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Amyloidosis is a disorder characterized by misfolded precursor proteins that form depositions of fibrillar aggregates with an abnormal cross-beta-sheet conformation, known as amyloid, in the extracellular space of several tissues. Although there are more than 30 known amyloidogenic proteins, both hereditary and non-hereditary, cardiac amyloidosis (CA) typically arises from either misfolded transthyretin (ATTR amyloidosis) or immunoglobulin light-chain aggregation (AL amyloidosis). Its prevalence is more common than previously thought, especially among patients with heart failure and preserved ejection fraction (HFpEF) and aortic stenosis. If there is a clinical suspicion of CA, focused echocardiography, laboratory screening for the presence of a monoclonal protein (serum and urinary electrophoresis with immunofixation and serum free light-chain ratio), and cardiac scintigraphy with 99m technetium-labeled bone-tracers are sensitive and specific initial diagnostic tests. In some cases, more advanced/invasive techniques are necessary and, in the last several years, treatment options for both AL CA and ATTR CA have rapidly expanded. It is important to note that the aims of therapy are different. Systemic AL amyloidosis requires treatment targeted against the abnormal plasma cell clone, whereas therapy for ATTR CA must be targeted to the production and stabilization of the TTR molecule. It is likely that a multistep treatment approach will be optimal for both AL CA and ATTR CA. Additionally, treatment of CA includes the management of restrictive cardiomyopathy with preserved or reduced ejection fraction in addition to treating the amyloid deposition. Future studies are necessary to define optimal management strategies for AL CA and ATTR CA and confirm cardiac response to therapy.

17Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually 18 exclusive experimental strategies optimizing either time or spatial resolution. To achieve both, 19we implement a GPU-supported, camera-based measurement strategy that highly resolves 20 spatial structures (~60 nm), temporal dynamics (≤ 2 ms), and molecular brightness from the 21 exact same data set. We demonstrate the applicability and advantages of multi-parametric 22 measurements to monitor the super-resolved structure and dynamics of two different 23 biomolecules, the actin binding polypeptide LifeAct, and the epidermal growth factor receptor 24 (EGFR). Simultaneous super-resolution of spatial and temporal details leads to an improved 25 precision in estimating the diffusion coefficient of LifeAct in dependence of the cellular actin 26 network. Multi-parametric analysis suggests that the domain partitioning of EGFR is primarily 27 determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-28 EGFR interactions but is largely independent of EGFR-actin interactions. These results 29 demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques 30 on the same data set enables robust physicochemical parameter estimation and provides new 31 biological knowledge that cannot be obtained from sequential measurements. 32 33 Key Words 34 35 Super-resolution, Imaging fluorescence correlation spectroscopy, Number and brightness 36 analysis, Super-resolution radial fluctuations, FCS diffusion law, Epidermal growth factor 37 receptor 38 egfr 39 Simultaneous spatiotemporal super-resolution microscopy

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