Protocol for the Design and Assembly of a Light Sheet Light Field Microscope

Protocol Protocol for the Design and Assembly of a Light Sheet Light Field Microscope

Jorge Madrid-Wolﬀ 1 and Manu Forero-Shelton 2,* 1 Biomedical Computer Vision Group, Universidad de los Andes, Bogota 111711, Colombia 2 Biophysics Laboratory, Universidad de los Andes, Bogota 111711, Colombia * Correspondence: [email protected]; Tel.: +57-1-339-4949 (ext. 2621)

Received: 16 May 2019; Accepted: 1 July 2019; Published: 4 July 2019

Abstract: Light field microscopy is a recent development that makes it possible to obtain images of volumes with a single camera exposure, enabling studies of fast processes such as neural activity in zebrafish brains at high temporal resolution, at the expense of spatial resolution. Light sheet microscopy is also a recent method that reduces illumination intensity while increasing the signal-to-noise ratio with respect to confocal microscopes. While faster and gentler to samples than confocals for a similar resolution, light sheet microscopy is still slower than light field microscopy since it must collect volume slices sequentially. Nonetheless, the combination of the two methods, i.e., light field microscopes that have light sheet illumination, can help to improve the signal-to-noise ratio of light field microscopes and potentially improve their resolution. Building these microscopes requires much expertise, and the resources for doing so are limited. Here, we present a protocol to build a light field microscope with light sheet illumination. This protocol is also useful to build a light sheet microscope.

Keywords: light ﬁeld microscopy; light sheet ﬂuorescence microscopy; LSFM; 3D imaging; fast imaging; open science

1. Introduction Recent advances in fluorescent indicators such as calcium markers and transgenic model organisms have allowed the study of rapid dynamic biological processes in vivo like neural signaling, cardiovascular activity, and bacterial populations. Fast image acquisition methods have emerged in recent years to record these processes. Some of the fast methods use parts with little to no inertia in order to obtain slices at high speeds [1–6], while other methods such as extended depth of field [7,8] and integral imaging [9] capture the whole volume using a single camera exposure. Among these methods, Light Field Microscopy [10] allows for the fast acquisition of volumetric information of the sample from single camera exposures, at the expense of reduced spatial resolution. Nonetheless, light field microscopy has enabled functional imaging of neuronal activity at single-neuron resolution in the entire organism or portions of the larval zebrafish brain [11–13], adult Drosophila melanogaster [14], and mammalian brains [15]. This up-and-coming technique requires custom-made optical setups, which are intricate and non-trivial to design and align. Here we show a protocol to build and align such a microscope and include some basic design guidelines.

2. Experimental Design The major difference between a conventional microscope and a light field microscope is the inclusion of microlenses, which must be matched to the objective lens in f-number [16], making objective interchanges cumbersome for a single experiment. In this setup we have included a second detection arm to help with sample navigation, which is one of the main reasons why objectives are changed during an experiment. This arm has a shorter tube lens that results in a lower magnification

Methods Protoc. 2019, 2, 56; doi:10.3390/mps2030056 www.mdpi.com/journal/mps Methods Protoc. 2019, 2, 56 2 of 15 and a larger field of view that facilitates finding the area of interest in a sample, or the sample itself. We also use this portion of the detection to align the system. In the context of light field this is particularly important since it is hard to interpret light field data by eye, as it requires additional software or hardware and is not usually done in real time, although methods have been demonstrated [17]. In order to minimize light losses and maintain signal-to-noise ratios, the light sheet detection arm uses the long-wavelength tail of GFP´s emission spectrum for detection. A dichroic (Semrock, Rochester, NY, USA, FF560-FDi02) splits the light field from the light sheet detection arms around 560 nm. A bandpass (Chroma, Bellows Falls, VT, USA, ET525/50 m) filters the GFP signal for the light field, and a second bandpass between (Chroma, Bellows Falls, VT, USA, ET575/50 m) filters the light for the light sheet detection. Alternatively, a 90–10 beam splitter (Thorlabs, Newton, NJ, USA, BS076) and a single GFP fluorescence filter may be used. Several aspects should be considered in order to design the detection arm of a light field system. First, the f-numbers of the objective and tube lens combination must be matched. Mismatch will result in overlapping of the microlenses’ projections on the camera chip or sub-optimal use of the number of pixels. If the f-number of the detection system is smaller than that of the microlenses there will be light rays that will cross over from one microlens’ detection pixels to another’s, significantly compromising the reconstruction. Conversely, if the microlenses have a much smaller f-number than the detection system, there will be an increased number of pixels to which no light arrives. This will compromise the axial resolution of the system as less pixels will be available for sampling the impinging light rays. The f-numbers of microscope objectives are relatively standard, so matching is done by selecting from off-the-shelf microlens arrays (which come in a variety of f-numbers), and, if necessary, by adjusting the f-number of the objective-tube lens system. This is done by using a tube lens of a different focal length than the objective’s standard. Although this will influence image quality, changes in the order of 50% are manageable. Using a tube lens of a different focal length will result in a modified effective magnification, affecting the final resolution of the system. Second, we consider the desired resolution. Lateral resolution of the resulting reconstructions will be given by the number of microlenses in the array. Axial resolution depends on the number of resolvable spots on the camera chip behind each lenslet, as described by Levoy et al. [10]. Using a camera with a high pixel density is recommended to adequately sample the incoming rays. Recently, more complex methods such as compressive sensing [18], multiplexing [19], and speckle-based structured illumination [20] have further improved the axial resolution in light field microscopy imaging. Then one should consider the design of the illumination. The height of light sheets should be comparable to the field of view (FoV) of the detection, which can be calculated as Camera chip size FoV = (1) M where M is the magnification of the detection. Thus, the height of the light sheet (its extent along the y-axis, may be computed in terms of the original beam width and the lenses’ focal lengths as f beam expander .long fscan lens fill. obj. heightlight sheet = wout (2) fbeam expandershort fcylindrical lens ftube lens where wout is the beam radius at the output of the laser (typically ~500 µm). Note that the width of the beam at the galvanometric mirror

