A guide to light sheet fluorescence microscopy for multi-scale …€¦ · Supplementary Note 1 |...

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Supplementary Information A guide to light sheet fluorescence microscopy for multi-scale imaging Rory M. Power 1,2,3 , Jan Huisken 1,2,3 1 Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstr. 108, 01307 Dresden, Germany 2 Morgridge Institute for Research 330 N Orchard St, Madison, Wisconsin 53715, USA 3 Department of Biomedical Engineering, University of Wisconsin 1415 Engineering Drive, Madison, Wisconsin 53706, USA Supplementary Notes Supplementary Note 1 DIY Light Sheet Microscope Development Supplementary Note 2 Commercially Available and Construction Protocols for Light Sheet Microscopes Supplementary Note 3 Matching Imaging Technologies to the Sample Supplementary Note 4 Delivery of Illumination to the Sample Supplementary Note 5 Spatial Resolution and Field of View Supplementary Note 6 Objective Lenses for Light Sheet Fluorescence Microscopy Supplementary Note 7 Multi-View Imaging of Large Samples Supplementary Note 8 Delivering the Required Temporal Resolution Supplementary Figures Supplementary Figure 1 Relative rates of signal for one and two-photon excitation: n1p/n2p Supplementary Tables Supplementary Table 1 Commercial available light sheet microscopes Supplementary Table 2 Reported protocols for replication of light sheet microscopes Supplementary Table 3 Microscope geometry and sample mounting for live imaging applica- tions with light sheet fluorescence microscopy Supplementary Table 4 Reported light sheet microscopes, and applicability to various biological systems. Supplementary Table 5 A selection of objective lenses suitable for light sheet fluorescence mi- croscopy Nature Methods: doi:10.1038/nmeth.4224

Transcript of A guide to light sheet fluorescence microscopy for multi-scale …€¦ · Supplementary Note 1 |...

Page 1: A guide to light sheet fluorescence microscopy for multi-scale …€¦ · Supplementary Note 1 | DIY Light Sheet Microscope Development Developing a light sheet microscope can be

Supplementary Information

A guide to light sheet fluorescence microscopy for multi-scale imaging Rory M. Power 1,2,3, Jan Huisken 1,2,3

1 Max Planck Institute of Molecular Cell Biology and Genetics

Pfotenhauerstr. 108, 01307 Dresden, Germany 2 Morgridge Institute for Research

330 N Orchard St, Madison, Wisconsin 53715, USA 3 Department of Biomedical Engineering, University of Wisconsin

1415 Engineering Drive, Madison, Wisconsin 53706, USA

Supplementary Notes

Supplementary Note 1 DIY Light Sheet Microscope Development Supplementary Note 2 Commercially Available and Construction Protocols for Light Sheet

Microscopes Supplementary Note 3 Matching Imaging Technologies to the Sample Supplementary Note 4 Delivery of Illumination to the Sample Supplementary Note 5 Spatial Resolution and Field of View Supplementary Note 6 Objective Lenses for Light Sheet Fluorescence Microscopy Supplementary Note 7 Multi-View Imaging of Large Samples Supplementary Note 8 Delivering the Required Temporal Resolution

Supplementary Figures Supplementary Figure 1 Relative rates of signal for one and two-photon excitation: n1p/n2p

Supplementary Tables

Supplementary Table 1 Commercial available light sheet microscopes Supplementary Table 2 Reported protocols for replication of light sheet microscopes Supplementary Table 3 Microscope geometry and sample mounting for live imaging applica-

tions with light sheet fluorescence microscopy Supplementary Table 4 Reported light sheet microscopes, and applicability to various biological

systems. Supplementary Table 5 A selection of objective lenses suitable for light sheet fluorescence mi-

croscopy

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Supplementary Note 1 | DIY Light Sheet Microscope Development

Developing a light sheet microscope can be surprisingly simple with a few basic principles and a specific application in mind. Not only will this ultimately produce results superior to a generic system, it can also be highly cost effective and may be reconfigured later for other applications at minimal additional cost. The highly economical nature of light sheet microscopy has even lead to the suggestion of ‘SPIM farms’ as high-ly parallelized imaging systems based on the OpenSPIM architecture 1. The OpenSPIM project in particular has played a key part in expanding the reach of LSFM but also in demonstrating to non-specialists that only minimal training is needed to construct a research quality instrument. That being said, providing full pro-tocols for construction can go only so far in educating one in the principles of microscope design. Con-versely, academic texts typically focus on theory with little discussion of the practicalities associated with microscope development, which surely contains aspects of mechanical, electrical and optical engineering, computational science and a solid grounding in biology.

