Development of a Lensless Radiomicroscope for Cellular-Resolution Radionuclide Imaging.

The action of radiopharmaceuticals takes place at the level of cells. However, existing radionuclide assays can only measure uptake in bulk or in small populations of single cells. This potentially hinders the development of effective radiopharmaceuticals for disease detection, staging, and treatment. Methods: We have developed a new imaging modality, the lensless radiomicroscope (LRM), for in vitro, cellular-resolution imaging of ß- and α-emitting radionuclides. The palm-sized instrument is constructed from off-the-shelf parts for a total cost of less than $100, about 500 times less than the radioluminescence microscope, its closest equivalent. The instrument images radiopharmaceuticals by direct detection of ionizing charged particles via a consumer-grade complementary metal-oxide semiconductor detector. Results: The LRM can simultaneously image more than 5,000 cells within its 1 cm2 field of view, a 100-times increase over state-of-the-art technology. It has spatial resolution of 5 µm for brightfield imaging and 30 µm for 18F positron imaging. We used the LRM to quantify 18F-FDG uptake in MDA-MB-231 breast cancer cells 72 h after radiation treatment. Cells receiving 3 Gy were 3 times larger (mean = 3,116 µm2) than their untreated counterparts (mean = 940 µm2) but had 2 times less 18F-FDG per area (mean = 217 Bq/mm2), a finding in agreement with the clinical use of this tracer to monitor response. Additionally, the LRM was used to dynamically image the uptake of 18F-FDG by live cancer cells, and thus measure their avidity for glucose. Conclusion: The LRM is a high-resolution, large-field-of-view, and cost-effective approach to image radiotracer uptake with single-cell resolution in vitro.

Radioluminescence in biomedicine: physics, applications, and models.

The electromagnetic spectrum contains different frequency bands useful for medical imaging and therapy. Short wavelengths (ionizing radiation) are commonly used for radiological and radionuclide imaging and for cancer radiation therapy. Intermediate wavelengths (optical radiation) are useful for more localized imaging and for photodynamic therapy (PDT). Finally, longer wavelengths are the basis for magnetic resonance imaging and for hyperthermia treatments. Recently, there has been a surge of interest for new biomedical methods that synergize optical and ionizing radiation by exploiting the ability of ionizing radiation to stimulate optical emissions. These physical phenomena, together known as radioluminescence, are being used for applications as diverse as radionuclide imaging, radiation therapy monitoring, phototherapy, and nanoparticle-based molecular imaging. This review provides a comprehensive treatment of the physics of radioluminescence and includes simple analytical models to estimate the luminescence yield of scintillators and nanoscintillators, Cherenkov radiation, air fluorescence, and biologically endogenous radioluminescence. Examples of methods that use radioluminescence for diagnostic or therapeutic applications are reviewed and analyzed in light of these quantitative physical models of radioluminescence.

Theoretical investigation of ultrasound-modulated Cerenkov luminescence imaging for higher-resolution imaging in turbid media.

Cerenkov luminescence imaging (CLI) is an optical technique for imaging radiolabeled molecules in vivo. It has demonstrated utility in both the clinical and preclinical settings and can serve as a substitute for nuclear imaging instrumentation in some cases. However, optical scattering fundamentally limits the resolution and depth of imaging that can be achieved with this modality. In this Letter, we report the numerical results that support the potential for ultrasound-modulated Cerenkov luminescence imaging (USCLI), a new imaging modality that can mitigate optical scattering. The technique uses an acoustic field to modulate the refractive index of the medium and, thus, the intensity of Cerenkov luminescence in a spatially precise manner. This mechanism of contrast has not been reported previously. For a physiologically compatible ultrasound peak pressure of 1 MPa, ∼0.1% of the Cerenkov signal can be modulated. Furthermore, our simulations show that USCLI can overcome the scattering limit of resolution for CLI and provide higher-resolution imaging. For an F18 point source centered in a 1 cm3 simulated tissue phantom with a scattering coefficient of µs'=10 cm-1, <2 mm full width at half-maximum lateral spatial resolution is possible, a resolution three times finer than the same phantom imaged with CLI.

Quantitative assessment of Cerenkov luminescence for radioguided brain tumor resection surgery.

Cerenkov luminescence imaging (CLI) is a developing imaging modality that detects radiolabeled molecules via visible light emitted during the radioactive decay process. We used a Monte Carlo based computer simulation to quantitatively investigate CLI compared to direct detection of the ionizing radiation itself as an intraoperative imaging tool for assessment of brain tumor margins. Our brain tumor model consisted of a 1 mm spherical tumor remnant embedded up to 5 mm in depth below the surface of normal brain tissue. Tumor to background contrast ranging from 2:1 to 10:1 were considered. We quantified all decay signals (e±, gamma photon, Cerenkov photons) reaching the brain volume surface. CLI proved to be the most sensitive method for detecting the tumor volume in both imaging and non-imaging strategies as assessed by contrast-to-noise ratio and by receiver operating characteristic output of a channelized Hotelling observer.

