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Abstract
In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.
Highlights
In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes
laser scanning fluorescence microscopy (LSM) techniques are optimized and acquisition parameters are chosen to display a given structure of interest. This approach works well for many applications but is disadvantageous in circumstances where structures of contrasting brightness cannot be displayed simultaneously. This is true for neuronal imaging, where cell bodies are significantly larger than neuronal processes, and where there is heterogeneity in the density of cell populations resulting in high intra-scene dynamic range
The Photomultiplier tubes (PMT) used in LSM have a limited detection dynamic range, typically three orders of magnitude, which determines the range of variance in the detectable fluorescence signal and the maximum and minimum intensities that can be simultaneously detected within a field of view[5]
Summary
In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. Single photon counting is impractical for high-resolution imaging at high SNR, restricting its use to small fields of view and longer dwell times[10] Another limiting factor is the readout rate (pixel clock rate), which gives the speed at which data can be retrieved from the detection scheme[10]. Recently has the use of sophisticated photon counting circuity or the implementation of field programmable-gate arrays in combination with statistical processing substantially improved their dynamic range, extending photon-counting operation to higher-emission rate regimes[11,12] These methods are still early in development, far from being commercially available, and have only been applied in a few specialized studies[6,8,11,13]
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