Here we attempt to distil the various aspects of light sheet microscope design, focusing on the optical con-figuration, arrangement in space and the relationship thereof with the sample preparation. This is not in-tended as a protocol for microscope construction or to illustrate how various beam paths can be configured (these topics are dealt with elsewhere1–5). Rather, the aim is to draw attention to some of the key decisions that should be made before embarking upon this path. It is worth noting that we do so with a bias towards in vivo imaging where light sheet microscopy has had the greatest impact and future potential. For fixed and cleared samples some of the principles may hold but the goals of anatomical biology are sufficiently abstracted from those of more biologically dynamic fields that the two are best considered in isolation.

Supplementary Note 2 | Commercially Available and Construction Protocols for Light Sheet Micro-scopes

In our discussion of tuning a given microscope to a specific application it is first necessary to consider the reverse where a single system is expected to handle many varied imaging tasks. Although there is no reason why a DIY microscope cannot be designed in this manner, this is typically the domain of commercial sys-tems. Construction protocols can also simplify the assembly of a basic light sheet microscope or replication of existing research instruments. Both cases are summarized in Supplementary Tables 1 and 2, respectively. See Supplementary Note 3 for a description of each of the geometries.

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Supplementary Table 1 | Commercial available light sheet microscopes

A selection of commercially available light sheet microscopes that can provide a useful resource and learning tool in large multi-user facilities but lack the customizability of DIY systems.

Manufacturer, Model Illumination Detection Geometry Optimum sample

3i, Lattice light sheet microscope

1-sided dithered/structured illumination lattice light sheet (NA max 0.65, water)

1-sided widefield detec-tion (25× /1.1, water)

Upright configuration (60/30˚ degree to vertical -illumination/detection)

Small and highly transpar-ent e.g. C. elegans embryo, superficial zebrafish tissues, cells.

Applied Scientific Instrumentation, iSPIM

1-sided static Gaussian light sheet (NA max 0.8, water)

1-sided widefield detec-tion (40×/0.8, water)

Upright configuration (45/45˚ degree to vertical illumination/detection)

Small samples or tissues prepared on coverslips. e.g. C. elegans embryos

Applied Scientific Instrumentation, diSPIM

2-sided static Gaussian light sheet (NA max 0.8, water). Objectives shared for illumina-tion and detection

2-sided widefield detec-tion (40×/0.8, water). Objectives shared for illumination and detec-tion

Upright configuration (45/45˚ degree to vertical -illumination/detection)

Small samples or tissues prepared on coverslips. e.g. C. elegans embryos

LaVision, Ultramicroscope II

2-sided static Gaussian light sheet (ultra-low NA, air)

1-sided widefield detec-tion (1.26× - 12.6×/0.5, air)

Upright configuration (90/0˚ degree to vertical -illumination/detection)

Very large, optically cleared samples

Leica, TCS SP8 DLS (Add on module for existing SP8 micro-scopes)

2-sided scanned Gaussian light sheet (NA max 0.15, air)

1-sided widefield detec-tion (10×/0.3, water or 25×/0.95, air)

Upright configuration (90/0˚ degree to vertical -illumination/detection).

Samples prepared on coverslips

Luxendo, MuVi-SPIM

2-sided scanned Gaussian light sheet (NA max 0.3, water)

2-sided widefield or confocal line detection (16×/0.8, or 25×/1.1, water).

Flat configuration (optical paths parallel with optical table surface)

Samples mounted vertically in FEP tubes or gelated cylinders

Zeiss, Lightsheet Z.1

2-sided scanned and pivoted Gaussian light sheet (NA max 0.2, air into water)

1-sided widefield detec-tion (5×/0.16, n=1.45 or 10×/0.5, 20×/1.0, 40×/1.0 or 63×/1.0 water)

Flat configuration (optical paths parallel with optical table surface)

Samples mounted vertically in FEP tubes or gelated cylinders

Supplementary Table 2 | Reported protocols for replication of light sheet microscopes

Both the OpenSPIM and OpenSPIN have been designed as open source initiatives with all part files and full infor-mation regarding construction freely available online 1,2. The diSPIM and Bessel beam plane illumination microscopes are based on existing research microscopes, which have either developed into commercial systems (Applied Scientific Instrumentation, iSPIM, diSPIM) or the designs have been superseded and subsequently commercialized (3i, Lattice light sheet microscope). In either case this may require non-disclosure agreements to obtain part files and software 3,4. The eduSPIM system has been designed to be operated by non-specialists for education purposes and with maintenance performed remotely 6,7.

Protocol Illumination Detection Geometry Optimum sample

OpenSPIM 1 1-sided static Gaussian light sheet (NA max 0.3)

1-sided widefield detection (20×/0.5, water). Can be used with or without 0.5x magnifi-cation adapter

Flat configuration (opti-cal paths parallel with optical table surface)

Samples mounted verti-cally in FEP tubes or gelated cylinders

OpenSPIN 2 1-sided static or scanned Gaussi-an light sheet (NA max 0.3, air (uncorrected)).

1-sided widefield detection (4× 0.13, air (uncorrected) or 16×/0.8, water).

