Abstract
.Significance: Fluorescence polarization (FP) and fluorescence anisotropy (FA) microscopy are powerful imaging techniques that allow to translate the common FP assay capabilities into the in vitro and in vivo cellular domain. As a result, they have found potential for mapping drug–protein or protein–protein interactions. Unfortunately, these imaging modalities are ratiometric in nature and as such they suffer from excessive noise even under regular imaging conditions, preventing accurate image-feature analysis of fluorescent molecules behaviors.Aim: We present a high dynamic range (HDR)-based FA imaging modality for improving image quality in FA microscopy.Approach: The method exploits ad hoc acquisition schemes to extend the dynamic range of individual FP channels, allowing to obtain FA images with increased signal-to-noise ratio.Results: A direct comparison between FA images obtained with our method and the standard, clearly indicates how an HDR-based FA imaging approach allows to obtain high-quality images, with the ability to correctly resolve image features at different values of FA and over a substantially higher range of fluorescence intensities.Conclusion: The method presented is shown to outperform standard FA imaging microscopy narrowing the spread of the propagated error and yielding higher quality images. The method can be effectively and routinely used on any commercial imaging system and could be also translated to other microscopy ratiometric imaging modalities.
Highlights
IntroductionDirect in vivo fluorescence intensity measurements are crucial for measuring fluorescence marker concentration or other intrinsic parameters, but they can be quite challenging due to the absence of an internal reference and the presence of various nonrelevant artifacts, e.g., tissue scattering and absorption, sample–detector path geometry, or fluctuations in the excitation source.[1,2]
By integrating high dynamic range (HDR) imaging with fluorescence anisotropy (FA) and parallelizing the HDR acquisition scheme for each individual polarization channel, we demonstrate here that HDR-based FA images with highly improved signal-to-noise ratio (SNR) can be obtained
Because the noise is intrinsically entangled to the light signal and because it is not uncommon for fluorescence images to suffer from low SNR, in in vivo imaging settings, FA image degradation can be quite severe and strategies to overcome, or at least, ameliorate image degradation are in need
Summary
Direct in vivo fluorescence intensity measurements are crucial for measuring fluorescence marker concentration or other intrinsic parameters, but they can be quite challenging due to the absence of an internal reference and the presence of various nonrelevant artifacts, e.g., tissue scattering and absorption, sample–detector path geometry, or fluctuations in the excitation source.[1,2]. Membrane potentials,[4,5] and other parameters.[6] Depending on the specific imaging modality, the fluorescence signal is typically measured via excitation or emission under different or the same conditions of polarizations and wavelengths, and the ratiometric quantity is directly or indirectly calculated by taking the ratio of the two measurements. FA measurements instead are utilized for measuring equilibrium binding constants, molecular interactions, and enzymatic activity
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