ABSTRACT
Population dynamics of patterned neuronal firing are fundamental to information processing in the brain. Multiphoton microscopy in combination with calcium indicator dyes allows circuit dynamics to be imaged with single-neuron resolution. However, the temporal resolution of fluorescent measures is constrained by the imaging frequency imposed by standard raster scanning techniques. As a result, traditional raster scans limit the ability to detect the relative timing of action potentials in the imaged neuronal population. To maximize the speed of fluorescence measures from large populations of neurons using a standard multiphoton laser scanning microscope (MPLSM) setup, we have developed heuristically optimal path scanning (HOPS). HOPS optimizes the laser travel path length, and thus the temporal resolution of neuronal fluorescent measures, using standard galvanometer scan mirrors. Minimizing the scan path alone is insufficient for prolonged high-speed imaging of neuronal populations. Path stability and the signal-to-noise ratio become increasingly important factors as scan rates increase. HOPS addresses this by characterizing the scan mirror galvanometers to achieve prolonged path stability. In addition, the neuronal dwell time is optimized to sharpen the detection of action potentials while maximizing scan rate. The combination of shortest path calculation and minimization of mirror positioning time allows us to optically monitor a population of neurons in a field of view at high rates with single-spike resolution, â¼ 125 Hz for 50 neurons and â¼ 8.5 Hz for 1,000 neurons. Our approach introduces an accessible method for rapid imaging of large neuronal populations using traditional MPLSMs, facilitating new insights into neuronal circuit dynamics.
Subject(s)
Microscopy, Fluorescence, Multiphoton/instrumentation , Microscopy, Fluorescence, Multiphoton/methods , Neurons/cytology , Neurons/physiology , Software , Action Potentials/physiology , Animals , Mice , Mice, Inbred C57BLABSTRACT
High-risk prostate cancer poses a significant challenge to the treating physician and much debate exists regarding the ideal treatment approach. The purpose of this article is to enable physicians to identify patients with high-risk localized prostate cancer and evaluate whether monotherapy is sufficient for these patients. We review the current data on use of surgery, radiation therapy and hormonal therapy independently and in combination. We also discuss emerging therapeutics for high-risk disease including neoadjuvant chemotherapy and protocols under current and future investigation.
