Transcript
Simultaneous whole‐animal 3D‐imaging of neuronal activity using light‐field microscopy
Robert Prevedel*, Young‐Gyu Yoon*, Maximilian Hoffmann, Nikita Pak, Gordon Wetzstein, Saul Kato, Tina Schrödel, Ramesh Raskar, Manuel Zimmer, Edward S. Boyden and Alipasha Vaziri * These authors contributed equally to this work. Correspondence should be addressed to E.S.B. (
[email protected]) or A.V. (
[email protected]). Tutorial for Building Light Field Microscope (Hardware) Overview An LFM system appended to an epifluorescence microscope can be implemented as follows. Any of these parts can be replaced by a proper alternative, so we briefly discuss the considerations.
The key components are microlens array, a microlens stage, a 1:1 relay lens and a camera. o Microlens array : Microlens array is the most important component for a LFM system. User may refer to Ref. 1, 2 to properly determine the focal length and the pitch. o Microlens stage : The volume reconstruction quality depends a lot on the alignment of the system, so user need to make sure that the microlens array is well aligned with respect to the image plane and the camera. In order to achieve good alignment, a microlens stage that allows both solid fixation and angle adjustment is needed. o 1:1 relay lens : Whereas the camera can be placed right behind the microlens array in theory, re‐imaging using a 1:1 relay lens provides a lot of mechanical
flexibility in practice. Image circle size, distortion and aberration should be considered when determining the relay lens. o Camera : As the volumetric imaging speed is solely determined by the frame rate of the camera (assuming that the speed is not limited by the number of photons), sCMOS camera is generally a reasonable choice as it can provide large field of view, large number of pixels and high frame rate. Other specification of cameras that are generally important for scientific imaging including read noise, dark current and dynamic range should also be considered.
Part List Vendor Zeiss Zeiss Andor Nikon OkoTech Thorlabs Thorlabs Thorlabs Thorlabs Thorlabs Thorlabs
Part Number Axiovert200
Description Microscope with imaging port Microscope Objective (20x 0.5 dry; 40x 0.95 dry) Zyla Zyla sCMOS camera 10tap Nikon AF‐S 105mm Macro‐lens (used in 1:1 relay setting) 2.8 G VR IF‐ED Micro OKO Tech Microlenses with lens' f# matched to objective (e.g. 150um pitch, f=3mm) APO-Q-P150-R1.37 K6XS 6‐Axis Locking Kinematic Optic Mount and CM1‐4ER/M Compact Clamping 4‐Port Prism/Mirror 30 mm Cage Cube M4 Tap KB75/M Complete 75 mm x 75 mm Kinematic Base Top and Bottom Plates Metric MT1/M 13 mm Translation Stage PT1/M 25 mm Translation Stage with Standard Micrometer M6 Taps various (min. 3x) posts, post holder and clamps dependent on beam height to mount camera and microlens array to translation stage
Alignment
Since adjacent pixels in light‐field images may contain vastly different implication on the volume, aligning the microscope well is crucial for getting high quality 3D volume.
sCMOS
Green Collimated Light Source
Dichroic Filter
Green Fluorescent Slide
Blue Excitation Light Source
This figure (not drawn to scale, relay lens is omitted) illustrates the setup for aligning LFM as well as taking calibration images for LFM. The light from collimated light source will appear as an array of spots on the image and the light from green fluorescent slide will appear as an array of circles. If the intensity of two light sources is adjusted such that one do not dominate another, both the array of spots and the array of circles will be visible as shown in the following figure.
As one can infer from this figure, the key features of the calibration image of well‐aligned LFM are as follows. ‐ The size of the spots will becomes small (in‐focus) ‐ The arrays of spots and circles appear uniform over whole field of view ‐ The centers of spots and circles coincide ‐ The array of circles is perfectly aligned with respect to the pixel array in terms of rotation Once the microscope is aligned, this calibration image can be used for finding rectification parameters, which are also crucial for obtaining high quality reconstruction results. References 1. M. Levoy, R. Ng, A. Adams et al., ACM Trans. Graph. 25 (3), 924 (2006). 2. M. Broxton, L. Grosenick, S. Yang et al., Optics Express 21 (21), 25418 (2013).