Flat configuration (opti-cal paths parallel with optical table surface)

Samples mounted verti-cally in FEP tubes or gelated cylinders

diSPIM 3 2-sided static Gaussian light sheet (NA max 0.8, water). Objectives shared for illumination and detection

2-sided widefield detection (40×/0.8, water). Objectives shared for illumination and detection

Upright configuration (45/45˚ degree to vertical -illumination/detection)

Small samples or tissues prepared on coverslips. e.g. C. elegans embryos

Bessel beam plane illu-mination microscope 4

1-sided scanned/structured illumination Bessel light sheet (NA max 0.8 or NA max 0.65, water)

1-sided widefield detection (40×/0.8 or 25×/1.1)

Flat configuration (opti-cal paths parallel with optical table surface)

Small and highly trans-parent e.g. C. elegans embryo, superficial zebrafish tissues, cells.

eduSPIM 6 1-sided static Gaussian light sheet (NA max 0.1, air)

1-sided widefield detection (20×/0.4, air), correction for defocus

Flat configuration (opti-cal paths parallel with optical table surface)

Fixed sample in sealed chamber for long-term educational applications

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Supplementary Note 3 | Matching Imaging Technologies to the Sample

The natural starting point in designing a light sheet microscope is a specific sample and application. This is particularly crucial for new or exotic model organisms, but even where light sheet imaging has been report-ed in relatively mainstream sample types, this is no guarantee that the imaging protocol is appropriate. A key advantage of a custom-built microscope is that the geometry can be selected to accommodate a single sample with a specific preparation protocol. A few particularly innovative examples are discussed in the final section of the main text (see Towards the Future of LSFM). Sample mounting could amount to an en-tirely separate study unto itself, however since most sample preparation protocols are devised outside the immediate sphere of LSFM development, this aspect of imaging has only been highlighted when it directly pertains to the microscope geometry.

While more conventional microscopes have traditionally defined the properties of a sample to be imaged; often dictating that it should be thin, transparent and ideally flat on a coverslip. The longer working dis-tances typical of light sheet microscopes (owing to comparatively good performance at low-moderate NA) have allowed easier imaging of truly 3D samples and have in most cases allowed the coverslip to be elimi-nated altogether. While traditional mounting protocols, which may have merit for some samples can still be accommodated 3, more typically LSFM has required a rethink in the way samples are prepared and posi-tioned for imaging. Although this usually provides a more physiologically relevant environment, the influ-ence of mounting goes under-reported, particularly for long-term imaging where the sample may traverse several developmental stages, growing considerably as it does so 8.

In many cases the term sample mounting may be somewhat of a misnomer since samples are often only gently supported/enclosed 8 or indeed allowed to move freely 9. Regardless the main methods can be sum-marized as embedded, usually in a hydrogel cylinder; hanging, where the sample is supported by a hook, rood or by the suction of a pipette; enclosed, for example in a refractive index matched tube, or flow through, adopting a microfluidic approach 10. There are of course others, which may be specific for a given sample or application. The crucial point, particularly for unfamiliar samples is that one should first estab-lish how to isolate, position and orient the sample without unduly affecting normal biological function or development. Naturally, one should also bear in mind the optical consequences of the mounting protocol; hydrogels and perfluorinated polymers have a refractive index close to that of water and so are ideal for imaging in aqueous media. Once the optimum sample preparation protocol has been developed one may design a microscope to function specifically in this context. This should include an easy way to introduce the prepared sample and subsequently position it within the co-aligned field of views and allow sufficient space for any associated life support systems (e.g. temperature and permeated gas control). The size of the sample may dictate that multi-view imaging is a requirement and one must consider whether time con-straints allow this to be implemented without adding additional optical pathways. One must also consider the interaction of the sample and it’s mounting with gravity. For example, gelated cylinders will bend unless the axis of rotational symmetry and gravity are parallel. Moreover, samples free of rigid support may settle in tubes and so some consideration has to be given to how to maintain buoyancy in the context of flow-through systems.

Light sheet geometries can be categorized as with more conventional microscopes. For the sake of discus-sion, we define the geometry according to the alignment of optical pathways with regard to the underlying optical table surface and the facing of the objective lenses. This provides three clear categories: the first be-ing flat, where the optical pathways and table surface are parallel. The second and third are both defined by the optical table surface and optical pathways being perpendicular. These are then categorized as upright or inverted where the objectives point down or up respectively (imaging from above and below). There is some confusion as to this nomenclature since some upright systems have come to be referred to as inverted. Regardless, in the upright/inverted cases the optical pathway and table need only be perpendicular in a sin-gle axis. In the other axis, the angle made with the normal to the table surface may vary. For example, the dual-view inverted SPIM (diSPIM) (actually an upright system) has both optical pathways at 45˚ either side of the surface normal 3, whereas the inverted light sheet microscope (truly inverted) has the detection path at 30˚ and the illumination path at 60˚ respectively 11.

