Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

Abstract

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems.

Insight into diacetylene photopolymerization in Langmuir‐Blodgett films using simultaneous AFM and fluorescence microscopy imaging

“…In most modern applications, optical microscopy resolution is on the order of 200‐300 nm laterally, and 500 nm in depth. The resolving power of optical imaging is fundamentally limited by diffraction to approximately half the wavelength of the light used . However, its ability to image with chemical specificity (usually a sample is labeled with a fluorescent tag and fluorescence optical microscopy is used) allows researchers to identify specific structures or molecules within a sample.…”

“…The resolving power of optical imaging is fundamentally limited by diffraction to approximately half the wavelength of the light used. 3 However, its ability to image with chemical specificity (usually a sample is labeled with a fluorescent tag and fluorescence optical microscopy is used) allows researchers to identify specific structures or molecules within a sample. Optical microscopy 0 s chemical specificity and ability to image live processes within the depth of a sample are well complemented by the higher resolution capabilities of the AFM.…”

Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

“…Moreover, we developed the open source NyxSense (Supp. Note 3) software that implements ad hoc data analysis algorithms based on multi-dimensional phasor [30][31][32] fingerprinting. NyxSense enables quantitative unmixing of biochemical information from the NyxBits platform and multi-colour FLIM, delivering an unprecedented level of multiplexing.…”

Photon-statistics in sensitized emission FRET and FLIM: a comparative theoretical analysis

“…A higher precision leads directly to an improvement in the capability to resolve smaller biochemical differences in living cells. From a theoretical standpoint, this improvement in biochemical resolving power can also be understood for the general analysis of Fisher information in multi-dimensional or multiparametric detection systems (see for example the photon partitioning theorem in [56,57]). From a practical point of view, dual-colour fast high-resolution FLIM might be increasingly accessible thanks to the ongoing revolution in time-resolved detection technologies and could provide yet unexplored ideal performances.…”