Changes In Living Cells Research Articles

Capturing cell morphology dynamics with high temporal resolution using single-shot quantitative phase gradient imaging.

Label-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to ). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining. We aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images. Here, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz., M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability. We validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves , and our analysis scheme accurately retrieves the phase with a high correlation coefficient ( ), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings. Label-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects.

Dissecting lysosomal viscosity fluctuations in live cells and liver tissues with an ingenious NIR fluorescent probe

Viscosity is a pivotal component in the cell microenvironment, while lysosomal viscosity fluctuation is associated with various human diseases, such as tumors and liver diseases. Herein, a near-infrared fluorescent probe (BIMM) based on merocyanine dyes was designed and synthesized for detecting lysosomal viscosity in live cells and liver tissue. The increase in viscosity restricts the free rotation of single bonds, leading to enhanced fluorescence intensity. BIMM exhibits high sensitivity and good selectivity, and is applicable to a wide pH range. BIMM has near-infrared emission, and the fluorescent intensity shows an excellent linear relationship with viscosity. Furthermore, BIMM possessing excellent lysosomes-targeting ability, and can monitor viscosity changes in live cells stimulated by dexamethasone, lipopolysaccharide (LPS), and nigericin, and differentiate between cancer cells and normal cells. Noticeably, BIMM can accurately analyze viscosity changes in various liver disease models with HepG2 cells, and is successfully utilized to visualize variations in viscosity on APAP-induced liver injury. All the results demonstrated that BIMM is a powerful wash-free tool to monitor the viscosity fluctuations in living systems.

(Invited) Carbon Nanotube Photoluminescence for Cancer Research and Diagnosis

New technologies are needed for the early detection of cancer and the identification of therapeutic vulnerabilities in cancers. Our laboratory develops optical nanosensor technologies using carbon nanotubes, including sensors that identify disease fingerprints in serum, diagnostic implants to facilitate longitudinal detection of cancer biomarkers, and assays for cancer drug development. These methods employ the photoluminescence of single-walled carbon nanotube (SWCNTs) and covalent sp3 quantum well defects on SWCNTs, aka organic color centers (OCCs), that introduce new chemical sensitivities. To introduce new detection technologies using OCC-modified SWCNTs, we developed new supramolecular functionalization methods, as well as new types of optical instrumentation to detect changes in near-infrared emission within biological and clinical samples. We harnessed OCC-modified SWCNTs for the detection of metabolic changes in live cells and tissues, disease biomarkers in situ via implants, and overall disease states, aided by machine learning processes.

A novel near-infrared fluorescent probe with hemicyanine scaffold for sensitive mitochondrial pH detection and mitophagy study

Mitochondria, as an energy-producing powerhouse in live cells, is considered to be directly linked to cellular health. However, dysfunctional mitochondria and abnormal mitochondria pH would possibly activate mitophagy, cell apoptosis and intercellular acidification process. In this work, we synthesized a novel near infrared fluorescent probe (FNIR-pH) for measurement of mitochondrial pH based on the hemicyanine skeleton as a fluorophore. The FNIR-pH probe functioned as a mitochondrial pH substrate and exhibited quick and sensitive turn-on fluorescence responses to mitochondrial pH in basic solution due to the deprotonation of hydroxy group in the structure. From pH 3.0 to 10.0, the FNIR-pH exhibited almost 100-fold increase in fluorescence intensity at 766 nm wavelength. The FNIR-pH also displayed superior selectivity to various metal ions, excellent photostability, and low cytotoxicity, which facilitated further biological application. Owing to the proper pKa value of 7.2, the FNIR-pH paved the way for real-time monitoring of mitochondria pH changes in live cells and sensitive sensing of mitophagy. Moreover, the FNIR-pH probe was also implemented for fluorescent imaging of tumor-bearing mice to validate its potential application for in vivo imaging of bioanalytes and biomarkers.

Metabolic profiling of transferred mitochondria using FLIM intensity based image segmentation (FIBIS).

