Volumetric two-photon fluorescence imaging of live neurons using a multimode optical fiber

Abstract

11An ability to optically visualize neurons and their subcellular components across brain regions is 12 integral to our understanding of neuronal function 1 . Multimode optical fibers (MMFs), combined 13 with wavefront control methods, have achieved minimally-invasive in vivo imaging of neurons in 14 deep-brain regions with diffraction-limited spatial resolution 2-5 . To date MMF systems have been 15 based on linear fluorescence and are therefore only able to perform two-dimensional imaging due to 16 an absence of confocal filtering 6 . Here we report a method to achieve optical sectioning in live brain 17 tissue by taking advantage of the nonlinearity of two-photon excited fluorescence (TPEF). In 18 addition, we provide a new approach for the determination of transmission matrix for TPEF, a 19 necessary step for the light shaping requirement of MMF imaging. Central to these advances is the 20 use of a laser source able to generate both continuous wave (CW) light and femtosecond pulses. This 21 configuration allows the system to be compact with minimal distal optics, enhancing suitability for 22 neuroscience applications. With our system we demonstrate TPEF imaging of neurons and their 23 dendrites in live brain slices through a single 60 μm-core MMF with a numerical aperture of 0.37. 24 Taken together these developments represent an important step towards three-dimensional (3D) deep-25 brain imaging in vivo. 26 27 132 RT, CCS, MJB and NE acknowledge support from the Biotechnology and Biological Sciences 133

Deconvolution for multimode fiber imaging: modeling of spatially variant PSF

“…Second, the ability to capture the morphology of subcellular objects, such as dendritic spines on neurons, is an important capability of MMF imaging systems, which will primarily be enabled through a substantial increase in NA. Third, advances in two-photon excited fluorescence microscopy through MMF will benefit from the use of high NA because of the nonlinear dependence between signal generation and NA [21,29]. In fact, even at equivalent NA the non-linearity of the two-photon process will result in increased spatial variations compared to one-photon fluorescence because the excitation volume is effectively the square of the illumination volume.…”

“…Organotypic hippocampal slices (350 µm) were prepared from male Wistar rats (P7-P8; Harlan UK) as previously described [ 21 , 25 ]. After dissection, slices were cultured on Millicell CM membranes and maintained in culture media at C for 7-14 days prior to use.…”

“…A near-infrared version of this experimental system with a continuous-wave laser operating at 830 nm was used for the data shown in Fig. 1-2 [21].…”

Spatiotemporal focusing through a multimode fiber via time-domain wavefront shaping

“…Unfortunately, combining ultrashort pulses with MMF imaging is non-trivial, as the modal interference and modal dispersion in a MMF results in a complex spatiotemporal output field [12,13]. Recently, several nonlinear optical imaging techniques through a single MMF probe have been demonstrated including two-photon excitation microscopy [14,15], 3D microfabrication based on two-photon polymerization [16], and coherent anti-Stokes Raman scattering (CARS) microscopy [17]. All these methods of nonlinear imaging require spatial-domain wavefront shaping and consequently control of many spatial modes on the MMF input.…”

Chromato-axial memory effect in step index multimode fibers

“…Interestingly, the transverse wave-vector conservation naturally holds in step-index (SI) MMF [16]. Furthermore, broadband light propagation through a MMF has recently been observed resulting in an axially extended focus allowing efficient volumetric imaging [30,31]. In this letter we experimentally characterize the χ-axial ME, so far unexplored, at the distal facets of SI-MMFs, and theoretically model it based on the study of the correlation function.…”