We devised a multimodal planar imaging system for in vivo mouse imaging, employing four modalities: optical imaging, green and red fluorescence reflectance imaging, radionuclide imaging and X-ray radiography. We are testing separately, and then in a combined way, each detection mode, via in vivo mouse imaging, with the final purpose of identifying small implanted tumor masses, of providing early tumor detection and following metastatic dissemination. We describe the multimodal system and summarize its main performance, as assessed in our research work in the various stages of the development, in fluorescence and radionuclide tests on healthy or tumor bearing mice. For gamma-ray detection we used a semiconductor pixel detector (Medipix1 or Medipix2) that works in single photon counting. Laser-induced fluorescence reflectance imaging was performed in vivo using a pulsed light source to excite the fluorescence emission of injected hematoporphyrin (HP) compound, a CCD camera, a low pass filter and a commercial image analysis system. The bimodal system was used for the acquisition of combined images of the tumor area (fluorescence: animal top view; radionuclide: bottom view). It was shown that the tumor area can be imaged in a few minutes, with a few millimeter resolution (1 mm pinhole diameter), radioactively (99mTc radiotracer), and with the fluorescence system and that, in one case, only one of the two modalities is able to recognize the tumor. A phantom study for thyroid imaging with 125I source embedded in a simulated tissue indicated a spatial resolution of 1.25 mm FWHM with a 1 mm pinhole.
Recently multimodal imaging systems have been devised because the combination of different imaging modalities results in the complementarity and integration of the techniques and in a consequent improvement of the diagnostic capabilities of the multimodal system with respect to each separate imaging modality.
We developed a simple and reliable HematoPorphyrin (HP) mediated Fluorescence Reflectance Imaging (FRI) system that allows for in vivo real time imaging of surface tumors with a large field of view. The tumor cells are anaplastic human thyroid carcinoma-derived ARO cells, or human papillary thyroid carcinoma-derived NPA cells. Our measurements show that the optical contrast of the tumor region image is increased by a simple digital subtraction of the background fluorescence and that HP fluorescence emissivity of ARO tumors is about 2 times greater than that of NPA tumors, and about 4 times greater than that of healthy tissues. This is also confirmed by spectroscopic measurements on histological sections of tumor and healthy tissues. It was shown also the capability of this system to distinguish the tumor type on the basis of the different intensity of the fluorescence emission, probably related to the malignancy degree.
The features of this system are complementary with those ones of a pixel radionuclide detection system, which allows for relatively time expensive, narrow field of view measurements, and applicability to tumors also deeply imbedded in tissues. The fluorescence detection could be used as a large scale and quick analysis tool and could be followed by narrow field, higher resolution radionuclide measurements on previously determined highly fluorescent regions.
Umberto Bernini, Alberto Colasanti, Giovanni Guida, Annamaria Kisslinger, Raffaele Liuzzi, Maria Quarto, Patrizia Riccio, Paolo Russo, Giuseppe Roberti, Fulvia Villani
CW transillumination of tissue phantoms at 1064 and 820 nm has been performed, by measuring the profile of transmitted light transverse to the direction of the incident beam for various values of the scattering and absorption coefficients and for different viewing angles of the photodetector and sample thicknesses. The set of measurements, compared with the results of Monte Carlo simulations, allows to derive a CW optical characterization of tissue-like samples and to assess the possible resolution improvement of collimated detection with narrow viewing angles.
Monte Carlo (MC) simulations of photon transport in turbid media suffer a severe limitation represented by very high execution times in all practical cases. This problem could be approached with the technique of parallel computing, which, in principle, is very suitable for MC simulations because they consist in the repeated application of the same calculations to unrelated and superposing events. For the first time in the field of the optical and IR photon transport, we developed a MC parallel code running on the parallel processor computer CRAY-T3E (128 DEC Alpha EV5 nodes, 600 Mflops) at CINECA (Bologna, Italy). The comparison of several single processor runs (on Alpha AXP DEC 2100) and N-processor runs (on Cray T3E) for the same tissue models shows that the computation time is reduced by a factor of about 5*N, where N is the number of used processors. This means a computation time reduction by a factor ranging from about 102 (as in our case) up to about 5*103 (with the most powerful parallel computers) that could make feasible MC simulations till now impracticable.
