Myoglobin is a protein that is expressed quite unevenly among different cell types. Nevertheless, it has been widely acknowledged that the Fe3+ state of myoglobin, metmyoglobin (metMb) has a broad functional role in metabolism, oxidative/nitrative regulation and gene networks. Accordingly, real-time monitoring of oxygenated, deoxygenated and metMb proportions- or, more broadly, of the mechanisms by which metMb is formed, presents a promising line of research. We had previously introduced a Förster resonance energy transfer (FRET) method to read out the deoxygenation/oxygenation states of myoglobin, by creating the targetable oxygen (O2) sensor Myoglobin-mCherry. In this sensor, changes in myoglobin absorbance features that occur with lost O2 occupancy -or upon metMb production control the FRET rate from the fluorescent protein to myoglobin. When O2 is bound, mCherry fluorescence is only slightly quenched, but if either O2 is released or met is produced, FRET will increase- and this rate competing with emission reduces both emission yield and lifetime. Nitric oxide (NO) is an important signal (but also a toxic molecule) that can oxidize myoglobin to metMb with absorbance increases in the red visible range. mCherry thus senses both met and deoxygenated myoglobin, which cannot be easily separated at hypoxia. In order to dissect this, we treat cells with NO and investigate how the Myoglobin-mCherry lifetime is affected by generating metMb. More discriminatory power is then achieved when the fluorescent protein EYFP is added to Myoglobin-mCherry, creating a sandwich probe whose lifetime can selectively respond to metMb while being indifferent to O2 occupancy.
The biological relevance of nitric oxide (NO) in cells to processes of signaling, metabolic regulation, and disease treatment has become abundantly clear. NO or reactive oxygen species (ROS) can oxidize myoglobin to the met state (metMb; the Fe3+ state of myoglobin), a change accompanied with an altered absorbance profile in the visible region. Recent studies show that metMb has a broad functional role in metabolic pathways, oxidative/nitrative regulation and gene networks of many cells. Thus, real-time monitoring of the different charge states of myoglobin is a promising field of research. We previously introduced a Förster resonance energy transfer (FRET) sensor, EYFP-Myoglobin-mCherry, to measure the deoxygenation, oxygenation and met states of myoglobin, creating a simultaneous oxygen (O2) and NO sensor. In this sandwich probe, the mCherry binary chimera lifetime responds to oxygenated vs. deoxygenated myoglobin, while the yellow fluorescent protein (YFP) lifetime selectively responds to metMb (while indifferent to O2 concentration). We now use Citrine, a more robust YFP, in place of EYFP and append a mitochondrial targeting peptide sequence to specifically target mitochondria. We use fluorescence lifetime imaging (FLIM) of this mtCitrine-Myoglobin-mCherry sandwich probe while monitoring both oxygenation level and NO-induced met formation in mitochondria of mouse embryonic fibroblasts. We also test the NO response of Citrine alone to verify that the met sensitivity is specific to the Mb sandwich probe and not Citrine alone.
Fluorescence lifetime imaging (FLIM) of Myoglobin (Myo)-mCherry, is used for sensing oxygen partial pressure (pO2) in the intracellular environment. Herein, we present the potential sources of lifetime error such as sample oversaturation or dimeric Myo-mCherry configurations resulting in self-quenching fluorophores. We also provide a correction protocol for Myo-mCherry expression, adjusting parameters to account for second harmonic generation (SHG) components and dark counts that result in accurate mean lifetime values and pO2 in the cellular environment.
Molecular oxygen is an important reporter of metabolic and physiological status at the cellular and tissue level, and its concentration is used for the evaluation of many diseases (e.g.: cancer, coronary artery disease). The development of accurate and quantitative methods to measure O2 concentration ([O2]) in living cells, tissues and organisms is challenging and is subject of intense research. We developed a protein-based, fluorescent oxygen sensor that can be expressed directly in cells to monitor [O2] in the intracellular environment. We fused Myoglobin (Myo), a physiological oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence resonance energy transfer (FRET) pair, Myo-mCherry. The changes in the spectral properties of Myoglobin upon oxygen binding result in changes of the FRETdepleted emission intensity of mCherry, and this effect is detected by monitoring the fluorescence lifetime of the probe. We present here the preparation and characterization of a series of Myo-mCherry variants and mutants that show the versatility of our protein-based approach: the dynamic range of the sensor is tunable and adaptable to different [O2] ranges, as they occur in vitro in different cell lines, the probe is also easily targeted to subcellular compartments. The use of fluorescence overcomes the most common issues of data collection speed and spatial resolution encountered by currently available methods for O2-monitoring. By using Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we can map the oxygenation level of cells in vitro, providing a quantitative assessment of [O2].