fbeam expander .long 2wgalv. = 2wout (3) fbeam expandershort may not exceed the size of the mirror, otherwise the beam will be clipped. The width of the light sheet at its waist depends on the eﬀective numerical aperture of the illumination: Methods Protoc. 2019, 2, x FOR PEER REVIEW 3 of 16 Methods Protoc. 2019, 2, 56 3 of 15 𝑓 .. 𝑓 2𝑤 𝑓.. 𝑓 (4) 𝑁𝐴 =𝑁𝐴 . fb.e. 𝑃𝑢𝑝𝑖𝑙long f𝑤𝑖𝑑𝑡ℎ..tube lens 2wout fb.e. fscan lens = short where 𝑃𝑢𝑝𝑖𝑙 𝑤𝑖𝑑𝑡ℎ.. is the sizeNA of ethe f f . backNA pupil of the illumination objective. If the fraction right(4) Pupil widthill. obj. to NA is greater than 1, some clipping of the beam will occur at the pupil, which is manageable. The widthwhere ofPupil the widthlight sheetill. obj. atis theits waist size of will the be back given pupil by, ofand the also illumination its Rayleigh objective. length (the If the distance fraction at right which to itsNA widthis greater will have than doubled) 1, some clipping by of the beam will occur at the pupil, which is manageable. The width of the light sheet at its waist will be given by, and also its Rayleigh length (the distance at which its width will have doubled) by 𝜆 2𝑤 = (5) 2𝑁𝐴λ 2w0 = . (5) 2NAe f f .

2 𝜋𝑤πwo x𝑥 == (6)(6) R λ𝜆 where λ is the laser’s wavelength. Equation Equation (6) (6) may may be be used used to to compute the the extent of the light sheet along the optical axis of the detection.detection. As As the the lig lightht sheet widens, contrast decreases and optical sectioning becomes less effective. eﬀective. The The Rayleigh Rayleigh leng lengthth of of the the light sheet should be matched with the size of the sample and the FoV of the detection. InIn our our proposed proposed setup, shown shown in in Figure Figure 11,, wewe havehave aa beambeam whichwhich isis 44 mmmm atat thethe galvanometricgalvanometric mirror and slightly overfills overﬁlls the 12 mm wide back pupil of the illuminati illuminationon objective along the zz-axis-axis with an extensionextension ofof 13.213.2 mm mm to to produce produce light light sheets sheets which which are are 350 350µm μ highm high and, and, at best, at best, 1 µm 1 atμm their at theirwaist. waist. The FoVThe ofFoV the of LF the detection LF detection at M ate f f .𝑀= 50=50is 260× is µ260m μm260 × µ260m μ andm and 205 205µm μm164 × 164µm μ form thefor . × × × theorthographic orthographic detection. detection. Olarte Olarte et al. et provide al. provide more mo comprehensivere comprehensive details details on the on design the design of a light of a sheetlight sheetmicroscope microscope [21]. [21].

Figure 1. OpticalOptical setup setup of of a a light light sheet sheet light light field ﬁeld microscope. Methods Protoc. 2019, 2, 56 4 of 15

2.1. Materials Silver protected mirrors (Thorlabs, USA, PF10-03-P01) • Kinematic mounts (Thorlabs, USA, KM100) • Aperture irises (Thorlabs, USA, ID20) • f = 7.5 mm achromat (Thorlabs, USA, AC050-008-A-ML) • f = 40 mm achromat (Edmund Optics, Barrington, NJ, USA, 49–354) • 1” lens mount (Thorlabs, USA, LMR1/M) • 0.5” les mount (Thorlabs, USA, SMR05/M) • f = 65 mm f-θ lens (Sill Optics, Wendelstein, Germany, S4LFT0061/065) • f = 250 mm achromat (Edmund Optics, USA, 49–366) • f = 75 mm mounted achromatic cylindrical lens (Thorlabs, USA, LJ1703RM) • N PLAN 10x NA 0.25 dry objective (Leica Microsystems, Wetzlar, Germany) • f = 100 mm achromat (Edmund Optics, USA, 49–360) • Cage plate (Thorlabs, USA, CP02/M) • SM1 coupler (Thorlabs, USA, SM1T2) • SM1 to C-mount adapter (Thorlabs, USA, SM1A9) • 4” cage rods (Thorlabs, USA, ER4-P4) • Dichroic beam splitter (Semrock, USA, FF560-FDi02) • Cage cube (Thorlabs, USA, CM1-DCH/M) • Fluorescence band-pass filter for GFP (Chroma, USA, ET525/50 m) • Fluorescence band-pass filter for GFP (Chroma, USA, ET575/50 m) • LUMFLN 60x NA 1.1 water immersion objective (Olympus, Tokyo, Japan, LUMFLN 60XW) • f = 150 mm achromat (Edmund Optics, USA, 49–362) • 1:1 Relay pair (Thorlabs, USA, MAP10100100-A) • Lens tube for 1” optics (Thorlabs, USA, SM30L05) • Microlense array (RPC, USA, MLA-S100-f21) • 6-axis kinematic mount (Thorlabs, USA, K6XS) • 2.2. Equipment 488 nm CW diode laser (Oxxius, Lannion, France, LBX-488-50-CSB-PP) • Galvanometric mirrors (Edmund Optics, USA, 6215H) • CCD monochrome camera (Point Grey, BC, Canada, Black Fly 13H2M) • Micromanipulator (Sutter Instruments, Novato, CA, USA, MPC-200) • sCMOS camera (Hamamatsu, Japan, ORCA flash 4.0 V2) • xyz kinematic mount (Newport, RI, USA, 9063-XYZ-M) • 3. Procedure

3.1. Construction of Light Sheet Illumination Time for completion: 12 h (may be interrupted at any point). This section deals with the construction and alignment of a single-wavelength scanned-light sheet illumination. The light sheet will be a Gaussian beam produced by a cylindrical lens scanned by a galvanometric mirror.

