Abstract
In vivo functional imaging at single-neuron resolution is an important approach to visualize biological processes in neuroscience. Light sheet microscopy (LSM) is a cutting edge in vivo imaging technique that provides micron-scale spatial resolution at high frame rate. Due to the scattering and absorption of tissue, however, conventional LSM is inadequate to resolve cells because of the attenuated signal to noise ratio (SNR). Using dual-beam illumination and confocal dual-slit detection, here a dual-slit confocal LSM is demonstrated to obtain the SNR enhanced images with frame rate twice as high as line confocal LSM method. Through theoretical calculations and experiments, the correlation between the slit's width and SNR was determined to optimize the image quality. In vivo whole brain structural imaging stacks and the functional imaging sequences of single slice were obtained for analysis of calcium activities at single-cell resolution. A two-fold increase in imaging speed of conventional confocal LSM makes it possible to capture the sequence of the neurons' activities and help reveal the potential functional connections in the whole zebrafish's brain.
Highlights
Brain function relies on the activity of multiple neurons and their communication [1]
In light sheet based microscopy (LSM), only one plane of the sample is illuminated with a thin sheet of light, and the fluorescence is collected by a wide-field microscope, whose optical axis is orthogonal to the excited plane [22]
The length of z-step of these stacks is 1 μm. 3D point spread function (PSF) was extracted by reconstructing the image stacks using ImageJ (NIH, MD), as shown in Fig. 3(a) and 3(c)
Summary
Brain function relies on the activity of multiple neurons and their communication [1]. Thanks to the advent of calcium fluorescent label methods, in vivo imaging approaches such as laser scanning confocal microscopy (LSCM) [6], multiphoton microscopy (MPM) [7,8] and light sheet based microscopy (LSM) [9,10] have been developed to better visualize neuron’s calcium activities in living animals [2,11]. The other is sweeping the focused Gaussian, Bessel or Airy beam [23,24] across the imaging plane in digital scanned light sheet microscopy (DSLM) [25]. LSM is especially suitable for long-term in vivo imaging [26] Another advantage of LSM is that, the wide-field detection scheme allows for the high imaging speed of LSM and enables observation of instantaneous events such as blood cell flow [27] and neuron activity [9]
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