KEYWORDS: Neon, Kidney, Luminescence, Tomography, Fluorescence tomography, Sensors, Optical tomography, Monte Carlo methods, Signal to noise ratio, Multiplexing
Fluorescence Molecular Tomography (FMT) is a powerful optical imaging tool for preclinical research. Especially, its implementation with time-domain (TD) techniques allows lifetime multiplexing for simultaneously imaging multiple biomarkers and provides enhanced data sets for improved resolution and quantification compared to continuous wave (CW) and frequency domain (FD) methodologies. When performing time-domain reconstructions, one important aspect is the selection of a temporal sub-data set. Typically, such selection is performed a posteriori after dense temporal sampling during the acquisition. In this work, we investigate the potential to collect a priori sparse data sets for fast experimental acquisition without compromising FMT performances.
KEYWORDS: Fluorescence resonance energy transfer, Near infrared, In vivo imaging, Luminescence, Picosecond phenomena, Cancer, Quantum efficiency, In vitro testing, Photons, Iron
Transferrin (Tfn) is commonly used as a drug delivery carrier for cancer treatment. Tfn cellular internalization can be observed by Förster resonance energy transfer (FRET), which occurs when two fluorophores - donor and acceptor - are a few nanometers apart. Donor fluorescence lifetime can be used to sense and quantify FRET occurrence. In FRET state, the donor is quenched leading to a significant reduction in its lifetime. In this study, donor and acceptor near-infrared (NIR) fluorophore-labeled Tfn were used to quantify cellular internalization in breast cancer cell line (T47D). Based on donor lifetime, quantum yield and spectral data, seven NIR FRET pairs were chosen for this comparison. Performance of the different NIR FRET pairs was evaluated in vitro in multiwell plate settings and by analyzing the relationship between quenched donor fraction and acceptor:donor ratio. Additionally, we performed brightness comparison between each pairs. Several parameters, such as brightness, lifetime, R0 and FRET donor population values are used to identify the most suitable NIR FRET pair for in vivo studies in preclinical settings.
KEYWORDS: Luminescence, Tomography, Monte Carlo methods, Spatial resolution, Reconstruction algorithms, Fluorescence tomography, Inverse problems, Kidney, 3D modeling, Data modeling
Time domain florescence molecular tomography (TD-FMT) allows 3D visualization of multiple fluorophores based on
lifetime contrast and provides a unique data set for enhanced quantification and spatial resolution. The time-gate data set
can be divided into two groups around the maximum gate, which are early gates and late gates. It is well-established that
early gates allow for improved spatial resolution of reconstruction. However, photon counts are inherently very low at
early gates due to the high absorption and scattering of tissue. It makes image reconstruction highly susceptible to the
effects of noise and numerical errors. Moreover, the inverse problem of FMT is the ill-posed and underdetermined.
These factors make reconstruction difficult for early time gates. In this work, lp (0<p≤1) regularization based
reconstruction algorithm was developed within our wide-field mesh-based Monte Carlo reconstruction strategy. The
reconstructions performances were validated on a synthetic murine model simulating the fluorophores uptake in the
kidneys and with experimental preclinical data. We compared the early time-gate reconstructed results using l1/3, l1/2 and
l1 regularization methods in terms of quantification and resolution. The regularization parameters were selected by the Lcurve
method. The simulation results of a 3D mouse atlas and mouse experiment show that lp (0<p<1) regularization
method obtained more sparse and accurate solutions than l1 regularization method for early time gates.
KEYWORDS: Fluorescence resonance energy transfer, Near infrared, Fluorescence lifetime imaging, Tumors, In vivo imaging, In vitro testing, Luminescence, Imaging systems, Cancer, Target detection
One of the challenges in anti-cancer drug delivery systems is to quantitatively discriminate non-specific receptorindependent
tumor accumulation from receptor-mediated uptake into the tumor cells. To overcome this challenge, we
develop a new near infrared fluorescence resonance energy transfer fluorescence lifetime imaging (NIR FRET FLIM)
technique with wide-field illumination strategies to validate and characterize cellular uptake in both cancer cells and
normal cells with different donor-acceptor ratios in vitro and in vivo. Our results demonstrate that NIR FRET FLIM can
quantitatively distinguish receptor-bound from unbound donor in live animals with high sensitivity and high accuracy.
Thus, it has a great potential for the quantitative detection of targeted delivery systems for diagnostic and therapeutic
use.