Different types of retinal inhibition have distinct neurotransmitter release properties.

Neurotransmitter release varies between neurons due to differences in presynaptic mechanisms such as Ca(2+) sensitivity and timing. Retinal rod bipolar cells respond to brief dim illumination with prolonged glutamate release that is tuned by the differential release of GABA and glycine from amacrine cells in the inner retina. To test if differences among types of GABA and glycine release are due to inherent amacrine cell release properties, we directly activated amacrine cell neurotransmitter release by electrical stimulation. We found that the timing of electrically evoked inhibitory currents was inherently slow and that the timecourse of inhibition from slowest to fastest was GABAC receptors > glycine receptors > GABAA receptors. Deconvolution analysis showed that the distinct timing was due to differences in prolonged GABA and glycine release from amacrine cells. The timecourses of slow glycine release and GABA release onto GABAC receptors were reduced by Ca(2+) buffering with EGTA-AM and BAPTA-AM, but faster GABA release on GABAA receptors was not, suggesting that release onto GABAA receptors is tightly coupled to Ca(2+). The differential timing of GABA release was detected from spiking amacrine cells and not nonspiking A17 amacrine cells that form a reciprocal synapse with rod bipolar cells. Our results indicate that release from amacrine cells is inherently asynchronous and that the source of nonreciprocal rod bipolar cell inhibition differs between GABA receptors. The slow, differential timecourse of inhibition may be a mechanism to match the prolonged rod bipolar cell glutamate release and provide a way to temporally tune information across retinal pathways.

Slow changes in Ca2(+) cause prolonged release from GABAergic retinal amacrine cells.

The timing of neurotransmitter release from neurons can be modulated by many presynaptic mechanisms. The retina uses synaptic ribbons to mediate slow graded glutamate release from bipolar cells that carry photoreceptor inputs. However, many inhibitory amacrine cells, which modulate bipolar cell output, spike and do not have ribbons for graded release. Despite this, slow glutamate release from bipolar cells is modulated by slow GABAergic inputs that shorten the output of bipolar cells, changing the timing of visual signaling. The time course of light-evoked inhibition is slow due to a combination of receptor properties and prolonged neurotransmitter release. However, the light-evoked release of GABA requires activation of neurons upstream from the amacrine cells, so it is possible that prolonged release is due to slow amacrine cell activation, rather than slow inherent release properties of the amacrine cells. To test this idea, we directly activated primarily action potential-dependent amacrine cell inputs to bipolar cells with electrical stimulation. We found that the decay of GABAC receptor-mediated electrically evoked inhibitory currents was significantly longer than would be predicted by GABAC receptor kinetics, and GABA release, estimated by deconvolution analysis, was inherently slow. Release became more transient after increasing slow Ca(2+) buffering or blocking prolonged L-type Ca(2+) channels and Ca(2+) release from intracellular stores. Our results suggest that GABAergic amacrine cells have a prolonged buildup of Ca(2+) in their terminals that causes slow, asynchronous release. This could be a mechanism of matching the time course of amacrine cell inhibition to bipolar cell glutamate release.

Inhibition to retinal rod bipolar cells is regulated by light levels.

The retina responds to a wide range of light stimuli by adaptation of retinal signaling to background light intensity and the use of two different photoreceptors: rods that sense dim light and cones that sense bright light. Rods signal to rod bipolar cells that receive significant inhibition from amacrine cells in the dark, especially from a rod bipolar cell-activated GABAergic amacrine cell. This inhibition modulates the output of rod bipolar cells onto downstream neurons. However, it was not clear how the inhibition of rod bipolar cells changes when rod signaling is limited by an adapting background light and cone signaling becomes dominant. We found that both light-evoked and spontaneous rod bipolar cell inhibition significantly decrease with light adaptation. This suggests a global decrease in the activity of amacrine cells that provide input to rod bipolar cells with light adaptation. However, inhibition to rod bipolar cells is also limited by GABAergic connections between amacrine cells, which decrease GABAergic input to rod bipolar cells. When we removed this serial inhibition, the light-evoked inhibition to rod bipolar cells remained after light adaptation. These results suggest that decreased inhibition to rod bipolar cells after light adaptation is due to decreased rod pathway activity as well as an active increase in inhibition between amacrine cells. Together these serve to limit rod bipolar cell inhibition after light adaptation, when the rod pathway is inactive and modulation of the signal is not required. This suggests an efficiency mechanism in the retina to limit unnecessary signaling.