Name: autofluorescence imaging to evaluate cellular metabolism
Uploaded: 2021-11-15T13:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Cellular Metabolism at JoVE.com

Funding sources include the Cancer Prevention and Research Institute of Texas (CPRIT RP200668) and Texas A&#38;M University. Figure 1&#160;was created with BioRender.com.

The authors have no conflicts of interest to disclose.

Autofluorescence intensity and lifetime imaging have been widely used to assess metabolism in cells21,55. FLIM is high resolution and therefore resolves single cells, which is important for cancer studies because cellular heterogeneity contributes to tumor aggression and drug resistance7,39,41,44,45<su...

Metabolism is the cellular process of producing energy. Cellular metabolism encompasses multiple pathways, including glycolysis, oxidative phosphorylation, and glutaminolysis. Healthy cells use these metabolic pathways to generate energy for proliferation and function, such as the production of cytokines by immune cells. Many diseases, including metabolic disorders, cancer, and neurodegeneration, are characterized by altered cellular metabolism1. For example, some cancer cell types have elevated rates of glycolysis, even in the presence of oxygen, to generate molecules for the synthesis of nucleic acids, proteins, and lipids2,3. This phenomenon, known as the Warburg effect, is a hallmark of many cancer types, including breast cancer, lung cancer, and glioblastomas4. Because of the alterations of cellular metabolism associated with cancer progression, cellular metabolism can be a surrogate biomarker for drug response5,6. Moreover, understanding drug efficacy at a cellular level is crucial as cell heterogeneity can lead to differing drug responses in individuals7,8.
Technologies that identify and quantify changes in cellular metabolism are essential for studies of cancer and drug response. Chemical and protein analyses are used to evaluate the metabolism of cells or tissues but lack single-cell resolution and spatial information. Metabolic plate reader-based assays can measure pH and oxygen consumption in the sample over time and the subsequent metabolic perturbation by chemicals. The pH can be used to calculate the extracellular acidification rate (ECAR), which provides an insight into the glycolytic activity of the cells9. Whole-body imaging methods, including 2-[fluorine-18] fluoro-D-glucose positron emission tomography (FDG PET) and magnetic resonance spectroscopy (MRS), are noninvasive imaging modalities used clinically to identify tumor recurrence and drug efficacy through metabolic measurements10,11,12,13,14.
FDG-PET images the tissue uptake of FDG, a radiolabeled glucose analog. Increased uptake of FDG-PET by tumors relative to surrounding tissue is due to the Warburg effect12,13. MRS images common nuclei of molecules used for metabolism, such as 13C and 31P, and can obtain dynamic information about how metabolism changes in response to stimuli, such as exercise or eating14. Although FDG-PET and MRS can be used clinically, these technologies lack the spatial resolution to resolve intratumoral heterogeneity. Likewise, oxygen consumption measurements are made on a bulk population of cells. Autofluorescence imaging overcomes the spatial resolution obstacle of these technologies and provides a noninvasive method of quantifying cellular metabolism.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63282/63282fig01v2.jpg" /> 
Figure 1: NADH and FAD in common metabolic pathways. NADH and FAD are coenzymes used in glycolysis, the Krebs cycle, and the electron transport chain. Autofluorescence imaging of these molecules provides information about cellular metabolism. <a href="https://www.jove.com/files/ftp_upload/63282/63282fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions, including glycolysis, oxidative phosphorylation, and glutaminolysis (Figure 1). Both NAD(P)H and FAD are autofluorescent and provide endogenous contrast for fluorescence imaging1,15. NADPH has similar fluorescent properties to NADH. Because of this, NAD(P)H is often used to represent the combined signal of NADH and NADPH2,16.
Fluorescence lifetime imaging (FLIM) quantifies the fluorescence lifetime or the time for which a fluorophore is in the excited state. Fluorescence lifetimes are responsive to the microenvironment of the fluorophores and provide information about cellular metabolism17. NAD(P)H and FAD can exist within cells in either protein-bound or free conformations, each of which has a different lifetime. Free NAD(P)H has a shorter lifetime than protein-bound NAD(P)H; conversely, free FAD has a longer lifetime than bound FAD18,19. The lifetimes and lifetime component weights can be quantified from fluorescence lifetime decay data through Eq. (1)20:
I(t) = &#945;1e-t/&#964;1&#160;+ &#945;2e-t/&#964;2&#160;+ C&#160; (1)
Eq (1) represents the normalized fluorescence intensity as a function of time. The &#945;1 and &#945;2 in this equation represent the proportional components of short and long lifetimes (&#945;1+ &#945;2=1), respectively, &#964;1 and &#964;2 represent the short and long lifetimes, respectively, and C accounts for background light7,20. The amplitude-weighted lifetime, represented here as &#964;m, is calculated using Eq. (2).
&#964;m= &#945;1&#964;1+ &#945;2&#964;2&#160; (2)
A mean lifetime can be computed by averaging &#34;t&#34; over the intensity decay of the fluorophore, which for a two-exponential decay is shown by Eq. (3)17,21.
&#964;*m= (&#945;1&#964;12+ &#945;2&#964;22)/ (&#945;1&#964;1+ &#945;2&#964;2)&#160; (3)
A fluorescence intensity image can be computed from the lifetime image by integrating the fluorescence lifetime decay. Autofluorescence imaging is a nondestructive and label-free method that can be used to characterize the metabolism of live cells at a subcellular resolution. The optical redox ratio provides an optical analog metric of the chemical redox state of the cell and is calculated as the ratio of NAD(P)H and FAD intensities. Although the formula for calculating the optical redox ratio is not standardized22,23,24,25, it is defined here as the intensity of FAD over the combined intensities of NAD(P)H and FAD. This definition is used because the summed intensity in the denominator normalizes the metric between 0 and 1, and the expected result of the cyanide inhibition is a decrease in the redox ratio. The fluorescence lifetimes of free NAD(P)H and FAD provide insight into changes in the metabolic solvent microenvironment, including pH, temperature, proximity to oxygen, and osmolarity17.
Changes in the fluorescence lifetime of the bound fractions of NAD(P)H and FAD can indicate metabolic pathway utilization and substrate-specific metabolism26. Component weights can be interpreted for changes in the free to the bound fraction of the coenzymes18,19. Altogether, these quantitative autofluorescence lifetime metrics allow the analysis of cellular metabolism, and autofluorescence imaging has been used for identifying neoplasms from normal tissues27,28, characterizing stem cells29,30, evaluating immune cell function31,32,33,34,35, gauging neurological activity36,37,38, and understanding drug efficacy in cancer types such as breast cancer and head and neck cancer21,39,40,41,42. High-resolution autofluorescence imaging can be combined with image segmentation for single-cell analysis and quantification of intrapopulation heterogeneity43,44,45,46,47.
NAD(P)H and FAD can be imaged on single-photon or multiphoton fluorescence microscopes configured for intensity or lifetime imaging. For single-photon microscopes, NAD(P)H and FAD are typically excited at wavelengths of 375-405 nm and 488 nm, respectively, due to common laser sources at these wavelengths48. In two-photon fluorescence excitation, NAD(P)H and FAD will excite at wavelengths of approximately 700 to 750 nm and 700 to 900 nm, respectively15,49. Once the fluorophores are excited, NAD(P)H and FAD emit photons at wavelengths between ~410 nm to ~490 nm and ~510 nm to ~640 nm, respectively15. The NAD(P)H and FAD maxima emission wavelengths are approximately 450 nm and 535 nm, respectively48.
Because of their different excitation and emission wavelengths, the fluorescence of the two metabolic coenzymes can be spectrally isolated. An understanding of the spectral characteristics of NAD(P)H and FAD is necessary for the design and optimization of autofluorescence imaging protocols. Cyanide is an electron transport chain (ETC) complex IV inhibitor. The effects of cyanide on cellular metabolism and the autofluorescence intensities and lifetimes of NAD(P)H and FAD within cells are well characterized27,40. Therefore, a cyanide perturbation experiment is an effective means of validating NAD(P)H and FAD imaging protocols. A successful cyanide experiment provides confidence that the NAD(P)H and FAD imaging protocol can be used to assess the metabolism of unknown groups or perturbations.