1. Trace the beam path: Trace the beam path from the laser (Oxxius, LBX-488-50-CSB-PP) to the place where the sample will go by means of mirrors (Thorlabs, PF10-03-P01) in kinematic mounts (Thorlabs, KM100), as in Figure2. Place the galvanometric mirror (Edmund Optics, 6215H) in the path. Mark the beam path at its end (ideally downstream from the sample’s position) with a pair of irises (Thorlabs, ID20). Do not change the position of the irises afterwards. Methods Protoc. 2019, 2, 56 5 of 15 Methods Protoc. 2019, 2, x FOR PEER REVIEW 5 of 16

Methods Protoc. 2019, 2, x FOR PEER REVIEW 6 of 16

Figure 2.2. TracingTracing thethe beam beam path path with with a paira pair of mirrors,of mirrors the, the galvanometric galvanometric mirror. mirror. Record Record the path the withpath awith pair a ofpair irises. of irises.

2. ExpandExpand the beam : Expand the laser beam as shown in Figure3 3.. MountMount aa pairpair ofof lenses,lenses, oneone ofof shortshort negative negative focal length (Thorlabs, f = 7.57.5 mm, mm, AC050-008-A-ML) AC050-008-A-ML) and a second lens of positive longlong focal focal length length (Edmund (Edmund Optics, f = 4040 mm, mm, 4 49–354)9–354) in lens mounts (Thorlabs, SMR05SMR05/M/M and LMR1/M,LMR1/M, respectively). First, First, insert insert the the short lens in the beam path. Finely adjust the positionposition andand orientation orientation of of this this lens lens to make sure that the center of the beam still passes through the centercenter of of the the irises. Insert Insert the the lens lens of of larger larger focal length approximately 47.5 mm (the sum of the focalfocal lengths) lengths) downstream downstream from the first first lens. Fine Finelyly adjust its position and orientation to make suresure that that the center of the beam still passes throughthrough the center of the irises. Alternatively, cage platesplates (Thorlabs,(Thorlabs, CP02CP02/M)/M) and and cage cage rods rods (Thorlabs, (Thorlabs, ER3-P4) ER3-P4) may may be used be used to ensure to ensure that the that lenses the lensesare aligned are aligned along along the optical the optical axis. axis. Adjust Adjust its position its position along along the opticalthe optical axis axis to ensure to ensure that that an anFigureexpanded expanded 3. Beam collimated collimated expansion. beam Adjustbeam is achieved. isthe achieved. distance This between This may may be th verifiede be two verified lenses by measuring to by guarantee measuring the that width the a collimated width of the of beam the beambeamat two isat pointsachieved. two points downstream Adjust downstream their and lateral ensuring and posi ensuringtions that so itthat remainsthat the it beam remains constant is not constant deviated. by focusing by focusing the beam the at beam infinity at infinity(i.e., several (i.e., several meters away),meters oraway), with or the with aid the of a aid shearing of a shearing interferometer interferometer (Thorlabs, (Thorlabs, SI050). SI050). 3.3. CRITICAL CRITICAL STEP:STEP: InsertInsert thethe scanscan lens:lens: PlacePlace the scan lens (We use an f-θ lenslens asas scanscan lens. A regularregular achromat achromat may may be usedbe used as well). as (Sillwell). Optics, (Sill S4LFT0061Optics, S4LFT0061/065)/065) a focal length a downstreamfocal length downstreamfrom the galvanometric from the galvanometric mirror, as in Figure mirror,4. Finelyas in Fi adjustgure its4. Finely position adjust to ensure its position that rotations to ensure of thatthe mirror rotations produce of the translations mirror produce of the translations beam from theof the main beam optical from axis the while main remaining optical axis parallel while remainingto it. Additionally, parallel checkto it. Additionally, that translations check correspond that translations to rotations correspond following to therotations equation following the equation θmirror z = fscan lens tan𝜃 (7) 𝑧=𝑓 tan 2 (7) 2 4. Optional Step: Produce a calibration function for the galvanometric mirror: The controlling software of some galvanometric mirrors often indicate the voltage applied to the device, but not the induced angle of rotation. Manufacturers should include the relationship θ(V) in their documentation. Collect data for a beam displacement vs. voltage calibration function, namely z(V). Methods Protoc. 2019, 2, 56 6 of 15 Methods Protoc. 2019, 2, x FOR PEER REVIEW 6 of 16

Figure 3. Beam expansion. Adjust Adjust the distance between th thee two lenses to guarantee that a collimated Methodsbeam Protoc. is achieved.2019, 2, x FOR Adjust PEER their REVIEW lateral positionspositions so that the beam is not deviated. 7 of 16

3. CRITICAL STEP: Insert the scan lens: Place the scan lens (We use an f-θ lens as scan lens. A regular achromat may be used as well). (Sill Optics, S4LFT0061/065) a focal length downstream from the galvanometric mirror, as in Figure 4. Finely adjust its position to ensure that rotations of the mirror produce translations of the beam from the main optical axis while remaining parallel to it. Additionally, check that translations correspond to rotations following the equation

𝜃 𝑧=𝑓 tan (7) 2

Figure 4. The scan lens should be one focal distance awayaway from thethe galvanometricgalvanometric mirror. If placed correctly, rotationsrotations ofof thethe mirrormirror willwill produceproduce displacementsdisplacements butbut notnot rotationsrotations of of the the beam. beam.

5.4. OptionalInsert the Step tube: Produce lens: Place a calibration the tube lens function (Edmund for Optics,the galvanometric f = 250 mm, mirror: 49–366) The and controlling adjust its softwareposition untilof some a collimated galvanometric beam mirrors is obtained, often as indicate in Figure the5 .voltage applied to the device, but not the induced angle of rotation. Manufacturers should include the relationship 𝜃𝑉 in their documentation. Collect data for a beam displacement vs. voltage calibration function, namely z(V). 5. Insert the tube lens: Place the tube lens (Edmund Optics, f = 250 mm, 49–366) and adjust its position until a collimated beam is obtained, as in Figure 5.

Figure 5. Placing the tube lens. The tube lens should produce a beam expander with the scan lens. If placed correctly, a well collimated beam will be achieved and, at the its focal plane, which will coincide with the illumination objective’s back focal plane, rotations of the mirror will produce rotations (Δ𝜃), but not translations (Δz) of the beam. Methods Protoc. 2019, 2, x FOR PEER REVIEW 7 of 16

Figure 4. The scan lens should be one focal distance away from the galvanometric mirror. If placed correctly, rotations of the mirror will produce displacements but not rotations of the beam.

