Maddalena Autiero

Preliminary tests of a prototype system for optical and radionuclide imaging in small animals

We have assembled a prototype system for multimodal (radionuclide and optical) in vivo planar imaging of small animals (mice) using single photon emission radiotracers (Tc-99m) and a fluorescent marker (hematoporphyrin). Preliminary tests of the separate (optical and radionuclide) prototype imaging systems are presented, aimed at assessing their features and at determining the experimental protocol for in vivo imaging. Tests were performed on anesthetized healthy or tumor bearing mice. The gamma radiation detector is a small area (11 /spl times/ 11 mm/sup 2/) hybrid pixel detector based on the Medipix1 ASIC readout technology (64 /spl times/ 64 square pixels of 170 /spl mu/m by side), bump-bonded to a 300 /spl mu/m thick silicon detector. High spatial resolution in radioimaging (in the order of 1 mm) is achieved in vivo with a pinhole tungsten collimator (0.35 mm diameter, 90/spl deg/ acceptance angle, field of view of over 20 mm at 10 mm source distance). A future setup will use the Medipix2 hybrid detector (256 /spl times/ 256 square pixels, 55 /spl mu/m by side) bump-bonded to a 1 mm thick CdTe pixel detector. The laser-induced in vivo fluorescence imaging system comprises a pulsed light source (Nd:YAG laser, /spl lambda/=532 nm, energy/pulse = 30 mJ, pulse width = 50 ps, repetition rate = 10 Hz) used to excite the fluorescence emission (600-760 nm) of injected hematoporphyrin compound, a low sensitivity CCD camera and a commercial image analysis system. Images of normal and tumor regions are acquired by using a cut-on filter (/spl lambda/>600 nm). Digital image subtraction then enhances the tumor contrast with respect to the background. The final experimental protocol, only partly implemented here, includes independent and then combined optical/radio imaging of control mice and of a solid tumoral area (human thyroid derived anaplastic carcinoma) after injection of the radiotracer and/or of the fluorophore. In this work, the accumulation of the radionuclide in selected organs and of the fluorophore in the tumor provides the signal contrast in the two imaging modalities. Fluorescence spectroscopy of excised tissue samples is also performed to help the interpretation of fluorescence images. Results of in vivo combined imaging on tumor in mice will be shown in a next paper.

Experimental study on in vivo optical and radionuclide imaging in small animals

We report on tests of a combined fluorescence and radionuclide planar imaging system for in vivo investigation on small animals. Combined images of anaesthetized mice bearing a surface solid tumor are presented. The fluorescent marker is a hematoporphyrin compound laser-excited with green light and imaged in the red fluorescence emission with a standard monochrome charge coupled device (CCD) camera. The gamma-ray (/sup 99m/Tc) pinhole imaging system uses a semiconductor pixel detector obtained by hybridizing a Silicon (300-/spl mu/m thick) or a CdTe (1-mm thick) pixel detector to the Medipix2 (55-/spl mu/m pitch) readout integrated circuit for single photon counting. The acquisition of combined images of the tumor area (fluorescence: animal top view; radionuclide: bottom view) shows that the tumor area can be imaged in a few minutes, with a few millimeter resolution (1-mm pinhole diameter), radioactively (/sup 99m/Tc MIBI, 74 MBq), and with the optical system. Combined imaging revealed also a different uptake of the two types of tumors studied (one grown from anaplastic human thyroid carcinoma-derived cells, the other from human papillary carcinoma-derived cells). Future progress will be toward a more compact optical setup and the use of a thicker CdTe detector.

Toward a Medipix2 coded aperture gamma microscope

We report on tests of a radionuclide imaging system for in vivo investigations in small animals with low energy gamma-rays as from /sup 125/I (27-35 keV). The system imaging optics features a high resolution coded aperture mask and a fine pitch silicon hybrid pixel detector of the Medipix2 series (55 /spl mu/m pitch). The coded aperture (no-two-holes-touching MURA 62/spl times/62) had 70 /spl mu/m holes in 75 /spl mu/m tungsten, and was used in a 2:1 magnification for a field of view of about 7 mm. Laboratory tests with a /sup 109/Cd 22-keV source and in vivo mouse thyroid imaging tests with /sup 125/I show a system resolution of about 110 /spl mu/m. This low energy, semiconductor-based, compact gamma camera is the basic imaging unit of a small animal single photon emission computed tomography system with deep sub-millimeter resolution.

Multimodal system for in vivo tumor imaging in mice.

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.

In vivo macroscopic HPD fluorescence reflectance imaging on small animals bearing surface ARO/NPA tumor

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.

In vivo tumor detection in small animals by hematoporphyrin-mediated fluorescence imaging.

OBJECTIVE Noninvasive in vivo imaging of human tumors implanted in mice provides a reliable and economic tool for the investigation of tumor progression and metastasis and of the effectiveness of the antiblastic drugs on them. The purpose of this study is to report on the performance achievable by the well-known and extensively investigated HP-FRI (HematoPorphyrin (HP)-mediated Fluorescence Reflectance Imaging) when a high-quality image-acquisition device is used. BACKGROUND DATA Previous articles of ours showed that HP-FRI still represents a useful, simple and reliable optical imaging technique to detect surface tumors. Therefore, it is particularly suitable to be used in combination with other imaging modalities in a multimodal imaging system endowed with diagnostic capabilities much better than each separate modality. MATERIALS AND METHODS Six-week-old Crl:CD-1 nude mice were subcutaneously inoculated with tumor cells. Tumor-bearing mice were irradiated in vivo by a frequency-doubled pulsed Nd:YAG laser (lambda = 532 nm). A cooled CCD digital camera recorded fluorescence light emitted by HP injected in mice through a cut-on long-wavelength pass filter. RESULTS The system we developed allows in vivo imaging of surface tumors on small animals with a large field of view, high photometric sensitivity, adequate space resolution, and short measurement time. The estimated spatial resolution is 730 microm for a fluorescence source placed about 0.5 mm under the mouse skin. The first exploration of the capabilities of this HP-FRI setup on few mice shows that it allows the detection of (a) both types of investigated tumors, (b) early stage and late stage but visually unrecognizable tumors, (c) the gross structure of tumors, and (d) the discrimination of necrotic and nonnecrotic tumor regions.