Bulk metabolic assays on cell populations have shown that mitochondria transfer trigger breast cancer cells turn towards oxidative phosphorylation (OXPHOS) in favor of proliferation, migration, and cell growth. Yet, the current methods lack single cell resolution of interaction between endogenous and exogenous mitochondria to help understand the consequence metabolic alterations and other cell fate decisions. The phasor approach to fluorescence lifetime imaging microscopy (FLIM) has been widely used to measure the free to bound fraction of reduced form of NADH to quantify metabolic changes in live cells but not at the single mitochondrial scale. Here, we developed the FIBIS algorithm as a robust approach to recognize mitochondria from NADH intensity and further analyze mitochondrial metabolic states to investigate mitochondria transfer. We demonstrate that the NADH autofluorescence perfectly localizes with Mito7-mRuby labeled mitochondria and is free from artifactual emission spectral overlaps. Our data indicates that there was a 40% and a 20% increased fraction of bound NADH after mitochondria transfer in MCF7 and MB231 cells, respectively. This suggests that there is an enhancement of OXPHOS for both breast cancer cells. This was in correlation with the results from the Seahorse XF analyzer where the oxygen consumption rate was significantly higher after uptake of isolated mitochondria from breast epithelial cells. We also used Mitometer to show that there were increasing numbers of mitochondria and branching which imply that the fusion of exogenous and endogenous mitochondria are highly adaptable and increase cellular respiration. In summary, the FIBIS enables single mitochondrial metabolic profiling and aids in understanding the effects of transferred mitochondria to recipient cells. Our results also indicate that transferred mitochondria in cancer cells enhances OXPHOS, increased branching, and mitochondrial numbers, thereby decreasing cell viability and increasing sensitivity to chemotherapy agents.

LEMONADE: Lego-like membrane tension analyzer based on self-assembled DNA elastic networks.

Membrane tension regulates and is affected by many cellular processes, including cell motility and membrane trafficking. However, the role of membrane tension in such processes is poorly understood because of difficulties in measuring dynamic membrane tension changes in live cells. Current approaches for membrane tension measurements can be broadly divided into two categories. In the first, fluorescence-based signals from small molecular probes that insert into cellular membranes are used. These allow convenient mapping of the signals throughout the cell membrane, but probe local membrane properties which are not always easy to relate to membrane tension. In addition, they are prone to artefacts due to membrane heterogenicities. The second type of approach relies on pulling a thin membrane tether from the cell surface. The restoring force acting on the tether is related to membrane tension and can be measured using optical tweezers. However, this approach requires specialized equipment and tension is measured at a single location. To overcome these challenges, we are developing a novel approach based on the self-assembly of an elastic network on the cell surface we call LEMONADE (Lego-like membrane tensionanalyzer based on self-assembled DNA elastic networks). The new membrane tension sensor relies on membrane-anchored Lego-like DNA nanostructures that are linked by elastic connectors with a FRET pair modification. Cell membrane area changes will result in transient FRET signals which can be detected throughout the cell surface using standard live fluorescence microscopy. We will report our progress in the design, assembly, and calibration of LEMONADE.

A novel aggregation-induced emission fluorescent probe with large Stokes shift for sensitive detection of pH changes in live cells.

The detection of intracellular pH is crucial for elucidating the pathological process of cancers, as well as for medical diagnostic applications. Here, we developed an aggregation-induced emission active pH-responsive fluorescent probe (TPE-DCP) for sensitively detecting cell pH changes. The probe shows obvious pH-sensing properties at ~615 nm, with a pKa value of 6.82 and a good linear pH response ranging from 8.5 to 4.5. TPE-DCP holds advantages such as excellent anti-interference performance, good photostability, and low cytotoxicity, and has been successfully used to image intracellular pH changes in cells.

A Sensitive Fluorescent Probe with Large Stokes Shift for Real‐Time Tracking Lysosomal pH Changes in Live Cells

AbstractThe lysosome is the typical acidic organelle involved in many biological processes, the fluctuation of its pH influences its normal digestive function and even the fate of the cells. To conveniently detect and visualize lysosomal pH changes during heatstroke, we designed and synthesized a lysosomal‐targeted ratiometric pH‐sensitive fluorescent probe TCF‐LysopH. TCF‐LysopH showed an ideal pKa value (pKa=6.12) in the lysosomal pH range during heatstroke (5.00–7.00), rapid response for the changes of pH in real‐time, reversible pH switching ability, good selectivity, low cytotoxicity, and excellent lysosome‐targeting ability. The large Stokes shift of 110 nm allows us to apply the probe for fluorescence imaging in MCF‐7 cells. The probe has much better photostability (only less than 20 % decrease in intensity for 15 min irradiation) than LysoTracker Green. The fluorescence intensity became higher with the increase of pH, which is consistent with the tendency of the lysosomal pH change during heatstroke. Importantly, TCF‐LysopH was applied to measure the change of lysosomal pH during heatstroke in live cells, the results prove once again that the pH of the lysosome is increased during heatstroke. Thus, TCF‐LysopH was successfully developed as a practical and useful tool to provide a better understanding of the complicated mechanisms between lysosome pH change and heatstroke.

