Experimental method and statistical analysis to fit tumor growth model using SPECT/CT imaging: a preclinical study.

BACKGROUND: Over the last decade, several theoretical tumor-models have been developed to describe tumor growth. Oncology imaging is performed using various modalities including computed tomography (CT), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT) and fluorodeoxyglucose-positron emission tomography (FDG-PET). Our goal is to extract useful, otherwise hidden, quantitative biophysical parameters (such as growth-rate, tumor-necrotic-factor, etc.) from these serial images of tumors by fitting mathematical models to images. These biophysical features are intrinsic to the tumor types and specific to the study-subject, and expected to add valuable information on the tumor containment or spread and help treatment plans. Thus, fitting realistic but practical models and assessing parameter-errors and degree of fit is important. METHODS: We implemented an existing theoretical ode-compartment model and variants and applied them for the first time, in vivo. We developed an inversion algorithm to fit the models for tumor growth for simulated as well as in vivo experimental data. Serial SPECT/CT scans of mice breast-tumors were acquired, and SPECT data was used to segment the proliferating-layers of tumors. RESULTS: Results of noisy data simulation and inversion show that 5 out of 7 parameters were recovered to within 4.3% error. In particular, tumor "growth-rate" parameter was recovered to 0.07% error. For model fitting to in vivo mice-tumors, regression analysis on the P-layer volume showed R2 of 0.99 for logistic and Gompertzian while surface area model yielded R2=0.96. For the necrotic layer the R2 values were 0.95, 0.93 and 0.94 respectively for surface-area, logistic and Gompertzian. The Akaike Information Criterion (AIC) weights of the models (giving their relative probability of being the best Kullback-Leibler (K-L) model among the set of candidate models) were ~0, 0.43 and 0.57 for surface-area, logistic and Gompertzian models. CONCLUSIONS: Model-fitting to mice tumor studies demonstrates feasibility of applying the models to in vivo imaging data to extract features. Akaike information criterion (AIC) evaluations show Gompertzian or logistic growth model fits in vivo breast-tumors better than surface-area based growth model.

New compartment model analysis of lean-mass and fat-mass growth with overfeeding.

OBJECTIVES: Mathematical models of lean- and fat-mass growth with diet are useful to help describe and potentially predict the fat- and lean-mass change with different diets as a function of consumed protein and fat calories. Most of the existing models do not explicitly account for interdependence of fat-mass on the lean-mass and vice versa. The aim of this study was to develop a new compartmental model to describe the growth of lean and fat mass depending on the input of dietary protein and fat, and accounting for the interdependence of adipose tissue and muscle growth. METHODS: The model was fitted to existing clinical data of an overfeeding trial for 23 participants (with a high-protein diet, a normal-protein diet, and a low-protein diet) and compared with the existing Forbes model. RESULTS: Qualitatively and quantitatively, the compartment model data fit was smoother with less overall error than the Forbes model. The root means square error were 0.39, 0.93 and 0.72 kg for the new model, the Forbes model, and the modified Forbes model, respectively. Additionally, for the present model, the differences between some of the coefficients (on the cross dependence of fat and lean mass as well as on the intake diet dependence) across different diets were statistically significant (P < 0.05). CONCLUSIONS: Our new Dey-model showed excellent fit to overfeeding data for 23 normal participants with some significant differences of model coefficients across diets, enabling further studies of the model coefficients for larger groups of participants with obesity or other diseases.

Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue.

Applications of fluorescence based imaging techniques for detection in cellular and tissue environments are severely limited by autofluorescence of endogenous components of cells, tissue, and the fixatives used in sample processing. To achieve sufficient signal-to-background ratio, a high concentration of the probe needs to be used which is not always feasible. Since typically autofluorescence is in the nanosecond range, long-lived fluorescence probes in combination with time-gated detection can be used for suppression of unwanted autofluorescence. Unfortunately, this requires the sacrifice of the large portion the probe signal in order to sufficiently filter the background. We report a simple and practical approach to achieve a many-fold increase in the intensity of a long-lived probe without increasing the background fluorescence. Using controllable, well separated bursts of closely spaced laser excitation pulses, we are able to highly increase the fluorescence signal of a long-lived marker over the endogenous fluorescent background and scattering, thereby greatly increasing detection sensitivity. Using a commercially available confocal microscopy system equipped with a laser diode and time correlated single photon counting (TCSPC) detection, we are able to enhance the signal of a long-lived Ruthenium (Ru)-based probe by nearly an order of magnitude. We used 80 MHz bursts of pulses (12.5 ns pulse separation) repeated with a 320 kHz repetition rate as needed to adequately image a dye with a 380 ns lifetime. Just using 10 pulses in the burst increases the Ru signal almost 10-fold without any increase in the background signal.

Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange.

Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 µM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only ∼1.76 ns fluorescence lifetime. By contrast at high concentrations (4-30 mM) favoring formation of AO aggregates, a very different absorption with maximum at ∼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (∼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98±0.07×10(-7)c m(2)/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ∼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.

Generating multiple-pulse bursts for enhanced fluorescence detection.

The signal-to-background ratio is the limiting factor for fluorescence based detection, sensing, and imaging. A typical background signal will include direct scattering of excitation and Raman scattering of the sample as well as autofluorescence from the sample and additives. To improve the signal-to-background ratio, fluorophores of high brightness and/or high concentration of the fluorophores need to be used. Most of the background is instantaneous and short-lived (picosecond to nanosecond time scale), and using long-lived fluorescence probes combined with time-gated detection allows for significant suppression of unwanted background. Unfortunately, this approach requires substantial sacrifice of the probe signal in order to sufficiently filter the background unless the fluorescence lifetime of the probe is very long. However, long lived probes like ruthenium bipyridyl have relatively low brightness compared to probes that have shorter, 10-30 ns fluorescence lifetimes.We recently presented an approach based on bursts of multiple pulses that allowed for high probe signal amplification using long-lived ruthenium based probe (Ru) and an 80 MHz repetition-rate laser excitation. Unfortunately, Ru represents an extreme case for probe lifetime, and a probe with a shorter lifetime of 20 ns will require excitation from a pulsed source with much higher repetition rate to significantly enhance its signal. Such high repetition rates are not possible to generate with most of today's available electronics. In this report we present new approaches to optimize and generate bursts of pulses with high repetition rate within the burst and no need for new or improved electronics. The high repetition rates originate from a low-repetition source and are highly tunable. We demonstrate that a burst of 2-10 pulses spaced 3 ns apart (corresponding to a 'burst repetition rate' of 330 MHz) allows for high signal enhancement of the 20 ns probe over the sub-nanosecond/nanosecond background. Such an approach can be applied for any sensing format, allowing much higher sensitivity for detection. Since the energy of a single pulse is spread over a few pulses in the burst, the fluorophore's photostability also improves.

Polarization properties of fluorescent BSA protected Au25 nanoclusters.

BSA protected gold nanoclusters (Au25) are attracting a great deal of attention due to their unique spectroscopic properties and possible use in biophysical applications. Although there are reports on synthetic strategies, spectroscopy and applications, little is known about their polarization behavior. In this study, we synthesized the BSA protected Au25 nanoclusters and studied their steady state and time resolved fluorescence properties including polarization behavior in different solvents: glycerol, propylene glycol and water. We demonstrated that the nanocluster absorption spectrum can be separated from the extinction spectrum by subtraction of Rayleigh scattering. The nanocluster absorption spectrum is well approximated by three Gaussian components. By a comparison of the emissions from BSA Au25 clusters and rhodamine B in water, we estimated the quantum yield of nanoclusters to be higher than 0.06. The fluorescence lifetime of BSA Au25 clusters is long and heterogeneous with an average value of 1.84 µs. In glycerol at -20 °C the anisotropy is high, reaching a value of 0.35. However, the excitation anisotropy strongly depends on the excitation wavelengths indicating a significant overlap of the different transition moments. The anisotropy decay in water reveals a correlation time below 0.2 µs. In propylene glycol the measured correlation time is longer and the initial anisotropy depends on the excitation wavelength. BSA Au25 clusters, due to long lifetime and high polarization, can potentially be used in studying large macromolecules such as protein complexes with large molecular weight.

Two Photon Induced Luminescence of BSA Protected Gold Clusters.

In this short letter, we have synthesized the BSA protected Au25 nanoclusters and studied their two photon luminescence behavior. We demonstrate that BSA Au25 nanoclusters can be used as a probe with two photon excitation capability. Our results show a quadratic relation between excitation power and emission intensity whereas with one photon excitation shows a linear dependence. The emission spectrum of BSA Au25 nanoclusters with one photon and two photon excitation shows no appreciable change. Due to its long wavelength emission (650 nm) and two photon excitation, BSA Au25 can be potentially used as a probe for deep tissue imaging.