Cerenkov luminescence (CL), optical radiation induced by PET radiotracers, has shown promise as a means to visualize tumor margins during surgery. However, detecting this faint optical signal under ambient lighting conditions represents a major challenge. We have developed an ambient light CL imaging system that uses a sensitive imaging detector, custom electronic control board, and an LED illumination array. By gating both LED illumination and imaging detector, we have demonstrated that is possible to image faint Cerenkov-emitting sources in a perceptually well-lit room, without harm to the sensitive detector.
System performance was characterized by imaging 18F radionuclide solution contained in 10 mm well plates, ranging in activity from >1 MBq to <1 kBq, under visible light conditions with irradiances ranging from 0 to >30 µW/cm2. Both detector and LED illumination were gated at 30 Hz with 10 ms active duty cycles. Contrast-to-noise ratio (CNR) was computed from ROIs drawn over activity-containing wells. Though CNR decreased with increasing illumination levels, an activity of 240 kBq, was unambiguously detectable with gated illumination of 37 µW/cm2 (equivalent to typical indoor light levels) and an activity of <24 kBq was unambiguously detectable with gated illumination of 2 µW/cm2. Furthermore, we have characterized sources of noise for the imaging system, which have provided insight into strategies for optimization in anticipation of use in an intraoperative setting.
Traumatic brain injury (TBI) represents a major treatment challenge in both civilian and military medicine; on the cellular level, its mechanisms are poorly understood. As a method to study the dysfunctional repair mechanisms following injury, laser induced shock waves (LIS) are a useful way to create highly precise, well characterized mechanical forces. We present a simple model for TBI using laser induced shock waves as a model for damage. Our objective is to develop an understanding of the processes responsible for neuronal death, the ways in which we can manipulate these processes to improve cell survival and repair, and the importance of these processes at different levels of biological organization. The physics of shock wave creation has been modeled and can be used to calculate forces acting on individual neurons. By ensuring that the impulse is in the same regime as that occurring in practical TBI, the LIS model can ensure that in vitro conditions and damage are similar to those experienced in TBI. This model will allow for the study of the biochemical response of neurons to mechanical stresses, and can be combined with microfluidic systems for cell growth in order to better isolate areas of damage.
Axonal injury and stress have long been thought to play a pathogenic role in a variety of neurodegenerative diseases. However, a model for studying single-cell axonal injury in mammalian cells and the processes of repair has not been established. The purpose of this study was to examine the response of neuronal growth cones to laser-induced axonal damage in cultures of embryonic rat hippocampal neurons and induced pluripotent stem cell (iPSC) derived human neurons. A 532-nm pulsed Nd:YVO4 picosecond laser was focused to a diffraction limited spot at a precise location on an axon using a laser energy/power that did not rupture the cell membrane (subaxotomy). Subsequent time series images were taken to follow axonal recovery and growth cone dynamics. After laser subaxotomy, axons thinned at the damage site and initiated a dynamic cytoskeletal remodeling process to restore axonal thickness. The growth cone was observed to play a role in the repair process in both hippocampal and iPSC-derived neurons. Immunofluorescence staining confirmed structural tubulin damage and revealed initial phases of actin-based cytoskeletal remodeling at the damage site. The results of this study indicate that there is a repeatable and cross-species repair response of axons and growth cones after laser-induced damage.
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