Multiplane 3D superresolution optical fluctuation imaging

By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of $65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.Comment: 7 pages, 3 figure

Development of a bioluminescent nitroreductase probe for preclinical imaging

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.publishedVersio

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging

Purpose: Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with 111indium, via the chelate diethylene triamine pentaacetic acid (DTPA). Procedures: The necrosis avid properties of the radiotracer [111In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo. Results: Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [111In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis. Conclusions: The radiotracer [111In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy

Azacyanine dyes and use thereof

The application provides fluorescent dyes, which are cyanine dyes that incorporate additional aza moieties in the indolenium heterocycles and/or in the methine chains connecting them. Symmetrical and unsymmetrical chemically reactive azacyanine dyes are described for conjugation, as well as their bioconjugates for in-vitro and in-vivo assays and fluorescence imaging

Catching ghosts with a coarse net: use and abuse of spatial sampling data in detecting synchronization.

Synchronization of population dynamics in different habitats is a frequently observed phenomenon. A common mathematical tool to reveal synchronization is the (cross)correlation coefficient between time courses of values of the population size of a given species where the population size is evaluated from spatial sampling data. The corresponding sampling net or grid is often coarse, i.e. it does not resolve all details of the spatial configuration, and the evaluation error-i.e. the difference between the true value of the population size and its estimated value-can be considerable. We show that this estimation error can make the value of the correlation coefficient very inaccurate or even irrelevant. We consider several population models to show that the value of the correlation coefficient calculated on a coarse sampling grid rarely exceeds 0.5, even if the true value is close to 1, so that the synchronization is effectively lost. We also observe 'ghost synchronization' when the correlation coefficient calculated on a coarse sampling grid is close to 1 but in reality the dynamics are not correlated. Finally, we suggest a simple test to check the sampling grid coarseness and hence to distinguish between the true and artifactual values of the correlation coefficient

A System for In Vivo Imaging of Hepatic Free Fatty Acid Uptake

Alterations in hepatic free fatty acid (FFA) uptake and metabolism contribute to the development of prevalent liver disorders such as hepatosteatosis. However, detecting dynamic changes in FFA uptake by the liver in live model organisms has proven difficult. To enable noninvasive real-time imaging of FFA flux in the liver, we generated transgenic mice with liver-specific expression of luciferase and performed bioluminescence imaging with an FFA probe. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. By using this method, we detected a decrease in FFA accumulation in the liver after mice were given injections of deoxycholic acid and an increase after they were fed fenofibrate. In addition, we observed diurnal regulation of FFA hepatic uptake in living mice. Our imaging system appears to be a useful and reliable tool for studying the dynamic changes in hepatic FFA flux in models of liver disease

A System for In Vivo Imaging of Hepatic Free Fatty Acid Uptake

Alterations in hepatic free fatty acid (FFA) uptake and metabolism contribute to the development of prevalent liver disorders such as hepatosteatosis. However, detecting dynamic changes in FFA uptake by the liver in live model organisms has proven difficult. To enable noninvasive real-time imaging of FFA flux in the liver, we generated transgenic mice with liver-specific expression of luciferase and performed bioluminescence imaging with an FFA probe. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. By using this method, we detected a decrease in FFA accumulation in the liver after mice were given injections of deoxycholic acid and an increase after they were fed fenofibrate. In addition, we observed diurnal regulation of FFA hepatic uptake in living mice. Our imaging system appears to be a useful and reliable tool for studying the dynamic changes in hepatic FFA flux in models of liver disease