Orthogonally-tunable and ER-targeting fluorophores detect avian influenza virus early infection

Cell-based assays can monitor virus infection at a single-cell level with high sensitivity and cost-efficiency. For this purpose, it is crucial to develop molecular probes that respond selectively to physiological changes in live cells. We report stimuli-responsive light-emitters built on a T-shaped benzimidazole platform, and consecutive borylation reactions to produce a library of homologs displaying systematic changes in fluorescence quantum yield and environmental sensitivity. We find that certain fluorophores localize selectively at the endoplasmic reticulum, and interact with proteins involved in the stress signaling pathways. Notably, the mono-borylated compound responds selectively to the stress conditions by enhancing fluorescence, and detects avian influenza virus infection at the single-cell level. Our findings demonstrate the unprecedented practical utility of the stress-responsive molecular probes to differentiate cellular states for early diagnosis.

A benzothiazole-based fluorescence probe for imaging of peroxynitrite during ferroptosis and diagnosis of tumor tissues.

Ferroptosis, as a new regulated mode of cell death, is mainly characterized by iron-dependent accumulation of lipid peroxides related to reactive oxygen species (ROS). However, the changes of peroxynitrite (ONOO-) during ferroptosis are not very clear due to the limited reports. On the other hand, ONOO- has become an endogenous toxicant leading to cell apoptosis and necrosis. Thus, it is urgent to develop molecular tools with high selectivity and sensitivity to monitor intracellular ONOO-. Herein, we presented a fluorescent probe, BTMO-PN, by introducing a phenylboronic acid ester as the ONOO- recognition site into benzothiazolyl derivative. BTMO-PN exhibited a rapid and marked fluorescence enhancement signal toward ONOO-, owing to the ONOO--triggered the cleavage of phenylboronic acid ester to release strongly fluorescent BTMO. Moreover, BTMO-PN could image endogenous and exogenous ONOO- changes in live cells. Importantly, using BTMO-PN, we demonstrated the up-generation of ONOO- levels in cancer cells during ferroptosis. Furthermore, BTMO-PN has successfully been applied for distinguishing tumor tissues from normal tissues with excellent contrast, making it great potential for cancer diagnosis by detecting the ONOO- changes.

A benzothiophene-quinoline-based targetable fluorescent chemosensor for detection of viscosity and mitochondrial imaging in live cells

Mitochondria are the sites of respiration in cells, and they participate in many indispensable biological processes. Because variations in mitochondrial viscosity can lead to dysfunctions of mitochondrial structure and function (and even induce malignant diseases), new sensors that can accurately monitor changes in mitochondrial viscosity are essential. To better investigate these changes, we report the development and evaluation of a novel benzothiophene-quinoline-based fluorescent chemosensor (BQL) that was designed especially for monitoring mitochondrial viscosity. BQL demonstrated a large Stokes shift (minimizing interference from autofluorescence) and a good response to viscosity (using the TICT principle). Moreover, BQL demonstrated little to no pH-dependency, polarity-dependency, or interference from other analytes. Thus, BQL has an excellent specificity for viscosity. BQL was used to monitor viscosity changes in mitochondria induced by ion carriers, and was used to report on viscosity in real time during mitophagy. To sum up, BQL provided a new approach for detecting viscosity in living cells and in vivo. BQL should prove to be an excellent tool for the analysis of viscosity changes in live cells.

Fluorescence imaging of epigenetic genome modifications

Epigenome contains a lot of information about cell state. Epigenetic analysis includes primarily sequence-based methods, which provide detailed data on distribution of modifications along the genome, but are poorly applicable for screenings. Specific fluorescence labeling and imaging of epigenetic modifications is an attractive complementary approach. It is currently based mainly on histone modifications study. We expect that inclusion of DNA modifications into imaging-based study would empower the method. In this review we discuss methods for fluorescence imaging of DNA modifications (mainly 5-methylcytosine). It opens an easy way to single cell analysis and high-throughput screening. Moreover, tracking epigenome changes in live cells becomes possible with genetically encoded probes.