Some medical applications involving the interaction between light and biological tissues require both the knowledge of optical characteristics of tissues and a realistic treatment of light transport into them. In this work we describe a transillumination technique testing water solutions of Intralipid by the transmitted radiation intensity. The experimental apparatus includes a diode laser ((lambda) emiss equals 820 nm), a detection fiber, a PMT and a Digital Signal Analyzer. The performances of this very simple and cheap system are comparable to those ones obtained with more sophisticated apparatus; the results show that this technique could represent a preliminary step toward the realization of an user-friendly and cheap laser system for measuring optical parameters of tissues.
The growing number of laser applications in medicine and biology has determined a renewed interest on the study of the light transport in turbid media such as biological tissues. One of the most powerful methods used to describe this kind of process is given by the Monte Carlo techniques. We have developed a FORTRAN90 code, running on an Alpha Vax AXP DEC 2100 to simulate the transport of a photon beam with a Gaussian temporal and spatial profile through a multilayered sample. The code provides the sample transmittance and reflectance (both time and space resolved) that can be compared to the experimental data. Monte Carlo calculations have been performed to simulate time-resolved transillumination through water latex and intralipid water solutions with optical properties similar to those of biological tissues. The comparison of Monte Carlo results with experimental data and with analytical solutions to diffusion equation shows a good agreement, suggesting that Monte Carlo techniques are indeed a powerful tool for predictions on light transport in turbid media.
In last years NIR radiation was extensively used to detect targets hidden in optically turbid media. We performed transillumination measurements on tissue-like phantoms (absorbing objects embedded in Intralipid and black ink solutions) to determine the intensity of transmitted radiation. The experimental apparatus include a diode laser ((lambda) emiss equals 820 nm, CW power equals 250 mW), a detection fiber (diameter equals 600 mm), a PMT and a Digital Signal Analyzer (bandwidth equals 1 GHz). At variance of very complex systems that reject highly scattered photons by time-resolved detection, the developed system performs this selection by collimation. The experimental data were compared with diffusion approximation predictions.
The optical properties of controlled size latex particles suspended in water have been investigated by using two different time-resolved transmittance set-ups. Least-mean square fitting between experimental data and analytical solution to diffusion approximation equation has given values of optical parameters in good agreement with the predictions of the Mie theory. In this way, the validity of these predictions was checked in the investigated experimental conditions and the data analysis confirms that time-resolved transmittance can be a reliable technique to measure the optical properties of scattering solutions.
KEYWORDS: Luminescence, Resistance, Time metrology, Control systems, Quantitative analysis, Information operations, Carbon dioxide, Fluorometers, Data modeling, Quantum efficiency
A fluorimetric technique to measure transport parameters of fluorescent drugs through cellular membranes is described. This method eliminates procedures that would lead to errors in the measurement of drug accumulated by cells, and measures the fraction of free drug in cells. The use of a simple three-compartment model in conjunction with fluorescence measurements performed on the extracellular medium and on Triton-permeabilized cells at different times during daunorubicin incorporation allows determination of the kinetic parameters of the transport through cellular membranes. With this technique we found that LoVo cells have a greater daunorubicin uptake, a similar input rate constant and a lower output rate constant than the drug resistant LoVo/DX cells. Preliminary photoactivation measurements of these two cell lines with these compounds showed that phototoxic effects are related to the amount of drug bound to cellular sites.
