive index difference between the object and the surrounding solution. The observed fluorescence intensity gradients do not reflect the fluorophore concentration in the solution. It is suggested to apply a Gaussian fit or smoothing to the observed fluorescence intensity gradient and use this as a basis to recover the fluorophore concentration in the proximity of the refractive index step change. The method requires that the reference and sample objects have the same geometry and refractive index. The best results were obtained when the sample objects were also used for reference since small differences such as uneven surfaces will result in a different extent of aberration.

Life Cycle and Lensing of a Macular Microcyst

“…The distortion and shadowing are caused by the curvature of the microcyst's structure and lateral changes in refractive index encountered by the incident beam. Lateral step changes like this are a known issue in microscopy [22], reducing brightness and resolution. If microcysts are present in a retina, this will affect quantification of cone mosaics in disease.…”

Morpholino Target Molecular Properties Affect the Swelling Process of Oligomorpholino-Functionalized Responsive Hydrogels

“…The close proximity of the glass fiber to the hydrogel affects the detected fluorescence intensity, i.e., the refraction of excitation and emission light through the fiber causes a decrease in the observed fluorescence that is the most pronounced at the fiber end face and reaches as far as 50 µm into the hydrogel/solution [33]. Due to this effect of the fiber, the fluorescence intensity profiles do not reflect the concentration of the target.…”

“…Due to this effect of the fiber, the fluorescence intensity profiles do not reflect the concentration of the target. To correct for this, an intensity profile I T0 in a solution of the non-binding target T0 was obtained for each individual hydrogel and used as reference for quantifying the effect of the fiber on the fluorescence intensity ( Figure 3) [33]. The concentration of the target inside the hydrogel relative to that in the immersing solution can then be obtained by dividing the fluorescence intensity profiles I T2 and I T10 by the reference profile I T0 for each individual hydrogel.…”

“…The relative concentration profiles are referred to as I TX/T0 where TX is one of the targets T2 or T10. the effect of the fiber on the fluorescence intensity ( Figure 3) [33]. The concentration of the target inside the hydrogel relative to that in the immersing solution can then be obtained by dividing the fluorescence intensity profiles IT2 and IT10 by the reference profile IT0 for each individual hydrogel.…”