4. Optional Step: Produce a calibration function for the galvanometric mirror: The controlling software of some galvanometric mirrors often indicate the voltage applied to the device, but not the induced angle of rotation. Manufacturers should include the relationship 𝜃𝑉 in their documentation. Collect data for a beam displacement vs. voltage calibration function, namely z(V). 5.Methods Insert Protoc. the2019 tube, 2, 56 lens: Place the tube lens (Edmund Optics, f = 250 mm, 49–366) and adjust7 of its 15 position until a collimated beam is obtained, as in Figure 5.

Figure 5. Placing the tube lens. The The tube lens should pr produceoduce a beam expander with the scan lens. If placed correctly,correctly, a a well well collimated collimated beam beam will will be achievedbe achieved and, atand, the at its the focal its plane, focal whichplane, will which coincide will Methodswithcoincide Protoc. the 2019 illuminationwith, 2 ,the x FOR illumination objective’sPEER REVIEW objective’s back focal plane,back focal rotations plane, of therotations mirror of will the produce mirror rotationswill produce (∆θ), 8 of 16 rotationsbut not translations (Δ𝜃), but not (∆ z)translations of the beam. (Δz) of the beam. 6. Insert the cylindrical lens: Place the cylindrical lens (Thorlabs, f = 75mm, LJ1703RM) and adjust 6. itsInsert position the cylindrical along the lens: opticalPlace axis the cylindricaluntil a sharp lens (Thorlabs, focused fline= 75mm, appears LJ1703RM) precisely and on adjust the galvanometricits position along mirror, the optical as in Figure axis until 6. Galvanometric a sharp focused mirrors line appears are very precisely small; on check the galvanometric that the beam ismirror, not clipped as in Figure by the6 mirror.. Galvanometric The orientation mirrors of are the verylens matters: small; check place that its flat the face beam pointing is not clipped toward itsby thefocus mirror. and Theensure orientation that a line of the along lens the matters: z-axis place will itsbe ﬂat produced face pointing at the toward back itspupil focus of andthe illuminationensure that a objective. line along the z-axis will be produced at the back pupil of the illumination objective.

FigureFigure 6.6. Place the cylindrical lens before the galvanometric mirror. Ensure that its focal plane lies on thethe mirror’smirror’s surface.surface.

7.7. Check the alignment : The focused line formed by the cylindrical lens should be relayed by thethe scan and tube lenses onto the backback focalfocal planeplane ofof thethe microscopemicroscope objective.objective. Check thatthat whilewhile rotatingrotating the galvanometric mirror, thethe lightlight sheetsheet rotatesrotates butbut isis notnot displaceddisplaced atat thisthis plane.plane. 8.8. InsertInsert the illumination objectiveobjective:: PlacePlace thethe illuminationillumination microscopemicroscope objectiveobjective (Leica(Leica NN PLANPLAN 10x NA 0.25).0.25). AdjustAdjust itsits positionposition along along the the optical optical axis axis until until a beama beam which which is collimated is collimated along along the they-axis y-axis but but highly highly divergent divergent along along the zthe-axis z-axis is achieved, is achieved, as in as Figure in Figure7. 7. Methods Protoc. 2019, 2, 56 8 of 15 Methods Protoc. 2019, 2, x FOR PEER REVIEW 9 of 16

Figure 7. PlacingPlacing the the illumination illumination objective. objective. If If placed placed co correctly,rrectly, a collimated collimated beam should be achieved along the y-axis, while strongly converging into a light sheetsheet alongalong thethe zz-axis.-axis.

3.2. Construction Construction of of the the Orthographic Orthographic Detection Time for completion: 3 h Time for completion: 3 h 1. Set up an orthographic detection system: Mount an achromatic tube lens (Edmund Optics, 1. Set up an orthographic detection system: Mount an achromatic tube lens (Edmund Optics, f = f = 100 mm, 49–360) in a cage plate (Thorlabs, CP02/M). Attach an inspection camera (Point Grey, 100 mm, 49–360) in a cage plate (Thorlabs, CP02/M). Attach an inspection camera (Point Grey, Black Fly 13H2M) to a cage plate by means of a SM1 coupler (Thorlabs, SM1T2) and a SM1 Black Fly 13H2M) to a cage plate by means of a SM1 coupler (Thorlabs, SM1T2) and a SM1 to C- to C-mount adapter (Thorlabs, SM1A9). Assemble the cage system with the use of four rods mount adapter (Thorlabs, SM1A9). Assemble the cage system with the use of four rods (Thorlabs, ER4). Adjust the distance from the tube lens to the camera sensor by obtaining a (Thorlabs, ER4). Adjust the distance from the tube lens to the camera sensor by obtaining a point- point-like image of a collimated light beam, or alternatively, by imaging a distant object, as in like image of a collimated light beam, or alternatively, by imaging a distant object, as in Figure 8. Figure8. 2. Insert the beamsplitter: Mount a dichoric beamsplitter (Semrock, FF560-FDi02) in a cage cube (Thorlabs, CM1-DCH/M). Attach the cube to the cage system built on step 1 so that the transmitted light from the beamsplitter reaches the inspection camera. Mind the orientation of the beamsplitter (refer to the manufacturer’s documentation for this). 3. Insert two fluorescence filters: Mount a fluorescence band-pass filter (Chroma, ET525/50 m) in a cage plate. Attach the cage plate to the cage cube from step 2 by means of cage rods. Light reflected from the dichroic filter should then go through the bandpass filter. Mind the orientation of the bandpass filter. Mount a second fluorescence band-pass filter (Chroma, USA, ET575/50 m), this time for the inspection camera. See Figure9. 4. Insert a detection objective: Mount an infinity-corrected microscope objective 140–170 mm in front of the achromatic tube lens to a cage plate, as in Figure 10. Use a dry long-working distance

objective for microscope construction, even if the apparatus is designed to work with water Figureimmersion 8. Building objectives. an orthographic We use a Leicadetection. 10 NAAdjust 0.25 the air distance objective between with externalthe tube M25lens threadsand the that camera until a sharp image of a distant object× is achieved, or a collimated beam appears focused. If necessary, use a neutral intensity filter to reduce the intensity of light reaching the camera to avoid damaging it. Methods Protoc. 2019, 2, x FOR PEER REVIEW 9 of 16

Figure 7. Placing the illumination objective. If placed correctly, a collimated beam should be achieved along the y-axis, while strongly converging into a light sheet along the z-axis.

