Near-infrared (NIR) fluorescence tomography of multiple fluorophores has previously been limited by the bandwidth of the NIR spectral regime and the broad emission spectra of most NIR fluorophores. We describe in vivo tomography of three spectrally overlapping fluorophores using fluorescence lifetime-based separation. Time-domain images are acquired using a voltage-gated, intensified charge-coupled device (CCD) in free-space transmission geometry with 750 nm Ti:sapphire laser excitation. Lifetime components are fit from the asymptotic portion of fluorescence decay curve and reconstructed separately with a lifetime-adjusted forward model. We use this system to test the in vivo lifetime multiplexing suitability of commercially available fluorophores, and demonstrate lifetime multiplexing in solution mixtures and in nude mice. All of the fluorophores tested exhibit nearly monoexponential decays, with narrow in vivo lifetime distributions suitable for lifetime multiplexing. Quantitative separation of two fluorophores with lifetimes of 1.1 and 1.37 ns is demonstrated for relative concentrations of 1:5. Finally, we demonstrate tomographic imaging of two and three fluorophores in nude mice with fluorophores that localize to distinct organ systems. This technique should be widely applicable to imaging multiple NIR fluorophores in 3-D.
Tau is a microtubule associated protein that is localized to the axon in neurons. During pathological conditions, including frontotemporal dementia (FTD), a shift in tau isoforms occurs that leads to enhanced expression of a form of tau with four (rather than three) microtubule binding repeats; this has been postulated to alter microtubule structure. Second harmonic generation (SHG) is a technique that allows the visualization of intact microtubules in axons of living neurons without the need for labeling or fixing. We examined how the presence of exogenous tau influences SHG in living neurons. Our results show that the presence of tau significantly enhances SHG, specifically in neuronal axons, despite the presence of tau throughout the entire cell. Our data also suggest that the presence or absence of the fourth microtubule binding repeat does not significantly alter tau's ability to enhance SHG. These results provide evidence that SHG is a useful, noninvasive tool to study tau-microtubule interactions in axons; further, it appears that tau overexpression, rather than specific isoforms, is the major contributor to tau-induced changes in axonal microtubule SHG signal.
The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.
A theoretical framework is presented that allows a lifetime based analysis of the entire temporal diffuse
fluorescence response curve from a turbid medium. Optimization studies using singular value decomposition
analysis show that direct time domain fluorescence reconstructions are optimally performed
using a few points near the peak and rise portions of the temporal response. It is also shown that the
initial portion of the fluorescent response curve offers superior contrast-to-noise performance, while the
late decay portions offer minimal cross-talk between multiple lifetime components.
Noninvasive molecular imaging of amyloid plaques in murine Alzheimer's disease models would accelerate drug
development and basic Alzheimer's research. Amyloid plaques differ from traditional fluorescent targets in size and
spatial distribution and therefore present a unique challenge for biomarker development and tomography. To study
imaging feasibility and establish biomarker criteria, we developed a digital mouse head model from a 100 &mgr;m-resolution,
digital, segmented mouse atlas1. The cortical region of the brain was filled with a spatially uniform distribution of
plaques that had different fluorescent properties from the surrounding brain tissue, similar to current transgenic mouse
models of Alzheimer's disease. Fluorescence was simulated with a Monte Carlo algorithm using different plaque
densities, detection geometries, and background fluorescence. Our preliminary results demonstrated that shielding
effects might require nonlinear reconstruction algorithms and that background fluorescence would seriously hinder
quantitative burden estimation. The Monte Carlo based approach presented here offers a powerful way to study the
feasibility of non-invasive imaging in murine Alzheimer's models and to optimize experimental conditions.
Fluorescence lifetime measurement is widely used in the biological sciences due to its inherent sensitivity and concentration independence. Frequency domain high-throughput plate readers and time-resolved energy transfer (TRET) plate readers are in common use and have been successful in a variety of applications ranging from basic biochemistry to drug discovery. Time-domain systems would have advantages due to their ability to distinguish both FRETing and non-FRETing populations, but have been difficult to develop due to inherent difficulties with background autofluorescence and lifetime component separation. Using a modified commercial lifetime plate reader, we demonstrate a method for removal of the complex auto-fluorescent background decay, described using a stretched exponential function (StrEF). We develop a generalized multi-exponential fitting algorithm (GeMEF), which progressively accounts for confounding lifetime components in FRET-based assays using a series of control experiments. We demonstrate the separability of FRET strength and efficiency and apply the technique to protein–protein interactions and protein conformational assays in a cell-based format. Presenilin 1 (PS1) is known to be important in Amyloid Precursor Protein (APP) processing in Alzheimer's disease. Using transfected cells, we demonstrate APP-PS1 interactions by FRET in a cell-based, 96-well plate format.
We discuss the application of time domain fluorescence techniques to the recovery of targets embedded in several cm thick biological tissue. Considering the general time domain problem first, a singular value analysis is used to study the optimal use of multiple frequency components extracted from time domain data. Furthermore, a computationally efficient algorithm is presented to tomographically reconstruct fluorophore locations using their decay amplitudes and validated using phantom experiments. The reconstruction algorithm presented here has wide applicability for non-invasive, diagnostic fluorescence imaging in small animals and other biological systems, given that fluorescence lifetime is a sensitive indicator of local tissue environment and elementary interactions at the molecular level.
Alzheimer's disease (AD) is characterized by the presence of aggregates of the amyloid- (A) peptide in the brain. These aggregates manifest themselves as senile plaques and cerebrovascular amyloid angiopathy (CAA). While traditional histochemical approaches can easily identify these deposits in postmortem tissue, only recently have specific ligands been developed to target A in living patients using positron emission tomography (PET). Successful detection of A pathology in patients will enable definitive preclinical diagnosis of AD, and enable quantitative evaluation of the efficacy of anti-A therapeutics developed to treat the disease. PET scanning, however, has several disadvantages including high cost, low availability, and the requirement for radioactive tracers. We describe recent progress in the development of techniques for imaging A deposits noninvasively using optical approaches. Successful development of an optical detection platform would enable inexpensive, accessible, nonradioactive detection of the A deposits found in AD.
Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimer's disease and Parkinson's disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.
Alzheimer's disease is characterized by the presence of neurofibrillary tangles and senile plaques in the brain. Clinical techniques are just becoming available for detecting plaques, allowing a definitive diagnosis of the disease. Using multiphoton microscopy and transgenic mouse models that develop senile plaques as they age, we have demonstrated chronic, in vivo imaging of these neuropathological lesions. We have used these tools to evaluate contrast agents with high affinity and specificity for senile plaques that would be suitable for non-invasive imaging with PET scanning if appropriately radiolabeled. These imaging tools should translate into early diagnostic procedures, as well as end-points for clinical trials aimed at clearing senile plaques from the brain. We have also developed FLIM for FRET determinations in vitro and in vivo between appropriate donor and acceptor fluorophores to examine the proximity of domains within a single protein. These results indicate that FRET measurements using FLIM can determine interactions of proteins on the nanometer scale, facilitating an understanding of both static and dynamic protein assemblies in neuropathological diseases.
Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.
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