Autofluorescent metabolic measurements of an optical redox ratio, NAD(P)H/FAD, have been utilized as a means of measuring cancer progression, treatment impact, subtype determination, and more. This optical redox ratio is traditionally measured through intensity with microscopy, but there is potential to adapt this technique for high throughput analysis using time-resolved flow cytometry with autofluorescence lifetime measurements. A fluorescence lifetime approach to these measurements allows for fluorophore concentration independent measurements that can provide new information to the field. The two fluorescent metabolites of interest that allow for redox analysis are NAD(P)H/NAD(P)+ and FADH2/FAD. Variations in the redox state and binding of these metabolites to their respective coenzymes, have been correlated to the cycles in which cells metabolize glucose into ATP, either oxidative phosphorylation (OXPHOS) or glycolysis. This technique will be used herein to study the metabolism of MCF-7 tamoxifen resistant and sensitive breast cancer cells using flow cytometry, first on a fluorescence intensity basis of the two metabolites. Our results show there is a clear shift towards an increased redox ratio for tamoxifen resistant cells, indicating a greater reliance on glycolysis as a means of metabolism. Future work will focus on adapting the intensity based redox ratio approach through high-throughput flow cytometry used here to a fluorescence lifetime based measurement.
Here we present a simple, high-throughput, time-resolved flow cytometer to detect changes in NAD(P)H autofluorescence lifetime. The lifetime of this metabolite is characterized by its binding state, when bound to its coenzyme the metabolite exhibits a longer lifetime (1 to 7 ns) indicating a preferential energy generation state of oxidative phosphorylation. A shorter lifetime, 0.1 to 1 ns, however, indicates an unbound coenzyme which has been correlated to the ATP energy pathway of glycolysis. The work here in is to demonstrate the capabilities of simple, time-resolved cytometry to show subtle changes in autofluorescence lifetime detection. Flow cytometry results are validated by the use of the Agilent Seahorse HS Mini. Results include the method of ATP energy generation measured by the Seahorse as a comparison to flow cytometry lifetime shifts. Measurements were made using an estrogen receptor positive breast cancer cell line and the same cell line under developed Tamoxifen resistance. The outcome of this work is a map of the metabolic profile of tamoxifen resistance in breast cancer.
We present a cytometric and metabolic screening tool that measures shifts in NAD(P)H autofluorescence and autofluorescence lifetimes from single cells based on metabolite-enzyme interactions. Short autofluorescence lifetimes of NAD(P)H (~0.1-1ns) indicate the metabolite is unbound from metabolic enzymes and the cell is favoring glycolysis for energy production. In contrast, longer autofluorescence lifetimes of NAD(P)H (~1-7ns) are an inference that the metabolite is bound to metabolic enzymes and the cell is respiring under oxidative phosphorylation. Using a simple time-resolved flow cytometer we are able to measure autofluorescence lifetimes of MCF-7 and T47D breast cancer cells, which we relate to metabolic changes within each cell line. In order to determine the resolution limits of our time-resolved instrument, we first treated cells under different conditions that directly alter the metabolic pathway that drives their energy production. We deprived cells of serum in their growth media, which drives the cell to utilize glycolysis as a metabolic pathway. By comparing normal to deprived cells, we were able to determine if our cytometry system is able to measure differences in the autofluorescence lifetimes. Results show a decrease in lifetime and autofluorescence intensity for both T47D and MCF7 following serum deprivation. Initial cytometric analysis illustrates consistent lifetime data with respect to fluorescence lifetimes and fluorescence intensities decreasing as expected. This study is a preliminary confirmation that our timeresolved cytometer can effectively detect autofluorescence signals, albeit with some limitations in lifetime resolution. Future work will include refinement of lifetime analysis, frequency domain approaches, and improving sensitivity.