3.2. Construction of the Orthographic Detection

Time for completion: 3 h 1. Set up an orthographic detection system: Mount an achromatic tube lens (Edmund Optics, f = Methods100 Protoc. mm,2019 49–360), 2, 56 in a cage plate (Thorlabs, CP02/M). Attach an inspection camera (Point Grey,9 of 15 Black Fly 13H2M) to a cage plate by means of a SM1 coupler (Thorlabs, SM1T2) and a SM1 to C- Methodsmount Protoc. adapter2019, 2, x FOR(Thorlabs, PEER REVIEW SM1A9). Assemble the cage system with the use of four 10 rods of 16 we couple via a thread adapter (Thorlabs, SM1A12) to a cage plate. Attach the cage plate to the (Thorlabs, ER4). Adjust the distance from the tube lens to the camera sensor by obtaining a point- two by means of cage rods. 2. likeInsert image the ofbeamsplitter: a collimated Mountlight beam, a dichoric or alternatively, beamsplitter by imaging(Semrock, a distant FF560-FDi02) object, as in in a Figurecage cube 8. (Thorlabs, CM1-DCH/M). Attach the cube to the cage system built on step 1 so that the transmitted light from the beamsplitter reaches the inspection camera. Mind the orientation of the beamsplitter (refer to the manufacturer’s documentation for this). 3. Insert two fluorescence filters: Mount a fluorescence band-pass filter (Chroma, ET525/50 m) in Methodsa Protoc.cage 2019plate., 2, Attachx FOR PEER the REVIEW cage plate to the cage cube from step 2 by means of cage rods. 10 Light of 16 reflected from the dichroic filter should then go through the bandpass filter. Mind the orientation 2. Insertof the thebandpass beamsplitter: filter. Mount Mount a asecond dichoric fluorescence beamsplitter band-pass (Semrock, filter FF560-FDi02) (Chroma, USA,in a cage ET575/50 cube (Thorlabs,m), this time CM1-DCH/M). for the inspection Attach camera. the cubeSee Figure to th e9. cage system built on step 1 so that the transmitted light from the beamsplitter reaches the inspection camera. Mind the orientation of the beamsplitter (refer to the manufacturer’s documentation for this). 3. Insert two fluorescence filters: Mount a fluorescence band-pass filter (Chroma, ET525/50 m) in a cage plate. Attach the cage plate to the cage cube from step 2 by means of cage rods. Light reflected from the dichroic filter should then go through the bandpass filter. Mind the orientation FigureFigure 8. Building anan orthographic orthographic detection. detection. Adjust Adjust the the distance distance between between the tube the lenstube and lens the and camera the ofcamerauntil the a sharpbandpassuntil a image sharp filter. of image a distant Mount of a object distant a second is achieved,object fluorescence is achieved, or a collimated band-passor a collimated beam filter appears beam (Chroma, focused. appears USA, Iffocused. necessary, ET575/50 If m),necessary,use this a neutral time use intensity fora neutral the inspection ﬁlter intensity to reduce filtercamera. the to intensityreduce See Figure th ofe intensity light 9. reaching of light the reaching camerato the avoid camera damaging to avoid it. damaging it.

Figure 9. Insert a beam splitter and two fluorescence filters before the tube lens.

4. Insert a detection objective: Mount an infinity-corrected microscope objective 140–170 mm in front of the achromatic tube lens to a cage plate, as in Figure 10. Use a dry long-working distance objective for microscope construction, even if the apparatus is designed to work with water immersion objectives. We use a Leica 10× NA 0.25 air objective with external M25 threads that we couple via a thread adapter (Thorlabs, SM1A12) to a cage plate. Attach the cage plate to the two by meansFigureFigure of 9. 9. cageInsertInsert rods. a a beam beam splitter splitter and and two two fluorescence ﬂuorescence filters ﬁlters before before the the tube tube lens. lens.

Figure 10. Place a detection objective.

5. UseUse homogeneous high-NA illumination: Place a Köhler Illumination module in front of the objective.objective. Maximize Maximize the the NA NA of of the the illumination illumination to to reduce reduce the the depth of field ﬁeld of the system and thusthus ensure ensure better better positioning positioning of the light field ﬁeld camera in the upcoming steps. 6. Focus a high contrast sample: Place a flat, high-contrast sample (ideally of known size) and obtain a sharp image of it on the inspection camera by adjusting its distance to the microscope objective, as in Figure 11. A Figuremicromanipulator 10. Place a detection (Sutter objective. Instruments, MPC-200) is useful for this task. Keep the sample in this fixed position. 5. Use homogeneous high-NA illumination: Place a Köhler Illumination module in front of the objective. Maximize the NA of the illumination to reduce the depth of field of the system and thus ensure better positioning of the light field camera in the upcoming steps. 6. Focus a high contrast sample: Place a flat, high-contrast sample (ideally of known size) and obtain a sharp image of it on the inspection camera by adjusting its distance to the microscope objective, as in Figure 11. A micromanipulator (Sutter Instruments, MPC-200) is useful for this task. Keep the sample in this fixed position. Methods Protoc. 2019, 2, 56 10 of 15

6. Focus a high contrast sample: Place a ﬂat, high-contrast sample (ideally of known size) and obtain a sharp image of it on the inspection camera by adjusting its distance to the microscope objective, as in Figure 11. A micromanipulator (Sutter Instruments, MPC-200) is useful for this Methodstask. Protoc. Keep 2019 the, 2, x sampleFOR PEER in REVIEW this ﬁxed position. 6 of 16 Methods Protoc. 2019, 2, x FOR PEER REVIEW 11 of 16

Figure 11. ImageImage a a high-contrast high-contrast sample. Displace Displace the samp samplele until a sharp image of it appears on the orthographic camera.