Abstract 156: Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter

Abstract Dysregulation of signal transduction pathways is associated with cancer initiation, progression, and recurrence. The PI3K/Akt signaling pathway has been extensively investigated as a therapeutic target due to its mechanistic association with several hallmarks of cancer. Studying dynamic changes in kinase activity can be difficult, and assays to measure these changes in live cells in a physiologically relevant environment are lacking. Standard approaches to monitoring Akt kinase activity are limited to end point assays which lack the ability to monitor the effects of treatments over time. Here we demonstrate the utility of the Incucyte® Kinase Akt Lentivirus Reagent, encoding a kinase translocation reporter based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent nuclear protein to denote the nuclear/cytoplasmic boundary. To demonstrate inhibition of Akt, A549 cells stably expressing the reporter were treated with compounds targeting the PI3K/Akt kinase pathway, including allosteric Akt inhibitors MK2206 and API-1, competitive Akt inhibitors AZD5363 and Ipatasertib, and upstream PI3K kinase inhibitors LY294002 and PI-103. Quantification of treatment responses using the Incucyte® Live Cell Analysis System showed concentration-dependent inhibition of Akt activity for all compounds with varying kinetic profiles over 24 hours. To study activation of Akt, HeLa cells were first cultured in the absence of serum to reduce Akt activity. After 4 hours of incubation in serum-free conditions, the cells were treated with either epidermal growth factor (EGF) or recombinant insulin-like growth factor (R3-IGF-1) to activate Akt. An increase in Akt activity was observed for both treatments. While R3-IGF-1-induced activation was sustained over the 12-hour time course, activation by EGF diminished over time. Cell lines with mutations in the tumor suppressor PTEN showed no response to serum starvation or activation with EGF or R3-IGF-1. In addition to monitoring Akt activity over time, the integrated red nuclear restricted protein of the reporter enables concurrent measurements of proliferation. The selective Akt inhibitor MK2206 decreased Akt activity in a similar concentration-dependent manner in both T-47D and MDA-MB-231 cell lines. In contrast, measurements of red object count from the same cells reveal differential effects of MK2206 on proliferation between the two cell lines, with concentration-dependent inhibition of T-47D cell growth but little effect on MDA-MB-231 cells. Overall, these data highlight the utility of the Incucyte® Kinase Akt Lentivirus Reagent to provide valuable kinetic measurements of Akt activity using live cells within a physiologically relevant environment. Citation Format: John N. Rauch, Susan K. Foltin, Libuse Oupicka, Matthew Dilsaver, Grigory S. Filonov, Gillian Lovell, Jasmine Trigg, Cicely L. Schramm. Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 156.

Improved Red Fluorescent Redox Indicators for Monitoring Cytosolic and Mitochondrial Thioredoxin Redox Dynamics.

Thioredoxin (Trx) is one of the major thiol-dependent antioxidants in living systems. The study of Trx functions in redox biology was impeded by the lack of practical tools to track Trx redox dynamics in live cells. Our previous work developed TrxRFP1, the first genetically encoded fluorescent indicator for Trx redox. In this work, we report an improved fluorescent indicator, TrxRFP2, for tracking the redox of Trx1, which is primarily cytosolic and nuclear. Furthermore, because mitochondria specifically express Trx2, we have created a new genetically encoded fluorescent indicator, MtrxRFP2, for the redox of mitochondrial Trx. We characterized MtrxRFP2 as a purified protein and used subcellularly localized MtrxRFP2 to image mitochondrial redox changes in live cells.

Endpoint and Kinetic Approaches for Assessing Transfection Efficacy in Mammalian Cell Culture.

The efficacy of transfection reagents and nanoparticles is often assessed by measuring levels of expressed reporter protein. Fluorescence and luminescence based assays provide sensitive, quantifiable and repeatable approaches. The genes expressing reporter protein can be integrated into the cells to create stable reporter cell lines or can be expressed from a transfected plasmid. Green fluorescent protein, luciferase, and secreted alkaline phosphatase are well-established reporters with versatile applications. Monitoring changes in live cells during and after transfection offer opportunities to reveal related mechanisms, efficacy, and bottlenecks of transfection.In this chapter, we describe the experimental setup and considerations for in vitro screening of delivery vectors. This can further be extended to measurements in reporter cell lines.