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Of course, these geometries should not constrain the choice of how to orient the microscope. For common sample preparations or model organisms however, one may wish to follow well-established protocols for light sheet imaging. For example, zebrafish and drosophila embryos are typically embedded or enclosed and vertically oriented such that they may be imaged in their entirety (at least for early developmental stages) using multi-view imaging 12,13. The nematode worm C. elegans presides over a legacy of coverslip-based preparations, which remain prevalent and can be imaged from above in an upright configuration (e.g. iSPIM, diSPIM 3,14). These three model organisms are arguably the most well studied using light sheet mi-croscopy and have all spurred the development of light sheet systems specifically geared to image them. In addition, any number of individual cell lines and their aggregates, which are each conceptually similar enough in their imaging have been studied along with a multitude of other model systems familiar or oth-erwise. These are summarized in Supplementary Table 3. Where more than one example exists, priority has been given to more generally representative approaches. Again, we focus only on live imaging applications that have been reported. A wealth of fixed tissues has also been imaged, although the choice of mounting and geometry is naturally less concerned with the continued health of the sample than practicalities relating to clearing and immersion media.

Taking the opposite approach, it may be equally useful to identify appropriate imaging technologies for a specific biological system or question by considering the type of studies each has enabled. Supplementary Table 4 documents the key LSFM platforms reported in the main text, the microscope geometry and defin-ing optical features as well as the approximate engineering challenge to replicate the design. Regarding the sample itself, the ideal size/type and reported applications are detailed.

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Supplementary Table 3 | Microscope geometry and sample mounting for live imaging applications with light sheet fluorescence microscopy

The examples given cannot cover the diversity of samples to which LSFM has been applied in its entirety, particularly as LSFM finds ever more application in model organisms that may have previously been deemed too light sensitive for time-lapse imaging Model Organism/Sample Microscope Geometry Sample Mounting

Zebrafish (danio rerio) (embryo/larva) Flat Typically enclosed in FEP tubes 8 or embedded in agarose cylinders 5 Fruit fly (drosophila melanogaster) (em-bryo/larva) Flat Typically embedded in agarose cylinders 15

Mouse (embryo)

Flat Inverted

Post-implantation either enclosed in hollow agarose cylinder 16 or mechanically held at the Reichert’s membrane using an acrylic role with various sized holes 17 Pre-implantation embryos supported on FEP foil 11

Mouse (adult) Upright (single objec-tive oblique) Head-fixed with cranial window 9

C. elegans (embryo/adult) (nematode worm) Upright Prepared on cover slips 14,18,19 Xenopus laevis (embryo) (African clawed frog) Flat No details given 20

Arabidopsis thaliana (Thale cress) Flat Freely growing in chamber 21 or grown within or on the surface of rigidified gels 22

Tribolium castaneum (red flour beetle) (embryo/larva) Flat Egg surface affixed at one end by agarose hemisphere 23

Volvox (green algae) (embryo) Flat No details given 24 Medaka fish (Oryzias latipes) (embryo) Flat Embedded in agarose cylinders 15

Chick (embryo) Upright Supported on silicone oil in a custom developmental chamber and fixed at vitelline membrane with o-ring to isolate hypoblast of em-bryo from external environment.

Flatworm (M. crozieri) (embryo) Flat Embedded in agarose cylinders 25 Snail (Crepidula fornicata) (embryo) Flat Mounted in seawater between coverslips 26 Sea urchin (Paracentrotus lividus) (embryo) Flat No details given 27 Phytoplankton (Gambierdiscus sp., Procen-trum sp.) Flat Flow-through 28

Cultured tissue slices Upright Cultured and transferred directly into perfused sample chamber 29

Cultured cells Upright Flat

Prepared on coverslips 19,30–32 Embedded in collagen matrix 33

Multi-cellular spheroids Flat Embedded in agarose 34–36

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Supplementary Table 4 | Reported light sheet microscopes, and applicability to various biological systems

The examples given are not exhaustive but offer an overview of the most significant technical advances. Each of the techniques is detailed in the main text. The microscope geome-tries are again denoted as flat (F), upright (U) or inverted (I). The ideal sample size is given to indicate the dimension of a cubic volume that could be effectively imaged using each of the techniques, of course the sample may be larger (allowing for steric limitations) and some relatively superficial sub-volume imaged. The limits are by no means hard rules but provide a useful guide to what can be imaged and what could be more easily imaged with a less advanced technique

LSFM Platform Design Purpose Defining Optical Features Geometry Challenge to Build Ideal Sample Size

Ideal Sample Type Reported applications

Selective Plane Illumination Microscopy 15/Multi-Directional SPIM 5 (SPIM/mSPIM)

Large Volume Imaging

Static/pivoted light sheet, 1/2-sided illumination F Simple 100 – 500 µm

Small transparent embryos/small embryos

Numerous

Digitally Scanned Light Sheet Microscopy 37 /Structured-Illumination DSLM 38 (DSLM/DSLM-SI)