Fluorescence lifetime microscopy (FLIM) and time-resolved cytometry (TRFC) are robust platforms that can resolve complex protein and cellular interactions. Flow cytometry has been a prominent staple in clinical and research for decades. In conventional form, flow cytometers can count cells and evaluate biophysical and biochemical attributes using fluorescence and inelastic scatter light. Cytometry has evolved beyond conventional paradigms are becoming polychromatic and mulitparametric apparatuses that can evaluate complex cellular interactions in real-time. A distancedependent technique known as Förster Resonance Energy Transfer, or FRET is a powerful quantitative tool that enables the ability to monitor binding interaction and morphological changes in the macro and microenvironment of cells. FRET measurements require sensitive instrumentation to capture and resolve subtle changes in biophysical and biochemical characteristics. TRFC captures a unique parameter known as fluorescence lifetime which is sensitive to microenvironmental changes. Past studies have demonstrated TRFC’s ability to resolve complex FRET interactions. Herein, we present the evolution of the TRFC modular platform that incorporates a microfluidic device. The microfluidic device in this contribution acoustically linearly focuses cells down the middle of the microcapillary, allowing for maximum optical excitation and optimizing optical geometries to maximize the capture of fluorescence.
Flow cytometers are invaluable tools that can quantitatively analyze and separate cells with respect to a cell’s biophysical and biochemical properties. Conventional cytometers collect these physical and chemical properties in the forms of inelastic light scatter and fluorescence. Specialized cytometers came to fruition after several advancements; smaller, more efficient photodetectors, tunable laser diodes, and the advent of microfluidics. Our work focuses on the latter topic. Microfluidic-based flow cytometry is robust in single cell and single molecule detection. Recent studies have leveraged significant quantitative analysis from multiplexing in phenotyping experiments, rare events in highcontent screening assays and sorting. Multiplexing requires multiple color channels to capture and resolve the presented spectral data. Color compensation is needed to resolve emission spectra overlap and becomes difficult when 10+ colors are used. Rare event detection requires large volumes of sample to the effect of 109 cells and greater. The task becomes time and resource consuming because conventional flow is limited by linear flow velocities (50,000 events/second) and requires extensive amounts of sheath fluid. Lastly, collecting these events by conventional flow requires careful separation by means of fluorescence activated cell sorting (FACS). Most cell sorters are capable of high yields but use piezoelectric transducers that are not as biocompatible as once thought. Herein we present a time-resolved acoustofluidic flow cytometer that can theoretically surpass the linear velocity constraint, use acoustic focus to elevate biocompatibility and reduce resource consumption and eliminate the need for multiple color channels.
Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. We exploit FRET to study the extent of activation of α4β1 integrin dimers expressed on the surface of leukocytes. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level.
Flow cytometry for single cell counting uses optical measurements to report multiple cell features such as cell morphology, cell phenotype, and microenvironmental changes. Time-resolved flow cytometry is a unique method that involves the detection of the average fluorescence lifetime as a cytometric parameter. Measuring the average fluorescence lifetime is helpful when discriminating between more than one emission signal from a single cell because of spectrally overlapping emission. In this contribution, we present preliminary measurements toward a study that advances simple time-resolved flow cytometry and introduces a technique to measure fluorescence lifetime values from single cells labeled with a Forster Resonance Energy Transfer (FRET) pair. Specifically, donor fluorophore fluorescein isothiocyanate (FITC) fluorescence lifetime is measured to identify its proximity to the acceptor fluorophore. We hypothesize that our time-resolved flow cytometry approach can resolve changes in FRET in order to study integrin structures on the surface of leukocyte cells. Our results show that FITC has an average lifetime of 4.2 +/-0.1 nsec, and an average fluorescence lifetime of 2.4 nsec +/-0.2 nsec when engaged in FRET. After the release of FRET (e.g. dequenched) the average fluorescence lifetime of FITC was measured to be 3.1 +/- 0.5 nsec. Phasor graphs reveal large distributions of fluorescence lifetimes on a per cell basis, suggesting the existence of multiple fluorescence lifetimes. These data suggest more than one integrin conformation occurs throughout the cell population. The impact of this work is the addition of quantitative information for FRET efficiency values and determination of FRET calculations using high-throughput data.