3.3. Construction of the Light Field Detection 3.3. ConstructionFigure 3. Beam of theexpansion. Light Field Adjust Detection the distance between the two lenses to guarantee that a collimated Timebeam foris achieved. completion: Adjust 10 their h lateral positions so that the beam is not deviated. Time for completion: 10 h 1.3. CRITICAL CRITICALSTEP: STEP: Insert Insert the the LF scan tube lens: lens Place: Mount the thescan main lens tube (We lens, use anan ff-=θ150 lens mm as scan achromat lens. 1. (EdmundA regularCRITICAL Optics, achromat STEP: 49–362), Insertmay in the abe cage LFused tube plate. as lens Insertwell).: Mount this(Sill cage the Optics, main plate tube inS4LFT0061/065) the lens, cage an rods f = 150 from a mm focal step achromat 3,length after (Edmunddownstreamthe bandpass Optics, from filter. 49–362), the The galvanometric light in a fieldcage systemplate. mirror, Insert is an as afocalthis in Fi cagegure detection plate 4. Finely in system, the adjust cage and rods its thus,position from the step detectionto 3,ensure after thethatobjective bandpass rotations and filter.theof the LF The tubemirror light lens produce field must system form translations a is 4f an system, afocal of the as detection inbeam Figure from system, 12; the that mainand is, the thus, optical back the focalaxis detection planewhile objectiveremainingof the detection and parallel the objective LF to tube it. Additionally, andlens themust front form focalcheck a 4 planef system,that oftranslations the as mainin Figure tube correspond 12; lens that must is, to coincide.the rotations back focal To following achieve plane ofthethis, the equation send detection a collimated objective beam and through the front the focal objective plane and of the adjust main the tube position lens ofmust the LFcoincide. tube lens To achieveuntil a collimatedthis, send a (although collimated expanded) beam through beam the exits ob it.jective and adjust the position of the LF tube 𝜃 lens until a collimated (although𝑧= expanded)𝑓 tanbeam exits it. (7) 2. Place a second camera: Attach a second camera that2 will record the light fields (Hamamatsu, ORCA flash 4.0 V2) to an xyz kinematic mount (Newport, 9063-XYZ-M), as in Figure 13. Place the camera downstream from the LF tube lens. Move it until a centered sharp image of the target appears in focus. While inspecting both cameras simultaneously, adjust the lateral and vertical position of the LF camera until both images are centered in the same place of the sample (one will be reflected by the beam splitter). Fix the camera to this position. 3. CRITICAL STEP: Acquire a reference image: Acquire an image of the sample at this moment. Measure it using image analysis software, such as ImageJ [22]. This image is important to guarantee that the right magnification is achieved when a relay pair of lenses is inserted in the upcoming steps. Keep the sample at its position until indicated. Do not pause the protocol until the end of step 4. The position of the sample may be lost, and magnification of the system may not be guaranteed. 4. Insert a relay pair: Move the main camera away from the main tube lens in order to give space to a relay lens pair. Insert the relay pair (Thorlabs, MAP10100100-A) in the light field detection; see

Figure 12. Insert the LF tube lens. Using collimated light, adjust the distance from the detection objective to the LF tube lens until a collimated beam results from the tube lens. This guarantees a 4f configuration between the objective and the tube lens. Methods Protoc. 2019, 2, x FOR PEER REVIEW 11 of 16

Figure 11. Image a high-contrast sample. Displace the sample until a sharp image of it appears on the orthographic camera.

3.3. Construction of the Light Field Detection

TimeMethods for Protoc. completion:2019, 2, 56 10 h 11 of 15

1. CRITICAL STEP: Insert the LF tube lens: Mount the main tube lens, an f = 150 mm achromat (EdmundFigure 14 .Optics, Ideally, 49–362), it should in be a cage mounted plate. so Insert that itthis may cage be movedplate in jointly the cage with rods the from main step camera, 3, after as theby attachingbandpass itfilter. to a The lens light tube field (Thorlabs, system SM30L05). is an afocal The detection relay pair system, is needed and thus, to project the detection the back objectivefocal plane and of the the LF microlenses tube lens must onto form the LF a 4 camera’sf system, chip, as in asFigure microlenses 12; that haveis, the a back very focal short plane focal oflength. the detection Adjust the objective positions and of thethe camera front focal and theplane relay of pairthe untilmain a tube sharp lens image must with coincide. the desired To achievemagniﬁcation this, send (that a collimated introduced beam by the through relay the pair) ob isjective achieved. and adjust Calculate the position the magniﬁcation of the LF tube by lensmeasuring until a collimated the size of the(although sample’s expanded) image and beam comparing exits it. it with the one obtained in step 3.

Methods Protoc. 2019, 2, x FOR PEER REVIEW 12 of 16

2. Place a second camera: Attach a second camera that will record the light fields (Hamamatsu, ORCA flash 4.0 V2) to an xyz kinematic mount (Newport, 9063-XYZ-M), as in Figure 13. Place the camera downstream from the LF tube lens. Move it until a centered sharp image of the target appears in focus. While inspecting both cameras simultaneously, adjust the lateral and vertical position of the LF camera until both images are centered in the same place of the sample (one will be reflected by the beam splitter). Fix the camera to this position. 3. CRITICAL STEP: Acquire a reference image: Acquire an image of the sample at this moment. Measure it using image analysis software, such as ImageJ [22]. This image is important to guarantee that the right magnification is achieved when a relay pair of lenses is inserted in the upcoming steps. Keep the sample at its position until indicated. Do not pause the protocol Figure 12. InsertInsert the the LF LF tube lens. Using Using collimated collimated lig light,ht, adjust adjust the the distance distance from the detection until the end of step 4. The position of the sample may be lost, and magnification of the system objective to the LF tube lens until a collimated be beamam results from the tube lens. This guarantees a 4 ff configurationconﬁgurationmay not be guaranteed. between the objective and the tube lens.