Large Volume Imaging

Scanned light sheet /with structured illumination F Simple 100 – 500 µm Small embryos Numerous

Individual Molecule Localization SPIM 34 (IML-SPIM) Super resolution Superimposed photo-activation and

imaging beam F Simple 1 – 20 µm Small embryos/ multiple cells Cellular spheroids

Reflected Light Sheet Microscopy 30 (RLSM) Single molecule Opposed ultra-high NA arrangement, 45˚ mirrored cantilever I Moderate 1 – 20 µm Single cells Transcription factor binding in single live cells

Single Objective SPIM 31 (soSPIM)

Super resolution, reduced steric constraints

Single ultra-high NA objective arrange-ment, 45˚ mirrored sample support, tunable lens for light sheet waist reposi-tioning

I Challenging 1 – 20 µm Single cells Live cells and Drosophila embryos

Axial Plane Optical Microscopy 39 (APOM) High resolution, reduced steric constraints

Single ultra-high NA objective arrange-ment, remote image rotation optics I Moderate 1 – 20 µm Single cells Pollen and mouse brain slices

πSPIM 40 High resolution Oblique high NA objective arrangement, near unity angular fill factor for illumina-tion and detection, image splitter

U Moderate 1 – 20 µm Single cells Endocytosis in live yeast

Bessel Beam Plane Illumination Microscopy 41 Isotropic high resolution over extended FOV

Scanned Bessel beam produced by physi-cal apertures, structured illumination F Moderate 10 – 100 µm Small embryos/

multiple cells Dynamics in live cells

Lattice Light Sheet Microscopy 19,42 Isotropic high/super resolution over extended FOV

Coherent Bessel beam array (lattice) light sheet, structured illumination, photo-activation capability, custom illumination optics

U Very Challenging 10 – 100 µm Small transparent embryos/ multiple cells

Sub-cellular dynamics, cell-cell/matrix inter-actions in live cells. In toto C. elegans embryo-genesis. Drosophila dorsal closure. Zebrafish neuromast

Real-Time Optimized Tiling Light Sheet SPIM 43 Isotropic high resolution over extended FOV

Coherent Bessel beam array (lattice) light sheet with dynamic light sheet tiling F Very Challenging 10 – 100 µm

Small transparent embryos/ multiple cells

In toto C. elegans embryogenesis. Imaging of zebrafish early embryogenesis

Axially Swept Light Sheet Microscopy 44 (ASLM)

Isotropic high resolution over extended FOV

Remotely refocused, swept light sheet and confocal line detection F Moderate 10 – 100 µm Small embryos/

multiple cells Cell-matrix interactions

RESOLFT Light Sheet Microscopy 32

Isotropic high resolution over extended FOV

Superimposed photo-activation, depletion and imaging beams U Challenging 10 – 100 µm Small embryos/ few

cells Structural proteins in live cells

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Two-Photon SPIM 45 (2P-SPIM) Large volume imaging 2 illumination paths, two-photon excita-

tion F Moderate 250 µm – 1 mm Embryos Long term in toto Drosophila embryogenesis

Multi-Color Two-Photon Light Sheet Microscopy 46

Large volume, multi-color imaging

2 illumination paths, multi-color two photon excitation by spectral overlap, image splitter

F Challenging 250 µm – 1 mm Embryos Imaging of Drosophila embryogenesis, beating zebrafish heart

Multi-View SPIM 47/ Confocal Multi-View SPIM 48 (MuVi-SPIM/C-MuVi-SPIM) Large volume imaging 2 illumination and 2 detection paths /

confocal line detection F Moderate-Challenging 250 µm – 1 mm Embryos Long term in toto Drosophila embryogenesis

Simultaneous Multi-View Light Sheet Micros-copy 49,50 (SiMView/AutoPilot) Large volume imaging

2 illumination and detection paths, one/two-photon excitation/real-time adaptive realignment

F Challenging/Very Challenging 250 µm – 1 mm Embryos Long term in toto Drosophila/zebrafish

embryogenesis. Zebrafish functional imaging

4-Lens SPIM 12 Large volume imaging 2 illumination and 2 detection paths, pivoted light sheets F Moderate-

Challenging 250 µm – 1 mm Embryos Long term in toto zebrafish embryogenesis

Isotropic Multi-View Light Sheet Microscopy 13 (IsoView)

Large volume imaging, isotropic resolution

4 objectives each for illumination and detection, confocal line detection F Very Challenging 250 µm – 1 mm Embryos

Long term in toto Drosophila embryogenesis. In toto Drosophila functional imaging. Whole brain zebrafish functional imaging

Inverted SPIM 14 (iSPIM)

High resolution, sample adapted imag-ing

Pivoted light sheet U Simple 50 – 300 µm Small embryos/ cultured tissue

Long term in toto C. elegans embryogenesis. Tissue slice functional imaging

Dual-View Inverted SPIM 18 (diSPIM)