The long fluorescence lifetime of quantum dots (QDs) is not often utilized in high-throughput bioassays, despite of the potential for the lifetime to be an optimum parameter for multiplexing with spectrally overlapping excitable species that have short fluorescence lifetimes. The limitation of currently available instruments that can rapidly resolve complex decay kinetics of QDs contributes to this dearth. Therefore work in our laboratory is focused on developing unique and reliable frequency-domain flow cytometry (FDFC) systems as well as QDs applications where fluorescence dynamics are exploited. In this paper we demonstrate both by simulation and experimental validation, the viability of rapidly capturing the fluorescence lifetime of QDs from single QDs-labeled cells and microspheres by employing a home-built FDFC system. With FDFC theory we simulated measurements of long-lived QDs decays and evaluated the potential to discriminate multi-exponential decay profiles of QDs from typical cellular autofluorescence lifetimes. Our FDFC simulation work included calculations of fluorescence phase-shifts at multiple modulation frequencies extracted from square wave modulation signals (i.e. similar to heterodyning frequency-domain spectroscopy). Experimental work to support the result from our simulations involved acquiring measurements from real samples and processing them for multi-frequency phase shifts. Additionally the average excited-state lifetimes of QDs (streptavidin conjugated CdSe/Zns and oleic acid coated CdSxSe1-x/ZnS) measured were found to be greater than 15 ns. The average lifetime results were consistent with published literature values as well as verified with independent time domain measurements. This work opens the possibility of developing powerful bioassays using FDFC based on the long fluorescence lifetime of QDs.
Digital excited state lifetime measurements in cytometry were performed on multi-tagged Chinese Hamster Ovary
(CHO) cells in order to discriminate between spectrally overlapping fluorescent species. Fluorescence lifetime was
determined through digital Fourier analysis with a specialized data acquisition system subsequent to multi-frequency
intensity modulation by a solid-state laser excitation source. This work demonstrates that square wave modulation
coupled with digital lifetime signal processing can lead to separation of ethidium bromide (EB) and propidium
iodide (PI), in cells stained with both dyes.
By driving the square wave modulation of the laser at 2 MHz, we were able to access the multiple harmonics present
within that wave. In an offline analysis, the phase differences of scatter and fluorescence channels were examined at
each harmonic of the primary frequency. The phase difference revealed approximate fluorescence lifetimes of 27.1-ns and 13.0-ns for the EB and PI, respectively. Although the absolute lifetime of each species was not resolved to
high accuracy, this work shows a clear separation of the lifetime value calculated at each harmonic. The calculated
values that most closely corresponded to the single-dye and
multiple-dye average lifetimes were found at the
fundamental harmonic frequency (2 MHz) as well as the 4th harmonic (14MHz) frequency. At 2 and 14MHz the
average lifetime was 27.1ns and 13.0ns, respectively.
Autofluorescence emission is commonly measured in flow cytometry and is used as a negative control in protocols that
explore binding of exogenous fluorophores to cell receptors or other targets of interest. The presence of intrinsic
fluorophores however may burden complex cytometry applications. For example it may be difficult to resolve
fluorescence signals from multi-intensity and multi-color measurements when the de-convolved fluorescence in question
falls close to the autofluorescence background. One possible solution to intensity and spectral overlap problems in flow
cytometry is to acquire fluorescence decay kinetic measurements. To this end we focus on advancing time-dependent
flow cytometry and conduct measurements of endogenous fluorescence lifetime. Instrument developments to a phase-sensitive
flow cytometry (PSFC) system were coupled with lifetime measurements of intrinsic fluorophores from viable
cell samples. The average lifetime of >300,000 individual rat fibroblast cells was measured at discrete wavelengths
ranging from 457- to 785-nm using a 10-MHz intensity-modulated excitation beam. AC amplitude, DC, and phase-shift
were resolved and the average lifetime from excitable endogenous species was measured. The lifetime results ranged
from 1- to 6-ns over the broad spectral range. Cataloging lifetime values prefaces the use of phase-sensitive techniques
in more complex systems and provides a priori measurements necessary for PSFC filtering known lifetime signals from
Raman, or other emission and scattering events.