We investigated the in vitro photoactivation properties of two chlorin derivatives, 8-cis-heptylchlorin dicarboxylic acid and 3-trans-heptylchlorin bisamidoglucose derivative, that exhibit a very strong absorbance peak in the red region of the spectrum and a value of the extinction coefficient (at the wavelength of the maximum absorbance) higher by one order of magnitude than the corresponding value for the porphyrins. The experiments of photoactivation were performed on a normal epithelial cell line (FRTL-5), using as an irradiation source an array of diodes emitting red light (lambda equals 675 nm). We found that, in the concentration range of 1 to 3000 ng/ml, photoactivation of chlorins greatly enhanced mortality of drug exposed irradiated cells (density of energy equals 0.25 mJ/cm2) with respect to the ones exposed to the drug but kept in the dark. This response is immediate and it looks line an 'all or none' effect. The comparison between the extinction coefficient and the efficacy of photoactivation of the two chlorin derivatives seems to indicate that they have similar values of the photophysical and photochemical quantities characterizing the processes responsible of the generation of singlet oxygen, the reactive species to whom the photoinduced toxicity of chlorins was generally imputed.
A system for the excitation and detection of autofluorescence induced by N2 laser light (337.1 nm, 1 divided by 1.5 mJ/pulse) on biological tissues in vivo is described. Spectra obtained with medium spectral resolution spectrograph were detected with a 512 by 512 CCD array. Spectral measurements performed on patients bearing different cutaneous diseases show that, in the wavelength range 420 - 480 nm, pathological skin tissues fluoresce less than healthy ones and these differences might be useful for diagnostic purposes.
A novel approach to gene trasfection, i.e. the introduction of an extraneous gene into a host cell, by the 'non-contact forces' of a laser microbeam, is presented here. By means of a large magnification (100x) objective, the blue microbeam of an Argon laser (488 nm) has been focused on the cell membrane in culture in the presence of a pH indicator (namely the phenol-red), which is an usual component of culture media. Due to the local high light absorption of phenol red, which shows an absorption peak at 475 nm, at the site of the beam impact the cell membrane melts forming small circular holes. Throughout the holes, DNA purposely added to culture medium, may penetrate the cytoplasm. The wall damage, whose extension may be regulated by controlling the irradiation time, disappear spontaneously (membrane repair) within 1-2 minutes. By this technique, thereafter indicated as 'optoporation', we have successfully transfected into murine NIH3T3 fibroblasts (beta) -galactosidase and chloramphenicol-acetyltransferase bacterial genes. These conditions of transfection by means of 'non contact' forces, are very mild and do not require any addition of extraneous, potentially toxic chemicals. In addition, since the radiation used is in the visible region, where nucleic acids and most proteins do not absorb, no further deleterious effects for the cell are expected.
Irradiation with 86 J/cm2 of cultures of Fisher-rate thyroid cells (FRTL5) in the presence of daunomycin derivatives at wavelengths between 488 and 595 nm i.e., in the visible- absorption bands of these drugs, is shown to enhance their cytotoxicity. Daunomycin, its 4- demethoxy derivative, 5-iminodaunomycin, and two amino-substituted 4-demethoxy derivatives of daunomycin are tested. While a 2-h exposure to the drugs in the dark produces 50 short-term cell mortality at dosages (LD50) in the range 23 to 138 (mu) g/ml, irradiation administered during the cell exposure to the drugs is found to lower the LD50 values down to the range 45 to 289 ng/ml. Furthermore, while the LD50 values for all drugs in the absence of photoactivation are similar, if light is administered those for the 4- demethoxy compounds are lowered by 3 orders of magnitude and those for the other derivatives by 2 orders of magnitude. Microfluorimetric investigations reveal that photoactivation causes fading of the drug fluorescence in the perinuclear cytoplasm. The effect is more pronounced for drugs with higher photosensitizing properties. The nonfluorescent photoproducts which are formed in the cells during photoactivation exhibit a cytotoxic activity that is, at long term, lower than that of the original drug. The authors cannot yet assess which excited-state property of anthracyclines plays the key role in the photosensitized reaction(s) responsible for both short-term cell kill and long-term toxic effects. The show, however, that such property is strongly affected by the removal of the methoxy group from the C4 position.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.