Isotropic high resolu-tion, sample adapted imaging

2 objectives each used for illumination and detection, pivoted light sheets U Moderate 50 – 300 µm Small embryos/

cultured tissue Long term imaging of C. elegans embryogene-sis

Ultramicroscope 51 Ultra-large volume imaging Ultra-low NA optics, 2 illumination paths U Simple 500 µm – 2 cm Cleared tissues Structural imaging of fixed and cleared mouse

brains/embryos and Drosophila Clarity Optimized Light Sheet Microscopy 52 (COLM)

Ultra-large volume imaging 2 illumination paths, adaptive realignment F Moderate 500 µm – 2 cm Cleared tissues Structural imaging of fixed and cleared mouse

brains (High-Speed Simultaneous Multi-View Light Sheet Microscopy 53 (hs-SiMView)

Large volume, ultrafast imaging

2 illumination and 2 detection paths, fast objective scanning, one/two-photon excitation

F Challenging 100 – 500 µm Small embryos In toto Drosophila functional imaging.

Electrically Tunable Lens SPIM 54,55 (ETL-SPIM) Ultrafast imaging Electrically tunable lens based refocusing,

image splitter, ultrafast camera F Simple 100 – 250 µm Small embryos Zebrafish vasculature and heart

Wavefront Coding Light Sheet Microscopy 56,57 Ultrafast imaging Cubic phase plate modulating element for

extended depth of field imaging F/U Moderate 100 – 250 µm Small embryos Zebrafish functional imaging

Spherical Aberration Assisted Light Sheet Microscopy 58 (SPED) Ultrafast imaging 2 illumination paths, spherical aberration

induced extended depth of field imaging F Simple 250 µm – 1 mm Embryos Zebrafish functional imaging

Oblique Plane Microscopy 59/Swept Confocally-Aligned Planar Excitation Microscopy 9 (OPM/SCAPE)

Ultrafast imaging, reduced steric con-straints

Single objective, image rotation optics in detection path U/I Moderate 100 – 250 µm Any

Functional imaging of rat cardiac myocytes. Functional imaging in cortex of freely behav-ing mice. Freely moving Drosophila larvae

Hyperspectral Light Sheet Microscopy 60 Multi-color imaging Spectrally dispersive de-scanned detection F Moderate 100 – 250 µm Small embryos Zebrafish and Drosophila embryos

Inverted Light Sheet Microscopy 11 Sample adapted imag-ing

Scanned light sheet, demagnified detec-tion U Moderate 100 – 250 µm Small embryos Pre-implantation mouse embryogenesis

Upright Light Sheet Microscopy 61 Sample adapted, large volume imaging 3 illumination paths U Moderate 250 µm – 1 mm Embryos Functional imaging of head-mounted behav-

ing zebrafish

Upright Two-Photon Light Sheet Microscopy 62 Sample adapted, large volume imaging

2 illumination paths, two-photon excita-tion U Moderate 250 µm – 1 mm Embryos Functional imaging of zebrafish

Optical Projection Tomography Integrated SPIM 63 (OPTiSPIM) Multi-modal imaging Ultra-low NA optics F Simple 500 µm – 2 cm Cleared tissues Structural imaging of fixed and cleared mouse

embryos Spiral Optical Projection Tomography SPIM 64 Multi-modal imaging Helical acquisition scheme F Simple 250 µm – 1 mm Embryos Long term in toto zebrafish embryogenesis

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Supplementary Note 4 | Delivery of Illumination to the Sample

The corrupting and pernicious influence of phototoxicity in live imaging generally goes underreported and it is worth addressing the subject at this juncture, so that the developer may understand the need for vigi-lance when designing a light sheet microscope. Many developments have been reported with little consider-ation of the biological system under study, and while light sheet fluorescence microscopy has become a catchall term for low phototoxicity the degree to which this is deserved depends largely on how the illumi-nation is delivered.

The importance of phototoxicity is starting to be recognized in live cell super-resolution microscopies in particular 65, where high light intensities are taken as an occupational necessity. Continuing this trend is crucial in validating the claim that light sheet fluorescence microscopy is effectively biologically harmless. It should serve as a cautionary note that many of the developments outlined in this review have moved in the opposite direction. While this may be excusable when the desired spatiotemporal resolution or penetration could not be achieved in any other way, it is far from a desirable practice and any associated biological re-sults should be assessed with some scrutiny.

In an age of continuously replenished genetically encoded reporters, photobleaching does not constitute a meaningful readout of the light dose and can only signal a diminishing time window for effective fluores-cence imaging. When photobleaching is apparent, it is likely that phototoxicity has long since rendered the sample unviable. Where possible, embryos should be removed from their mounting after imaging and al-lowed to develop normally, spawn progeny and otherwise go on to undergo an unperturbed life cycle. Iso-lated cells and tissues should be rigorously compared with controls and their rates of division, microtubule growth 65 or retraction/protrusion 66 can provide indicators of their continued health.

