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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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 &#181;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

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Bioengineering

4.0K 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

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10&#8211;60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...