GEM-IL: A highly responsive fluorescent lactate indicator

SummaryLactate metabolism has been shown to have increasingly important implications in cellular functions as well as in the development and pathophysiology of disease. The various roles as a signaling molecule and metabolite have led to interest in establishing a new method to detect lactate changes in live cells. Here we report our development of a genetically encoded metabolic indicator specifically for probing lactate (GEM-IL) based on superfolder fluorescent proteins and mutagenesis. With improvements in its design, specificity, and sensitivity, GEM-IL allows new applications compared with the previous lactate indicators, Laconic and Green Lindoblum. We demonstrate the functionality of GEM-IL to detect differences in lactate changes in human oncogenic neural progenitor cells and mouse primary ventricular myocytes. The development and application of GEM-IL show promise for enhancing our understanding of lactate dynamics and roles.

A Luminescence Assay to Quantify Cell Viability in Real Time.

Comprehensive understanding of cellular responses to changes in the cellular environment or by drug treatment requires time-dependent analysis ranging from hours to several days. Here, we describe a sensitive, nonlytic live-cell assay that allows continuous or 'real-time' monitoring of cell viability, growth, and cytotoxicity over an extended period of time. We illustrate the use of the assay for small drug molecule and antibody-dependent cytotoxicity studies using cancer cells in 384-well plates. We show that the ability to measure changes in live cells over time provides instantaneous information on the biological status of the cells, information about the mode of action of the drug, and offers an added advantage of preserving the cells for multiplexing with downstream applications.

X-ray structurally characterized quinoline based fluorescent probes for pH sensing: Application in intracellular pH imaging; DFT calculations and fluorescent labelling

Quinoline based fluorescent probes QNOH-MO and QNOH-OME were synthesized, and their photo-physical properties were carefully investigated. The probes were well characterized by 1H-NMR, 13C-NMR, ESI-MS, and single-crystal X-ray structure analysis. QNOH-MO and QNOH-OME can identify small changes in pH by colorimetric and fluorescence methods. In low pH range, these probes display a red fluorescence in both live and fixed cells. However, with increasing pH, the observed red fluorescence decreased with a simultaneous increase in green fluorescence. The pH-dependent switching from red to green fluorescence is well supported by theoretical calculations and 1H NMR titration experiments. Furthermore, the ability of these probes to image pH changes in live cells was also scrutinized. Moreover, QNOH-OME was successfully utilized for fluorescent labelling of cholesterol molecule which is an important component of biological membranes and is used in the liposomal formulation for drug delivery applications. Additionally, QNOH-MO was quaternized to induce an intrinsic cationic property to the molecule. The cationic probe QNOH-MO-CA exhibited improved water solubility and pH sensing efficiency.

Dynamic Signatures of Lipid Droplets as New Markers to Quantify Cellular Metabolic Changes.

The metabolic properties of live cells are very susceptible to intra- or extracellular perturbations, making their measurements challenging tasks. We show that the dynamics of lipid droplets (LDs) carry information to measure the lipid metabolism of live cells. Coherent anti-Stokes Raman scattering microscopy was used to statistically quantify LD dynamics in living cells in a label-free manner. We introduce dynamic signatures of cells derived from the LD displacement, speed, travel length, and directionality, which allows for the detection of cellular changes induced by stimuli such as fluorescent labeling, temperature change, starvation, and chemical treatment. Histogram fittings of the dynamic signatures using log-normal distribution functions provide quantification of changes in cellular metabolic states. The LD dynamics also enable separation of subpopulations of LDs correlated with different functions. We demonstrate that LD dynamics measured by chemical imaging are new markers to quantify the metabolic changes in live cells.

Mitochondria determine the sequential propagation of the calcium macrodomains revealed by the super-resolution calcium lantern imaging.

Despite the wide application of super-resolution (SR) microscopy in biological studies of cells, the technology is rarely used to monitor functional changes in live cells. By combining fast spinning disc-confocal structured illumination microscopy (SD-SIM) with loading of cytosolic fluorescent Ca2+ indicators, we have developed an SR method for visualization of regional Ca2+ dynamics and related cellular organelle morphology and dynamics, termed SR calcium lantern imaging. In COS-7 cells stimulated with ATP, we have identified various calcium macrodomains characterized by different types of Ca2+ release from endoplasmic reticulum (ER) stores. Finally, we demonstrated various roles of mitochondria in mediating calcium signals from different sources; while mitochondria can globally potentiate the Ca2+ entry associated with store release, mitochondria also locally control Ca2+ release from the neighboring ER stores and assist in their refilling processes.