In general, light exposure should be kept to an absolute minimum. The maximum acceptable light level does not deliver the highest quality image but rather the minimum signal-to-noise that still allows the nec-essary quantitative analyses to be performed. Stelzer makes the cogent argument that exposure levels in all light microscopies are beyond that which an organism has evolved to handle 67. In the most lenient case this means the solar luminosity per unit area at the equator (ca. 1 kW m-2 = 1 kJ s-1 m-2), however light sensitive organisms such as corals 68 or nematode worms 18 are adapted to levels far lower than even this. Moreover, studies typically show that the photon energy itself is extremely important; using fluorophores with red-shifted absorption spectra has been shown to increase total exposure times for irreparable photodamage many times over 65,69.

The way light is delivered should also be considered, photodamage is considered to largely originate from non-linear processes 70 and so spreading the light dose across time and space as much as possible is a sensi-ble precaution. To illustrate this point, we consider the cases associated with static and scanned light sheets under one- and two-photon excitation. Assuming that the light sheet with an average thickness across the field of view (FOV) of 10 µm and a length of 400 µm illuminates a volume of the sample equal to 10 × 400 × 400 µm3 (note these parameters are reasonable for a beam with NA = 0.02, l = 488 nm). At equatorial solar intensity, this equates to 4 µW of laser power over the area that the static light sheet would be incident on the sample (4000 µm2). For the scanned light sheet, the evolutionarily adapted laser power is lower still since the beam area reduces by a factor of 40 (100 µm2), permitting only 100 nW of laser power. Perhaps these limits stretch credulity somewhat; samples are regularly exposed to far higher light levels under rou-tine microscopic examination. Yet it is unclear whether for a given sample and wavelength, there is some threshold intensity below which photodamage can be considered effectively linear 71. If this is apparent, the limits are likely to be very low indeed. Recall that one of the key contributors to the low photodamage po-tential of LSFM is the increased parallelization resulting in lower peak intensities and longer dwell times per pixel. Clearly both the total and peak light dose are likely to be important, but so too are temporal dynamics relating to excited state decay times 66 and unraveling these interrelated contributions remains challenging. In light of this complexity, one should aim to minimize the peak power to avoid non-linear photodamage.

A direct comparison between one and two-photon excitation is more difficult to make. Two-photon mi-croscopy dominates in some fields such as mammalian neuroscience 72 but its application to embryos in particular has been more limited 73 and remains a questionable practice. Regardless, one can follow a similar

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line of enquiry to determine typical signal rates. To do so one must consider the fluorophore photo-physics under the two regimes, characterized by the molecular absorption cross-sections, s, and quantum efficien-cies, F. Taking EGFP as an example, one can find that the molecular cross-section and QEs for one and two-photon excitation (1p/2p) are: s1p = 2.26 × 10-20 m2, F1p = 0.6 (at l1p = 488 nm); s2p = 3.9 × 10-56 m4 s, F2p = 0.77 (at l2p = 927 nm) 74. Note these quantities are notoriously difficult to measure accurately, although the values for EGFP are representative of a range of fluorophores under one- and two-photon excitation 74.

The number of fluorescent photons produced per second per fluorophore can be described in either case by 75:

n1p =πNAill

2

hcλ1p

!

"##

$

%&&Pσ1pΦ1p

(S1)

n2 p =0.664Fpτ p

πNAill2

hcλ2 p

!

"##

$

%&&

2

P 2σ 2 pΦ2 p

(S2)

Under continuous wave illumination (one-photon case) P is the laser power, h is Planck’s constant, NAill is the focusing numerical aperture. With pulsed illumination (two-photon case) ⟨P⟩ is the average laser pow-er, Fp is the pulse frequency and tp is the pulse duration. Note the associated 0.664 pre-factor assumes a Gaussian temporal pulse distribution 75. Again, a direct comparison is difficult, multi-photon microscopies typically use far higher time-averaged laser power than the corresponding single-photon analogue. There is some justification for this: a single photon at 927 nm is likely to be far less damaging than a single photon at 488 nm. Non-linear photodamage in point-scanning multi-photon microscopy has been well studied 76,77 and we would welcome similar studies in an LSFM context to disentangle the contributing factors. Regard-less, taking typical values of Fp = 80 MHz, tp = 140 fs one can explore the laser power and NA dependence of the two modes as shown in Supplementary Figure 1.

Supplementary Figure 1 | Relative rates of signal for one and two-photon excitation: n1p/n2p.

As a function of laser power (red) or effective illumination NA (blue). The time averaged power (2p) = power (1p). For one-photon excitation the illumination NA = effective illumination NA. For the same NA but with two-photon excita-tion, the light sheet is on one hand thinner by a factor of √2 due to the non-linearity of the evolved signal 62 but also thicker by a factor of l2p/l1p. Therefore, the signal rate for the two-photon case has been calculated using NA2p=NA1p.( l2p/(l1p.√2)) to remove any contribution due to the different beam waists, but has been plotted for the equivalent one-photon excitation NA.