Figure 13. PlacePlace a asecond second camera camera and and acquire acquire a reference a reference image. image. Place Place a camera a camera after after the LF the tube LF lens, tube lens,making making sure sureto obtain to obtain a sharp a sharp image image of the of thesame same portion portion of ofthe the sample sample such such as as that that seen seen on the orthographic camera. Do not displace the sample. Acquire Acquire a a reference reference image image and and measure measure the the sample. sample. Use this image to ensure magniﬁcationmagnification whenwhen insertinginserting thethe relayrelay pair.pair.

4. Insert a relay pair: Move the main camera away from the main tube lens in order to give space to a relay lens pair. Insert the relay pair (Thorlabs, MAP10100100-A) in the light field detection; see Figure 14. Ideally, it should be mounted so that it may be moved jointly with the main camera, as by attaching it to a lens tube (Thorlabs, SM30L05). The relay pair is needed to project the back focal plane of the microlenses onto the LF camera’s chip, as microlenses have a very short focal length. Adjust the positions of the camera and the relay pair until a sharp image with the desired magnification (that introduced by the relay pair) is achieved. Calculate the magnification by measuring the size of the sample’s image and comparing it with the one obtained in step 3. Methods Protoc. 2019, 2, 56 12 of 15 MethodsMethods Protoc.Protoc. 20192019,, 22,, xx FORFOR PEERPEER REVIEWREVIEW 13 13 of of 16 16

FigureFigure 14. 14. InsertInsert a a relay relay pair. pair. Move Move the the camera camera backward backwardsbackwardss and place the relay relay pair. pair. An image image of of the the samplesample should should appear appear in in focus focus withwith theththee correctcorrect magniﬁcationmagnificationmagnification (from(from thethethe relay).relay).relay).

Insert the microlens array: 5.5. InsertInsert thethe microlensmicrolens array:array: MountMountMount aa a microlensmicrolens microlens arrayarray array (RPC,(RPC, MLA-S100-f21)MLA-S100-f21) inin aa 6-axis6-axis kinematic kinematic mountmountmount (Thorlabs,(Thorlabs, (Thorlabs, K6XS).K6XS). K6XS). InsertInsert Insert thethe microlensmicrolens arrayarray atat thethe imageimage planeplane ofof the the LF LF tube tube lens, lens, as as in in FigureFigureFigure 15.15.15. TheThe The microlensesmicrolenses microlenses willwill will sisi sittt onon on thethe the imageimage image planeplane plane ofof thethe mainmain tubetube lenslens whenwhen a a sharpsharp image image ofofof theirtheir edgesedges appearsappears onon toptop ofof the the sample. sample. AdAd Adjustjustjust thethe pitchpitch andand yaw yaw angles angles of of the the microlensmicrolens arrayarrayarray byby by meansmeans ofof thethe 6-axis6-axis kinematickinematic mountmount untiluntil thethe centralcentral regionregion of of the the microlens microlens array array is is uniformlyuniformlyuniformly in in focus.focus. RotateRotateRotate the thethe microlens microlensmicrolens array arrayarray to toto have havehave it asitit as alignedas alignedaligned as possible asas possiblepossible with withwith the pixelthethe pixelpixel grid gridgridof the ofof camera.thethe camera.camera. Move MoveMove the thethe camera cameracamera backwards backwardsbackwards and andand place placeplace the thethe relay relayrelay pair. pair.pair. An AnAn image imageimage of ofof thethe sample sample shouldshouldshould appearappear appear inin in focusfocus focus withwith thethe correctcorrect magnificationmagnification magniﬁcation (from (from the the relay). relay).

FigureFigure 15.15. InsertInsert thethe microlens microlens arrayarray at at the the image image plane plane of of thethe tubetube lens.lens. ThisThis is ensured ensured when when an an imageimage of of thethe microlensesmicrolenses appearsappears superimposedsuperimposed on on thethe imageimageimage ofofof thethethe sample.sample.sample. Then, Then, using using paraxial paraxial illumination,illumination, jointlyjointlyjointly move movemove the thethe relay relayrelay pair pairpair and andand the thethe camera cameracamera until untiluntil a grid aa gridgrid of dots ofof dotsdots appears appearsappears on the onon sensor. thethe sensor.sensor. This ThisensuresThis ensuresensures that the thatthat back thethe backback focal focalfocal plane plpl ofaneane the ofof lenslet thethe lensletlenslet array arrayarray is imaged. isis imaged.imaged.

6.6. OPTIONALOPTIONALOPTIONAL STEP:STEP: STEP: RemoveRemoveRemove thethe samplesample fromfrom thethe fieldfield ﬁeld ofof view.view. 7.7. CRITICALCRITICAL STEP:STEP: ImageImage thethe backback focalfocal planeplane ofof thethe microlenses:microlenses: ProvideProvide paraxialparaxial illumination,illumination, asas inin FigureFigure 15.15. ThisThis maymay bebe donedone byby closingclosing bothboth thethe fieldfield andand apertureaperture diaphragmdiaphragm Methods Protoc. 2019, 2, x FOR PEER REVIEW 6 of 16

Methods Protoc. 2019, 2, x FOR PEER REVIEW 14 of 16

of the Köhler illumination system or by sending an expanded well-collimated beam through the Figure 3. Beam expansion. Adjust the distance between the two lenses to guarantee that a collimated Methodsdetection. Protoc. 2019 , 2, 56 13 of 15 beam is achieved. Adjust their lateral positions so that the beam is not deviated. 8. Move the relay pair and the main camera away from the microlens array by its back focal length. Image the back focal plane of the microlenses. This will be achieved when a grid of the smallest 7.3. CRITICAL STEP: STEP: Insert Image the the scan back lens: focal Place plane the scan of thelens microlenses:(We use an f-θProvide lens as scan paraxial lens. spots is visible, such as in Figure 16. If possible, use light of a long wavelength to maximize the MethodsAillumination, Protoc. regular 2019 , 2achromat, asx FOR in Figure PEER may REVIEW 15 .be This used may beas donewell). by (Sill closing Optics, both theS4LFT0061/065) field and aperture a focal diaphragm 14length of 16 effects of diffraction on the lenslet array [23]. downstreamof the Köhler from illumination the galvanometric system or mirror, by sending as in anFigure expanded 4. Finely well-collimated adjust its position beam to through ensure of the Köhler illumination system or by sending an expanded well-collimated beam through the thatthe detection. rotations of the mirror produce translations of the beam from the main optical axis while detection. 8. remainingMove the relay parallel pair to and it. theAdditionally, main camera check away that from translations the microlens correspond array by to its rotations back focal following length. 8. Move the relay pair and the main camera away from the microlens array by its back focal length. theImage equation the back focal plane of the microlenses. This will be achieved when a grid of the smallest Image the back focal plane of the microlenses. This will be achieved when a grid of the smallest spots is visible, such as in Figure 16. If possible, use light of a long wavelength to maximize the spots is visible, such as in Figure 16. If possible, use𝜃 light of a long wavelength to maximize the effects of diffraction on the lenslet𝑧= array𝑓 [23].tan (7) effects of diffraction on the lenslet array [23]. 2