The extraction of fluorophore lifetimes in a biological sample provides useful information about the probe environment that is not readily available from fluorescence intensity alone. Cell membrane potential, pH, concentration of oxygen ([O2]), calcium ([Ca2+]), NADH and other ions and metabolites are all regularly measured by lifetime-based techniques. These measurements provide invaluable knowledge about cell homeostasis, metabolism and communication with the cell environment. Fluorescence lifetime imaging microscopy (FLIM) produces spatial maps with time-correlated singlephoton counting (TCSPC) histograms collected and analyzed at each pixel, but traditional TCSPC analysis is often hampered by the low number of photons that can reasonably be collected while maintaining high spatial resolution. More important, traditional analysis fails to employ the spatial linkages within the image. Here, we present a different approach, where we work under the assumption that mixtures of a global set of lifetimes (often only 2 or 3) can describe the entire image. We determine these lifetime components by globally fitting precise decays aggregated over large spatial regions of interest, and then we perform a pixel-by-pixel calculation of decay amplitudes (via simple linear algebra applied to coarser time-windows). This yields accurate amplitude images (Decay Associate Images, DAI) that contain stoichiometric information about the underlying mixtures while retaining single pixel resolution. We collected FLIM data of dye mixtures and bacteria expressing fluorescent proteins with a two-photon microscope system equipped with a commercial single-photon counting card, and we used these data to benchmark the gDAI program.
Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration ([O2]) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous [O2] within the intracellular environments. The mitochondrial [O2] at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria.
The precise location and nature of the artery water permeability barrier is unknown.
Water imaging in arteries with femtosecond coherent anti-Stokes Raman scattering microscopy
reveals that this barrier is formed by the endothelial basolateral membrane.
Fluorescence lifetime imaging microscopy is a technique in which the fluorescence lifetime(s) of a fluorophore
is measured at each spatially resolvable element of a microscope image. Imaging of fluorescence lifetimes enables
biochemical reactions to be followed at each microscopically resolvable location within the cell. FLIM has thus become
very useful for biomedical tissue imaging. Global analysis [1] is a method of recovering fluorescence decay parameters
from either time-resolved emission spectra to yield Decay-Associated Spectra [2], or equivalently, from FLIM datasets
to yield Decay-Associated Images. Global analysis offers a sensitive and non-invasive probe of metabolic state of
intracellular molecules such as NADH. Using prior information, such as the spatial invariance of the lifetime of each
fluorescent species in the image, to better refine the relevant parameters, global analysis can recover lifetimes and
amplitudes more accurately than traditional pixel-by-pixel analysis. Here, we explain a method to analyze FLIM data so
that more accurate lifetimes and DAIs can be computed in a reasonable time. This approach involves coupling an
iterative global analysis with linear algebraic operations. It can be successfully applied to image, e.g. metabolic states of
live cardiac myocytes, etc.
Fluorescence imaging in situ may provide highly specific identification of cell types and altered metabolic activity near the surface of tissue. Most approaches to developing the necessary analytical framework for quantitative 3-D use are based on numerical solutions of some form of transport equation. These are highly computer-intensive and can only be carried out for specified parameters. We apply a random walk model for photon migration which enables us to find an exact expression for the frequency-dependent fluorescent signal emitted from the site of a single fluorophore. Our general expression allows for broad variation of the degree of absorptivity, and is potentially important in providing a basis for the development of fluorescence image reconstruction algorithms.
We describe a new optical low-coherence reflectometer (interferometer) for depth profiling and lateral scanning without moving parts which can also be employed as a stationary FT-IR spectrometer. The reflectometer covers a range of 0.45 mm and 1 mm in the depth and lateral dimensions, respectively. The entire depth range is recorded simultaneously in one scan using a cooled 16-bit CCD camera; the lateral dimension is covered by scanning the probe beam sequentially across the sample with an acousto- optic deflector. The frequency shift generated by this deflector and an additional one placed in the reference arm creates an AC heterodyne signal with a frequency of 2kHz. Since the CCD camera cannot record the AC signal directly, a special readout scheme is employed. Stationary imaging was demonstrated using an artificial phantom. Using the same interferometer configured as a stationary FT-IR spectrometer, we measured the emission spectrum of a LED with a resolution of 0.74 nm at a central wavelength of 820 nm. We discuss the performance of the stationary CCD imaging system and compare it to that of a single-detector system employing moving parts.