KEYWORDS: Luminescence, Photon counting, Signal to noise ratio, Signal detection, Time resolved spectroscopy, Fluorescence lifetime imaging, Sensors, Optimization (mathematics), Fluorescence resonance energy transfer, Ranging
The large dynamic range of fluorescence emission collected is one of the major challenges in wide-field fluorescence
lifetime imaging. To overcome this challenge, we developed an active illumination strategy to acquire optimal
fluorescence signals over the sample imaged even in the presence of large fluorophore concentration distributions. We
validated the stability of our approach in a multi-well plate setting with fluorophore concentrations ranging <2 orders of
magnitude. We report the ability of our method to retrieve accurately the lifetime over this concentration range based on
optimized wide-field data. Our results demonstrate that active wide-field illumination can improve the signal-to-noise
ratio and weak-signal sensitivity for enhanced accuracy of fluorescence decay curve fitting and lifetime estimation at
high acquisition speed.
We report an application of Mesoscopic Fluorescence Molecular Tomography to 3D tissue engineering construct. Engineered thick tissue was hosting two 3D printed vasculatures. The channels were formed by live cells, expressing GFP and mCherry reporter genes, embedded in 3mm turbid media. Tissue and cells kept in a 3mm thick perfusion chamber during the entire imaging process which took less than 5 minutes.
In this paper, we present our investigation on multispectral autofluorescence lifetime imaging of RPE cells using two-photon
excitation. Morphological characters of RPE cells are obtained with high spatial resolution. Different
autofluorescence lifetime parameters have been compared at different emission bands. Spatial distribution of dominant
endogenous fluorophores in RPE cells, such as FAD, A2E and AGE etc have been obtained by the analysis of τm and
a1/a2 ratio in the whole emission spectrum.
In this paper, we present our investigation on the identification of endogenous fluorophores in photoreceptors using autofluorescence spectroscopy, which is performed with an inverted laser scanning confocal microscope equipped with an Argon ion laser and a GreNe laser. In our experiments, individual cones and rods are clearly resolved even in freshly prepared retina samples, without slicing or labeling. The experiment results show that autofluorescence spectrum of the photoreceptors has three peaks approximately at 525nm, 585nm and 665nm. Furthermore, the brightest autofluorescence originates from the photoreceptor outer segments. We can, therefore, come to a conclusion that the peaks at 525nm, 585nm are corresponding to FAD and A2-PE, respectively, which are distributed in the photoreceptor outer segments.
Transplantation technique of retinal pigment epithelium has been noticeable in recent years and gradually put into
clinical practice in treatment of retinal degenerative diseases. Generally, immunological, histochemical, and physical
methods are used to study the iris pigment epithelium (IPE) and retinal pigment epithelium (RPE) cells, which need
complex sample preparation. In this paper, we provided a simple autofluorescence microscopy to investigate the fresh
porcine IPE and RPE cells without any pretreatment. The results showed that the morphology and size of both were
similar, round and about 15 μm. The main flourophore in both cells was similar, i.e. lipofuscin. In additional, the
autofluorescence spectrum of RPE shifted blue after light-induced damage by laser illuminating. Because it was easier
for IPE to be damaged by laser than for RPE, and the power of one scanning operation to get a full image was strong
enough to damage IPE sample, we hadn't get any satisfied autofluorescence spectrum of IPE.
In this paper, a novel method for the differentiation of the retinal pigment epithelium (RPE) cells after light-induced
damage by two-photon excitation is presented. Fresh samples of RPE cells of pig eyes are obtained from local
slaughterhouse. Light-induced damage is produced by the output from Ti: sapphire laser which is focused onto the RPE
layer. We study the change of the autofluorescence properties of RPE after two-photon excitation with the same
wavelength. Preliminary results show that after two-photon excitation, there are two clear changes in the emission
spectrum. The first change is the blue-shift of the emission peak. The emission peak of the intact RPE is located at
592nm, and after excitation, it shifts to 540nm. It is supposed that the excitation has led to the increased autofluorescence
of flavin whose emission peak is located at 540nm. The second change is the increased intensity of the emission peak,
which might be caused by the accelerated aging because the autofluorescence of RPE would increase during aging
process. Experimental results indicate that two-photon excitation could not only lead to the damage of the RPE cells in
multiphoton RPE imaging, but also provide an evaluation of the light-induced damage.
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