<table><tbody><tr><td>2-deoxy-d-glucose (2-DG)</td><td>Sigma</td><td>AC111980000; AC111980010; AC111980050; AC111980250</td><td></td></tr><tr><td>Antibiotic Antimicrobial (pen-strep)</td><td>Gibco</td><td>15240096</td><td></td></tr><tr><td>Cell Samples</td><td>American Type Culture Collection</td><td>N/A</td><td>MCF-7 cancer line</td></tr><tr><td>CellProfiler</td><td>Broad Institute</td><td>N/A</td><td>Image analysis software</td></tr><tr><td>Conical Tube</td><td>VWR</td><td>89039-664</td><td>15 mL conical tube</td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>11965092</td><td>Culture media</td></tr><tr><td>FAD dichroic mirror</td><td>Semrock</td><td>FF495-Di03-25x36</td><td>495 nm</td></tr><tr><td>FAD emission filter</td><td>Semrock</td><td>FF01-550/88-25</td><td>550/88 nm</td></tr><tr><td>FAD excitation filter</td><td>Semrock</td><td>FF01-458/64-25</td><td>458/64 nm</td></tr><tr><td>FBS</td><td>ThermoFisher</td><td>16000036</td><td></td></tr><tr><td>Fluorescence Lifetime Microscope</td><td>3i</td><td>N/A</td><td></td></tr><tr><td>Glass bottom dish</td><td>MatTek Corp</td><td>P35G-1.0-14-C</td><td></td></tr><tr><td>Multiphoton Laser</td><td>Coherent</td><td>N/A</td><td>2P Coherent Laser, Tunable 680 nm-1080 nm</td></tr><tr><td>NAD(P)H dichroic mirror</td><td>Semrock</td><td>FF409-Di03-25x36</td><td>409 nm</td></tr><tr><td>NAD(P)H emission filter</td><td>Semrock</td><td>FF02-447/60-25</td><td>447/60 nm</td></tr><tr><td>NAD(P)H excitation filter</td><td>Semrock</td><td>FF01-357/44-25</td><td>357/44 nm</td></tr><tr><td>PBS</td><td>ThermoFisher</td><td>70011044</td><td></td></tr><tr><td>Potassium Cyanide</td><td>Sigma-Aldrich</td><td>380970</td><td></td></tr><tr><td>SlideBooks 6</td><td>3i</td><td>N/A</td><td>Image acquisition software</td></tr><tr><td>SPCImage</td><td>Becker &amp;amp; Hickl GmbH</td><td>N/A</td><td>Fluorescence lifetime analysis software</td></tr><tr><td>Stage Top Incubator</td><td>okoLab</td><td>N/A</td><td></td></tr><tr><td>Trypsin</td><td>Biosciences</td><td>786-262</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5128</td><td></td></tr><tr><td>YG beads</td><td>Polysciences</td><td>19096-2</td><td>Yg microspheres (20.0 &amp;micro;m)</td></tr></tbody></table>