In most of the optical methods proposed for imaging an absorbing object embedded in a turbid medium, data is collected using a single source and detector scanned mechanically across the surface of the medium. In this study we exploited destructive interference of diffusive photon- density waves originating from two sources to localize one absorbing (or fluorescent) object in a scattering medium. A frequency-domain instrument is described for scanning several laser- beam spots across the surface of a turbid medium using 1D (or 2D) acousto-optical deflectors and detecting the signals with a gated, intensified CCD camera at a modulation frequency of 246 MHz. The localization of multiple objects arranged in the form of a spatial grating was investigated theoretically with an analytic model by combining the magnitude and phase of the signals detected from the objects. A novel grating pattern comprising several destructively interfering lines, which acts as spatial frequency filter, is discussed. The results were compared with those obtained using a single-source/single-detector scanning configuration. We show that the FWHM (full-width half-maximum) of the signal detected using the single- source/single-detector configuration establishes a limiting spatial scale over which multiple objects can be resolved. Beyond this limit the resolution can only be increased under severe penalty of contrast and signal loss.
The authors studied the use of destructive interference of two diffusive photon-density waves for localization of an absorbing body and a fluorescent probe embedded in a scattering medium. The effect of the position of the embedded objects on the magnitude and phase of the light re-emitted from the medium was evaluated theoretically and experimentally. The objectives, accomplished with an asymmetrical laser-beam arrangement, were to reduce sensitivity to absorbing bodies located in superficial layers, while maintaining sensitivity to those lying deeper; and to establish a confined region of maximum sensitivity in which the distance of an absorbing body could be determined via phase measurement. Intensity and phase data were acquired with a modified frequency-domain spectrometer at modulation frequencies up to 600 MHz. Fluorescent probes were spatially localized with a symmetrical laser-beam arrangement. Magnitude and phase images acquired with a gated intensified CCD camera further defined the probe location. Simulations and experiments show potential applications to imaging.
The authors outline some examples of the advantages found in subdividing overall quenching into heterogeneous contributions. Subdivision is accomplished by overdetermination (global) and association (DAS, decay associated spectral) methods. In some cases, the subdivision of fluorescence leads to the unique identification of different fluorophores in different sites. Alternatively, the recovered components may reflect conformational heterogeneity at each site. For intrinsic protein fluorescence, it is often noted in the literature that single Trp proteins may be multiexponential. Genetic substitution in multi-Trp proteins, however, often leads to very strong (if not complete) lifetime-to-Trp assignment. Even if a single Trp experiences two or more microenvironments, it can be a useful reporter. The linkage of multiple lifetimes and amplitudes to changes in global conformation often reveals a more `sensitive' subpopulation or lifetime component that becomes a better indicator for important conformational states than aggregate intensity can provide. This has proven useful in studying pH transitions of proteins both in solution and embedded in membranes. Energy transfer is particularly useful in differentiating sites at different distances. Further, the disclosure of heterogeneity in distance is clearly superior to the reporting of a mean distance. This report surveys several systems that have been examined via emission DAS techniques, showing how each protein is better understood when viewed in terms of discrete spectral contributions. We conclude with an overview and some details about our construction of an EDAS (excitation-DAS) instrument; i.e., how excitation scans can be incorporated into a time-resolved instrument.
The folding of proteins into a (single?) compact globular structure is an important mystery in contemporary biochemistry. It has even been referred to as the "second half" of the genetic code (1), since folding often provides the ultimate limit to genetic engineering. A variety of spectroscopies have been applied over the years, and stopped-flow steady-state fluorescence has contributed its share of information. Time-resolved fluorescence, however, is a newcomer to this endeavor. Realizing that time-correlated single photon counting (TCSPC) can provide lifetime resolution with only a few thousand photons, Brand & coworkers began to examine HLADH lifetimes during denaturation nearly a decade ago (2). The arc-lamp based technology used then was adequate to resolve changes requiring many minutes, and the early inclusion of global analysis in the process then called "KINDK" (3) extended the accuracy available. It was clear then that the advent of high repetition rate sources such as the modelocked, cavity-dumped dye laser would lead to improved accuracy and faster collection times. The HLADH system was revisited as soon as such technology was inhand (4).
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