Randy J. Giedt

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue-green region of the visible-light spectrum that display fluorescence enhancement exceeding 11,000-fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans-cyclooctene(TCO)-tetrazine chemistry platform. By exploiting highly efficient through-bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No-wash, fluorogenic imaging of diverse targets including cell-surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.

Mitochondria-derived reactive oxygen species mediate heme oxygenase-1 expression in sheared endothelial cells.

Bovine aortic endothelial cells (ECs) respond to nitric oxide (NO) donors by activating the redox-sensitive NF-E2-related factor 2/antioxidant response element pathway and up-regulating heme oxygenase (HO)-1 expression. EC exposure to steady laminar shear stress causes a sustained increase in NO, a transient increase in reactive oxygen species (ROS), and activation of the HO-1 gene. Because steady laminar flow increases the mitochondrial superoxide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}) production, we hypothesized that mitochondria-derived ROS play a role in shear-induced HO-1 expression. Flow (10 dynes/cm2, 6 h)-induced expression of HO-1 protein was abolished when BAECs were preincubated and sheared in the presence of either NG-nitro-l-arginine methyl ester or N-acetyl-l-cysteine, suggesting that either NO or ROS up-regulates HO-1. Ebselen and diphenylene iodonium blocked HO-1 expression, and uric acid had no effect. The mitochondrial electron transport chain inhibitors, myxothiazol, rotenone, or antimycin A, and the mitochondria-targeted antioxidant peptide, Szeto-Schiller (SS)-31, which scavenges \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}, hydrogen peroxide (H2O2), peroxynitrite, and hydroxyl radicals, markedly inhibited the increase in HO-1 expression. These data collectively suggest that mitochondrial H2O2 mediates the HO-1 induction. MitoSOX and 2′,7′-dichlorofluorescin (DCF) fluorescence showed that mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} levels and intracellular peroxides, respectively, are higher in sheared ECs compared with static controls and, in part, dependent on NO. SS-31 significantly inhibited both the shear-induced MitoSOX and DCF fluorescence signals. Either phosphatidylinositol 3-kinase or mitogen-activated protein kinase cascade inhibitors blocked the HO-1 induction. In conclusion, under shear, EC mitochondria-derived H2O2 diffuses to the cytosol, where it initiates oxidative signaling leading to HO-1 up-regulation and maintenance of the atheroprotective EC status.

Mitochondrial fission in endothelial cells after simulated ischemia/reperfusion: role of nitric oxide and reactive oxygen species.

Ischemia (I)/reperfusion (RP)-induced endothelial cell (EC) injury is thought to be due to mitochondrial reactive oxygen species (mtROS) production. MtROS have been implicated in mitochondrial fission. We determined whether cultured EC exposure to simulated I/RP causes morphological changes in the mitochondrial network and the mechanisms behind those changes. Because shear stress results in nitric oxide (NO)-mediated endothelial mtROS generation, we simulated I/RP as hypoxia (H) followed by oxygenated flow over the ECs (shear stress of 10dyn/cm(2)). By exposing ECs to shear stress, H, H/reoxygenation (RO), or simulated I/RP and employing MitoTracker staining, we assessed the differential effects of changes in mechanical forces and/or O(2) levels on the mitochondrial network. Static or sheared ECs maintained their mitochondrial network. H- or H/RO-exposed ECs underwent changes, but mitochondrial fission was significantly less compared to that in ECs exposed to I/RP. I/RP-induced fission was partially inhibited by antioxidants, a NO synthase inhibitor, or an inhibitor of the fission protein dynamin-related protein 1 (Drp1) and was accompanied by Drp1 oligomerization and phosphorylation (Ser616). Hence, shear-induced NO, ROS (including mtROS), and Drp1 activation are responsible for mitochondrial fission in I/RP-exposed ECs, and excessive fission may be an underlying cause of EC dysfunction in postischemic hearts.

Mitochondrial Dynamics and Motility Inside Living Vascular Endothelial Cells: Role of Bioenergetics

The mitochondrial network is dynamic with conformations that vary between a tubular continuum and a fragmented state. The equilibrium between mitochondrial fusion/fission, as well as the organelle motility, determine network morphology and ultimately mitochondrial/cell function. Network morphology has been linked with the energy state in different cell types. In this study, we examined how bioenergetic factors affect mitochondrial dynamics/motility in cultured vascular endothelial cells (ECs). ECs were transduced with mitochondria-targeted green fluorescent protein (mito-GFP) and exposed to inhibitors of oxidative phosphorylation (OXPHOS) or ATP synthesis. Time-lapse fluorescence videos were acquired and a mathematical program that calculates size and speed of each mitochondrial object at each time frame was developed. Our data showed that inner mitochondrial membrane potential (ΔΨm), ATP produced by glycolysis, and, to a lesser degree, ATP produced by mitochondria are critical for maintaining the mitochondrial network, and different metabolic stresses induce distinct morphological patterns (e.g., mitochondrial depolarization is necessary for “donut” formation). Mitochondrial movement, characterized by Brownian diffusion with occasional bursts in displacement magnitude, was inhibited under the same conditions that resulted in increased fission. Hence, imaging/mathematical analysis shed light on the relationship between bioenergetics and mitochondrial network morphology; the latter may determine EC survival under metabolic stress.

Imaging windows for long-term intravital imaging: General overview and technical insights

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure.

Red Si–rhodamine drug conjugates enable imaging in GFP cells

Here we evaluated a series of Si-derivatized rhodamine (SiR) dyes for their ability to visualize a model drug in live cells. We show that a charge neutral SiR derivative (but not others) can indeed be used to follow the intracellular location of the model therapeutic drug in GFP cells.

Quantitating drug-target engagement in single cells in vitro and in vivo

Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.

Real-time high dynamic range laser scanning microscopy

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Single cell resolution in vivo imaging of DNA damage following PARP inhibition

Targeting DNA repair pathways is a powerful strategy to treat cancers. To gauge efficacy in vivo, typical response markers include late stage effects such as tumor shrinkage, progression free survival, or invasive repeat biopsies. These approaches are often difficult to answer critical questions such as how a given drug affects single cell populations as a function of dose and time, distance from microvessels or how drug concentration (pharmacokinetics) correlates with DNA damage (pharmacodynamics). Here, we established a single-cell in vivo pharmacodynamic imaging read-out based on a truncated 53BP1 double-strand break reporter to determine whether or not poly(ADP-ribose) polymerase (PARP) inhibitor treatment leads to accumulation of DNA damage. Using this reporter, we show that not all PARP inhibitor treated tumors incur an increase in DNA damage. The method provides a framework for single cell analysis of cancer therapeutics in vivo.

Single Cell Analysis of Drug Distribution by Intravital Imaging

Recent advances in the field of intravital imaging have for the first time allowed us to conduct pharmacokinetic and pharmacodynamic studies at the single cell level in live animal models. Due to these advances, there is now a critical need for automated analysis of pharmacokinetic data. To address this, we began by surveying common thresholding methods to determine which would be most appropriate for identifying fluorescently labeled drugs in intravital imaging. We then developed a segmentation algorithm that allows semi-automated analysis of pharmacokinetic data at the single cell level. Ultimately, we were able to show that drug concentrations can indeed be extracted from serial intravital imaging in an automated fashion. We believe that the application of this algorithm will be of value to the analysis of intravital microscopy imaging particularly when imaging drug action at the single cell level.