autofluorescence imaging to evaluate cellular metabolism

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.

The epithelial breast cancer cell line, MCF-7, was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. For fluorescence imaging, the cells were seeded at a density of 4 &#215; 105 cells per 35 mm glass-bottom imaging dish 48 h before imaging. The cells were imaged before and after cyanide treatment using the protocols stated above. The goal of the cyanide experiment is to confirm spectral isolation of NAD(P)H and FAD fluorescence and validate the imaging system and a...

Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Cellular Metabolism at JoVE.com

Autofluorescence Imaging to Evaluate Cellular Metabolism

This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced ...

autofluorescence-imaging-to-evaluate-cellular-metabolism

Research

JoVE Journal

Bioengineering

4.1K Views.  Texas A&M University-College Station. This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.

Video: Autofluorescence Imaging to Evaluate Cellular Metabolism

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting endogenous fluorescence. Viscosity is measured using a fluorescent molecular rotor.

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and ...

Cancer Research

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Biology

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection

Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the infested human or animal in the long term and, thus, impact agricultural and socio-economic outcomes. However, parasite-driven immune-regulatory effects have been described, with potential therapeutic relevance for autoimmune diseases. While the metabolism in both the host and parasites contributes to their defense and is the basis for nematode survival in the intestine, it has remained largely understudied due to a lack of adequate technologies. We have developed and applied NAD(P)H fluorescence lifetime imaging to explanted murine intestinal tissue during infection with the natural nematode Heligmosomoides polygyrus to study the metabolic processes in both the host and parasites in a spatially resolved manner. The exploitation of the fluorescence lifetime of the co-enzymes nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, which are preserved across species, depends on their binding status and the binding site on the enzymes catalyzing metabolic processes. Focusing on the most abundantly expressed NAD(P)H-dependent enzymes, the metabolic pathways associated with anaerobic glycolysis, oxidative phosphorylation/aerobic glycolysis, and NOX-based oxidative burst, as a major defense mechanism, were distinguished, and the metabolic crosstalk between the host and parasite during infection was characterized.

NADPH Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection

The present protocol describes the NAD(P)H fluorescence lifetime imaging of an explanted murine intestine infected with the natural parasite Heligmosomoides polygyrus, which allows one to investigate metabolic processes both in host and parasite tissues in a spatially resolved manner.

Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the ...

Immunology and Infection

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Author Spotlight: Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...

An Introduction to Cell Metabolism

In cells, critical molecules are either built by joining together individual units like amino acids or nucleotides, or broken down into smaller components. Respectively, the reactions responsible for this are referred to as anabolic and catabolic. These reactions require or produce energy typically in the form of a &#8220;high-energy&#8221; molecule called ATP. Together, these processes make up &#8220;Cell Metabolism,&#8221; and are hallmarks of healthy, living cells.JoVE&#8217;s introduction to cell metabolism briefly reviews the rich history of this field, ranging from early studies on photosynthesis to more recent discoveries pertaining to energy production in all cells. This is followed by a discussion of some key questions asked by scientists studying metabolism, and common methods that they apply to answer these questions. Finally, we&#8217;ll explore how current researchers are studying alterations in metabolism that accompany metabolic disorders, or that occur following exposure to environmental stressors.

Autofluorescence Imaging to Evaluate Cellular Metabolism

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Chapters in this video

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Cellular Redox Profiling Using High-content Microscopy

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

An Introduction to Cell Metabolism