Figure 16. Back focal plane of the microlenses. When imaging the back focal plane, a grid of dots will appear in the camera chip. Each dot should be located at the center of each microlens. Use the 6-axis kinematic mount to ensure that the yaw and pitch angles of the microlens array are correct (if they are not, some microlenses will be in focus, while others are not). Ensure that the microlens grid is as aligned with the pixel grid as possible. (Magnified view).

9. Place the high NA detection objective: Put the high magnification, high NA objective Figure 16. Back focal plane of the microlenses. When imagin imagingg the back focal plane, a grid of dots will (Olympus, LUMFLN 60XW) in the detection path. We use a custom 3D printed chamber to place appear in the camera chip.chip. Each dotdot should be located at the center of eacheach microlens.microlens. Use the 6-axis the sample. Record a light field, such as the one in Figure 17. kinematic mount to ensure that the yaw and pitch anglesangles of the microlens array are correct (if they are not, some microlenses will be in focus,focus, while othersothers are not).not). Ensure that the microlens grid is as 4. Expected Results aligned with the pixel gridgrid asas possible.possible. (Magnified(Magnified view). To produce 3D imagery from light fields, such as those illustrated in Figure 18, there is open software9. PlacePlace for thethe reconstructions highhigh NA NA detection detection [10,11,17,23,24]. objective: objective:Put In theouPutr high reconstructions,the magnification, high magnification, we high show NA highthat objective byNA using (Olympus,objective light sheets(Olympus,LUMFLN to selectively 60XW) LUMFLN illuminate in the 60XW) detection the in sample’sthe path. detection We volume, use path. a custom weWe mayuse 3D a separate printedcustom 3Dchamberfeatures printed toin chamber placecomplementary the to sample. place acquisitions,theRecord sample. awhich light Record field,may ain such light turn as field, ease the onelocalizationsuch in as Figure the oneand 17 .in improve Figure 17.resolution.

4. Expected Results To produce 3D imagery from light fields, such as those illustrated in Figure 18, there is open software for reconstructions [10,11,17,23,24]. In our reconstructions, we show that by using light sheets to selectively illuminate the sample’s volume, we may separate features in complementary acquisitions, which may in turn ease localization and improve resolution.

Figure 17. Registered light field field of 6 μµm fluorescentfluorescent beads in agar. The light field field should appear as a grid of non-overlapping circles.

Figure 17. Registered light field of 6μm fluorescent beads in agar. The light field should appear as a grid of non-overlapping circles. Methods Protoc. 2019, 2, 56 14 of 15

4. Expected Results To produce 3D imagery from light ﬁelds, such as those illustrated in Figure 18, there is open software for reconstructions [10,11,17,23,24]. In our reconstructions, we show that by using light sheets to selectively illuminate the sample’s volume, we may separate features in complementary acquisitions, which may in turn ease localization and improve resolution. Methods Protoc. 2019, 2, x FOR PEER REVIEW 15 of 16

Figure 18.18.Reconstructions Reconstructions of lightof light fields fields of 6µ mof fluorescent6μm fluorescent beads in beads agar. (Ain,B )agar. Reconstructions (A) and (B of) lightReconstructions fields from complementarilyof light fields from illuminated complementarily portions illuminated of the sample portions volume. of ( Cthe) Reconstruction sample volume. of the(C) lightReconstruction field from theof the volume light under field brightfrom the field volume illumination. under (brightD–F) Profiles field illumination. of illumination (D, patternsE), and for(F) (ProfilesA–C), respectively. of illumination In (patternsD,E), sets for of (A 5 µ,Bm), and wide (C light), respectively. sheets provide In (D optical) and (E sectioning), sets of 5 ofμm the wide volume. light Scalebarssheets provide 50 µm. optical sectioning of the volume. Scalebars 50 μm.

Author Contributions:Contributions: Conceptualization,Conceptualization, J.M.-W.J.M.W. andand M.F.-S.;M.F.S.; methodology, J.M.-W.J.M.W. and M.F.-S.;M.F.S.; resources, M.F.-S.;M.F.S.; writing—originalwriting—original draft draft preparation, preparation, J.M.-W. J.M.W. and M.F.-S.; and writing—reviewM.F.S.; writing—review and editing, and M.F.-S.; editing, visualization, M.F.S.; J.M.-W.;visualization, supervision, J.M.W.; M.F.-S.;supervision, funding M.F.S.; acquisition, funding M.F.-S. acquisition, M.F.S. Funding: Funding: This research was funded by the Department of Physics, grants on equipment and parts (2016 and 2017), the Vice-presidency for Research “Financiación a grupos” grant, and a “terminación de proyectos” grant all at Universidad2017), the Vice-presidency de los Andes. for Research “Financiación a grupos” grant, and a “terminación de proyectos” grant all at Universidad de los Andes. Acknowledgments: We thank Omar E. Olarte for useful discussion and Luis Gomez and Joseph Merchan for designAcknowledgments: and fabrication We of thank custom Omar mechanical E. Olarte parts. for useful discussion and Luis Gomez and Joseph Merchan for Conﬂictsdesign and of fabrication Interest: The of custom authors mechanical declare no conﬂictparts. of interest. Conflicts of Interest: The authors declare no conflict of interest. References

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