Multicore fiber bundles for imaging and stimulating optogenetically modified neurons have been largely adopted in neurophotonics research. They allow for directed, single-cell stimulation and imaging of neuronal activity. An inherent limitation of these bundles is the presence and detection of the empty space between individual fibers, resulting in a loss of significant amounts of data, and reduced image quality due to pixilation effects. We propose a novel approach and algorithm to depixelation and image reconstruction from fiber bundles that utilizes multiple image frames collected during on-axis fiber bundle rotation. The approach involves first acquiring the Fourier transform of a stationary, unrotated image, followed by its rotated counterparts. The phase information from each image is then acquired, cross-correlated, and the angle of rotation determined from this correlation. Rotated images are then weighed and summed to generate a final reconstructed, depixelated image. Simulations were initially performed using Matlab demo images. Experimentation was done with a resolution chart, and thereafter with a cell culture. 488 nm and 561 nm continuous wave laser sources (Coherent, Inc.) were used for imaging GCaMP6s and C1V1-mCherry, respectively, in hippocampal neuronal cultures. The light sources were coupled to a multicore fiber bundle (Schott, 1534702) containing 4,200, 7.5 µm fibers. Cell cultures were prepared from 2 day old transgenic mice (GCaMP6s, Jackson Labs) transfected with C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford). The results demonstrate this as an effective technique alongside fiber bundle imaging, serving as a useful and powerful tool for removing undesired artifacts associated with these fibers.
By combining optical and genetic methods, optogenetics has become a very important tool in neuroscience research for manipulating neuron activities. The rapid development of novel opsins and fluorescent indicators has introduced a large palette of biochemical probes for optogenetic stimulation and cellular imaging, which makes the all-optical neural circuit excitation and neural activity recording possible. Compared to visible-light illumination, two-photon excitation and imaging avoids the crosstalk from optogenetic probes and calcium sensors, and provides for deeper penetration and higher spatial-temporal resolution for single-cell-level precise manipulation. Two-photon interactions frequently necessitate the use of high-power sources with narrow bandwidth outputs. Although tunable sources, such as the titanium-sapphire laser, offer some degree of flexibility, multiple bulky and expensive lasers are required for simultaneous two-photon optogenetic stimulation and calcium imaging. Here, we propose to use fiber-based supercontinuum generation as a broadband coherent light source for two-photon excitation and imaging. A custom-made photonic crystal fiber is pumped by a Yb:KYW laser (1041 nm, 220 fs, 80 MHz) to generate a femtosecond output with a wide range of wavelengths, 900 - 1170 nm, which covers most of the two-photon excitation wavelengths of the molecules used in optogenetics, e.g. C1V1-2A-mCherry and GCaMP6s in our study. A pulse shaper is utilized to modulate the phases of partial wavelengths to tailor the temporal shape of the femtosecond pulse, which manipulates the absorption of optogenetic probes and provides a unique approach for controllable optogenetic excitation. Video-rate calcium imaging results suggest that spectral-temporal programmable supercontinuum pulses provide a powerful tool for neural network activity research.
Light delivery in in vivo optogenetic applications are typically accomplished via a single multimode fiber that diffuses light over a large area of the brain, and relies heavily on the spatial distribution of transfected light-sensitive neurons for targeted control.
In our investigations, an imaging fiber bundle (Schott, 1534702) containing 4,500 individual fibers, each with a diameter of 7.5 µm, and an overall outer bundle diameter of 530 µm, was used as the conduit for light delivery and optical recording/imaging in neuron cultures and in in vivo mouse brain. We demonstrated that the use of this fiber bundle, in contrast to a single multimode fiber, allowed for individually-addressable fibers, spatial selectivity at the stimulus site, precise control of light delivery, and full field-of-view imaging and/or optical recordings of neurons. An objective coupled the two continuous wave diode laser sources (561 nm/488 nm) for stimulation and imaging into the proximal end of the fiber bundle while a set of galvanometer-scanning mirrors was used to couple the light stimulus to distinct fibers. A micro lens aided in focusing the light at the neurons. In vivo studies utilized C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford) and GCaMP6s transgenic mice (Jackson Labs) for this all-optical approach.
Our results demonstrate that imaging fiber bundles provide superior control of spatial selectivity of light delivery to specific neurons, and function as a conduit for optical imaging and recording at the in vivo site of stimulation, in contrast to the use of single multimode fibers that diffusely illuminate tissue and lack in vivo imaging capabilities.
Current methods for light delivery in in vivo optogenetic applications are typically accomplished via a single multimode fiber that diffuses light over a large area of the brain, and relies on the spatial distribution of transfected light-sensitive neurons for targeted control.
In our investigations, an imaging fiber bundle (Schott) containing 4,500 individual fibers, each with a diameter of 7.5 µm, and an overall outer bundle diameter of 530 µm, served as a conduit for light delivery and optical recording/imaging. The use of this fiber bundle, in contrast to a single multimode fiber, allows for individually-addressable fibers, spatial selectivity at the stimulus site, more precise control of light delivery, and full field-of-view imaging and/or optical recordings of individual neurons in local neural circuits. An objective coupled the two continuous wave diode laser sources (561nm/488nm) (Coherent) for stimulation and imaging into the fiber bundle while a set of galvanometer-scanning mirrors was used to couple the light stimulus to distinct fibers within the proximal end of the imaging fiber bundle. In our study, C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford) and GCaMP6s transgenic mice (Jackson Labs) were utilized for this all-optical approach.
The results of our investigation demonstrate that imaging fiber bundles provide a new level of spatial selectivity and control of light delivery to specific neurons, as well as function as a conduit for optical imaging and recording at the in vivo site of stimulation, in contrast to the use of single multimode fibers that diffusely illuminate neural tissue and lack in vivo imaging capabilities.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.