Incoherent Holographic Lattice Light-Sheet (IHLLS)

IHLLS Principle

A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique [1-7] to replace the lattice light sheet (LLS) tube lens [8] with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches.

Our imaging technique, incoherent holography lattice light-sheet (IHLLS), uses the excitation technology of a lattice light-sheet (LLS) system. However, it allows us to construct the 3D amplitude volume of complex objects such as neurons with resolutions comparable or better than the conventional LLS’s resolution in the diffraction limited dithering mode and can achieve an extended field of view (FOV). 

IHLLS uses self-interference [9,10] of the emitted fluorescent light to create Fresnel holograms of a 3D object in combination with the phase-shifting concept from which a single channel on-axis interferometer creates three to four interference patterns but in which the are created by a single channel on-axis interferometer. The interferometer beam splitter is replaced by a phase SLM. In this arrangement, each spherical beam propagating from the points of each 3D object is split into two spherical beams with different radii of curvature. The interference patterns are added incoherently, to further create Fresnel holograms. These holograms are numerically processed by in-house diffraction software.

Implementation of IHLLS was addressed in two stages:


1.      Build a copy of the LLS detection arm but using incoherent light (IHLLS-1L). The optical design optimization is performed using Opticstudio. This enabled us to compute the focal length ( ) of the single diffractive lens that was uploaded on the SLM, to match pixel sizes to a specific resolution in both conventional LLS and in IHLLS 1L. Similarly, the correct distances were computed between each optical component.


2.      Upload onto the SLM two diffractive lenses super-imposed but with different focal lengths. These lenses are composed of randomly selected pixels (IHLLS 2L), to give access to various depths in the sample through which only the z-galvo was moved within the displacement range. This range is centered at the objective reference focus position (the center of the camera FOV in the z-axis). Again, using Opticstudio, we designed a re-configurable optical system such that the the two spherical beam displacement was equal at the camera plane to overlap precisely. 

IHLLS System

The LLS and IHLLS detection systems. (a) Schematics of both detection systems, LLS (blue arrow), IHLLS (red arrow); Both systems share the water immersed microscope objective MO (Nikon 25X, NA 1.1, WD 2 mm). The LLS system consists of a glass-based tube lens, LTL = 500 mm, a Semrock FF01-446/523/600/677-25 bandpass filter, BPF1, and a Hamamatsu ORCA-Flash 4.0 v3 sCMOS. The diffraction mask in the LLS excitation path was centered for all experiments on an anulus with higher NA, NAout = 0.55 and NAin = 0.485, therefore the beams used for excitation were more Bessel-like beams. The IHLLS system is equipped with a phase spatial light modulator SLM (1920 x1152, 9.2 µm pixel size, Meadowlark Inc.), lenses L1= L4 with focal lengths 175 mm, L2= L3 with focal lengths 100 mm; mirrors M1 (sliding mirror), M2, M3; polarizer P; band pass filters BPF2 centered at 575 nm (Chroma Tech., 23 nm bandpass width) for the excitation wavelength  = 488 nm; The camera in the incoherent arm is another Hamamatsu ORCA-Flash 4.0 v3 sCMOS;  (b) one diffractive lens of focal length, fSLM = 415 mm at the phase shift q1 = 0, and (c) two diffractive lenses with focal lengths fd1 =228 mm and fd2 = 2444 mm, at the phase shift q1 = 0. Pannels (d) and (e) show the scanning and detection geometries for the LLS and IHLLS 1L techniques. The vectors represent the x, y, z and s planes of the Bessel beams that were focused by an excitation objective lens (not showing) to form a lattice light sheet at the sample plan. z and x are moved by the z and x galvos. Pannels (f) and (g) show the scanning and detection geometries for the IHLLS 2L technique. While the z-galvo and z-piezo are moved along the z axis to acquire stacks in LLS (d, e), in IHLLS 2L only the z-galvo is moved at various z positions. The approach schemes for the three experiments are illustrated in panels (h) – (l). The first three approaches demonstrate the ability to combine LLSM imaging within situ electrophysiology (h-j); (h) Hippocampal neuron imaging demonstrates the ability to resolve very sparse synaptic inputs to the dendrites of pyramidal cells in intact slices in site; (i) Axons in intact lamprey spinal cord were imaged at high speed (330 frames/second using Ca2+ sensitive dye, to demonstrate both the spatiotemporal resolution; (j) Lamprey-FM demonstrates the ability to investigate stimulus evoked lipid vesicle fusion and intracellular transport; We then demonstrated holographic approaches to image in situ Lamprey presynaptic structures (k, l); (k) IHLLS 1L, used for settings and calibration; and (l) IHLLS 2L, used for holographic imaging. 

Video showing a demo of the scanning idea in both systems, LLS and IHLLS.

Visualization_LLS_IHLLS.avi, http://prism.osapublishing.org/Staff/Details/425069

IHLLS OpticStudio Design

Optical design of the IHLLS; a) Ray tracing using only one diffractive lens; b) The spot diagram from a); c) Ray tracing using two diffractive lenses, fp1 the first focus position; d) The spot diagram from c); e) Ray tracing using two diffractive lenses, fp2 the second focus position; f) The spot diagram from e). The distance d1 is the distance between the MO and a dummy surface with infinite radius needed to check the beam collimation after the MO. The distance d2 is the distance between the dummy surface and lens TL1. 

IHLLS APPLICATIONS

Amplitude and Phase Cells Imaging

Neuronal Cells Deformation Measurements

Multi-color Neuronal Cells Imaging

Multi-color Colocalization Neuronal Cells Imaging

Future Research

 

Alzheimer’s disease (AD) is a devastating disease of the mind in which neurons are lost leading to dementia. For neurons to survive, they must maintain functional outputs. This synaptic function is damaged in AD, and there are extensive data on the role that APOE genotype plays. This has been studied extensively in postsynaptic mechanisms, however, paradoxically, there is much less is known of presynaptic effects of APOE genotype even though loss of synaptic release is tightly coupled to synaptic maintenance and to neuronal survival. Changes in presynaptic Ca2+ homeostasis will profoundly change synaptic transmission, plasticity, and neuron survival. Presynaptic Ca2+ triggers transmission, but residual Ca2+ in the terminal also provides short-term plasticity. These mechanisms are crucial in bursts of activity that induce long-term plasticity which form memories. if synaptic activity is lost, so are synapses, causing dying back neuropathy and neuronal death. Thus, understanding presynaptic Ca2+ in disease is crucial while its study has been neglected because it is hard to do. I will use my optical imaging skills in the aging and degenerating brain in mouse models. In short, cell culture cannot be used to study aging neurons, but using very high spatiotemporal resolution imaging in slices from aging brains solves this problem. I will determine relationships between aging, APOE genotype and changes in synaptic transmission modulated by changes in presynaptic Ca2+ homeostasis.

References