The increasing need for highly polychromatic approaches to flow cytometry, coupled with rapid technological advances,
have driven the design and implementation of commercial instruments that measure up to 19 parameters using multiple
lasers for excitation, an intricate optical filter/mirror arrangement, and analysis using fluorescence compensation
approaches. Although such conventional multiparameter flow cytometers have proven highly successful, there are
several types of analytical measurements that would benefit from higher density of spectral information and a more
flexible approach to spectral analysis including, but certainly not limited to: spectral deconvolution of overlapping
spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, cellular autofluorescence
characterization, and flow based Raman spectroscopy. For these purposes, we have developed a high resolution spectral
flow cytometer using an EMCCD camera with 1600 by 200 pixels, which is capable of detecting less than 200
fluorescein molecules with a spectral resolution of less than 3 nm. This instrument will enable high throughput
characterization of single cell or particle emission spectra. For proof of principle instrument operation, we have begun
characterization of intrinsic cellular autofluorescence, which is the major source of background for cell-based
fluorescence assays. Specifically, we will describe recent work on the high resolution spectral characterization of
autofluorescence for several commonly used cell types. Autofluorescence emission is known to cover over almost the
entire spectrum from 300 to nearly 800 nm. These emissions are attributed to flavins, elastin, Indolamine dimers and
trimers, NADH and collagen among other molecules. We will show that several unique autofluorescence spectra arise in
the different cell lines thereby suggesting the possibility of discrimination of cell types based on intrinsic fluorescence.
KEYWORDS: Tumors, Signal to noise ratio, Imaging systems, Near infrared, Optical imaging, Tissues, Luminescence, Signal detection, Nuclear imaging, Tissue optics
The cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) (c(KRGDf)), which is known to target v3 integrin, is dual-labeled with a radiotracer, 111indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, for continuous-wave (cw) imaging of v3 positive human M21 melanoma in xenografts. Twenty-four hours after administration of the dual-labeled peptide at a dose equivalent to 90 µCi of 111In and 5 nmol of near-infrared (NIR) dye, whole-body gamma scintigraphy and cw imaging was conducted. Image acquisition time was 15 min for the gamma scintigraphy images and 800 ms for the optical images acquired using an NIR sensitive intensified charge-coupled device. The results show that while the target-to-background ratio (TBR) of nuclear and optical imaging were similar for surface regions of interest and consistent with the origin of gamma and NIR radiation from a common targeted peptide, the signal-to-noise ratio (SNR) was significantly higher for optical than nuclear imaging. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets. While tomographic reconstructions are necessary to probe TBR, SNR, and contrast for interior tissues, this work demonstrates for the first time the direct comparison of molecular optical and planar nuclear imaging for surface and subsurface cancers.
Several phantom and in vivo small animal imaging studies have been performed to detect the re-emitted fluorescence signal arising from micro to pico molar concentrations of fluorophore by employing band-pass and band-rejection filters. However, elimination of the back-reflected excitation light still remains a major challenge for further reducing the noise floor in fluorescence imaging. Furthermore, despite the well-known deterioration of interference filter performance as the angle of incidence deviates from zero degrees, most studies do not employ collimated light optical design required for efficient excitation light rejection using interference filters. In this study, we measured quantities in frequency domain data for the combination of three-cavity interference and holographic super notch filters. To assess excitation leakage, the “out-of-band (S (λx ) )” to “in-band (S (λm ) - S (λx ) )” signal ratio, AC amplitude (IAC ), and phase delay (δ-δ*) measured from a gain modulated, intensified CCD imaging system with and without collimating optics was evaluated. The addition of collimating optics resulted in a reduction of 82% to 91% of the out-of-band to in-band ratio for the phantom studies and an increase of 1.4 to 3.7 times of the target-to-background ratio (T:B) for small animal studies.
The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the αvβ3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi’s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi’s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.
KEYWORDS: Tumors, Signal to noise ratio, Cameras, Optical imaging, In vivo imaging, Near infrared, Image quality, Imaging systems, Luminescence, Melanoma
Fluorescence-enhanced optical imaging measurements and conventional gamma camera images on human M21 melanoma xenografts were acquired for a "dual-modality" molecular imaging study. The avb3 integrin cell surface receptors were imaged using a cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) [c(KRGDf)] probe which is known to target the membrane receptor. The probe, dual-labeled with a radiotracer, 111Indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, was injected into six nude mice at a dose equivalent to 90mCi of 111In and 5 nanomoles of near-infrared (NIR) dye. A 15 min gamma scan and 800 millisecond NIR-sensitive ICCD optical photograph were collected 24 hours after injection of the dual-labeled probe. The image quality between the nuclear and optical data was investigated with the results showing similar target-to-background ratios (TBR) based on the origin of fluorescence and gamma emissions at the targeted tumor site. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets measured by surface regions of interest.
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