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Clearly, under truly physiological light levels the signal evolved in the one-photon case is vastly superior, and very high laser power is required to compensate when using two-photon excitation. Similarly, when weakly focused the one-photon case produces a lot more signal in relative terms as would be expected. As an example, the relative signal rate approaches unity 1 for: P = 1 mW, ⟨P⟩ = 310 mW, NA1p = 0.03, NA2p = 0.1. Purely on the basis of Gaussian optics the two-photon excitation light sheet would be ca. 5 times short-er and one must also consider that the on-axis signal rate diminishes much more rapidly than in the one-photon case due to the non-linearity of the excitation.

Since the primary reason for using two-photon excitation in LSFM is to allow the light sheet to cover larger samples without too much spreading of the beam deep inside tissue, reducing the NA to increase the signal is not really viable. This leaves increasing the laser power as the only available route to deliver signal rates approaching that of the one-photon case, which may be excusable in some cases or samples but far from desirable. This analysis is largely supported by reported laser powers used for identical imaging tasks where both one and two-photon imaging has been performed. For example, Truong et al use 100 mW for two-photon excitation at 940 nm vs. 0.2 mW for one-photon excitation at 488 nm for identical imaging tasks in Drosophila 45. Similarly, Tomer et al. use 300 mW for two-photon excitation at 940 nm vs 0.36 mW for one-photon excitation at 488 nm, again for identical imaging tasks in Drosophila 49.

Since two-photon excitation hasn’t been delivered satisfactorily in a static light sheet mode, the comparison already assumes the more intense illumination required for a scanned light sheet. The choice of how to de-liver the illumination should therefore be a crucial consideration when developing a light sheet microscope. Before one opts for highly penetrating and potentially damaging two-photon excitation, the reduced paral-lelization of the scanned approach in general or indeed beam shaping schemes that appreciably illuminate out of focus regions 19,41 one should recall that the needs of the biology should always come first.

Supplementary Note 5 | Spatial Resolution and Field of View

Resolution and field of view (FOV) in light sheet microscopy are fundamentally intertwined (Box 2). One may approach, either from the viewpoint of a desired FOV or an achievable spatial resolution, although one should remain cognizant of the interplay between the two. Assuming the former, the detection magnifica-tion should be selected such that the magnified FOV is equal to the active area on the camera chip. This may or may not correspond to the entire chip; utilizing a smaller area is usually accompanied by some in-crease in frame rate, dependent on the specific architecture of the camera. Excluding custom optics, one is somewhat constrained by the choice of commercially available objectives (see Supplementary Note 6), as such it may not be possible to achieve the desired magnification using correctly referenced tube lenses. One option, at least for lenses that do not employ tube lens based color-correction, is to tune the magnification using adapters or an appropriate achromatic tube lens with a different focal length to that of the reference. Crucially, this also requires consideration of the objective field number when de-magnifying to ensure that the entire FOV can be imaged onto the camera in focus. For example, a ca. 4-megapixel sCMOS camera or ca. 1-megapixel EMCCD camera both measure approximately 20 mm along the diagonal. A field number of 25 mm can be considered high and would indicate that a maximum 0.8´ magnification (relative to that stated on the objective barrel: e.g. 8´ total for a 10´ lens) should be used to maintain flatness and avoid vi-gnetting. There are of course further considerations for objective lens selection; while some common mag-nifications may be available with a range of numerical apertures (NA), one may wish to prioritize NA over magnification, particularly when low light conditions are prevalent or the spatial resolution must be max-imized. However, the diffraction limit only defines the lateral resolution when the data is sufficiently spa-tially sampled and holds only when the pixel size, p, satisfies:

p ≤ 0.305λMNA

(S3)

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where l is the imaging wavelength, M is the magnification and NA is the numerical aperture. It is common practice to undersample (i.e. the inequality is not satisfied) when a large FOV is more important than achieving diffraction limited resolution, which will be degraded deep within tissue in any case. Equivalently, assuming a pixel size of 6.5 µm and l = 510 nm, one finds that the ratio of magnification to NA should be no less than ca. 42:1 to ensure that Nyquist sampling is achieved. Taking a few objectives commonly used in LSFM as examples (see Supplementary Note 6) we find that the 10×/0.3, 16×/0.8, 20×/0.5, 20×/1.0, 40×/1.0 and 25×/1.1 all undersample to varying degrees. We also find several lenses that are able to deliver proper sampling e.g. 40×/0.8, 63×/1.0, 100×/1.1.

The illumination optics similarly need to be tuned to give the appropriate FOV coverage although the con-straints are somewhat relaxed owing to the comparatively low NA utilized in typical light sheet micro-scopes. The desire is to produce a sufficiently weakly divergent light sheet to cover the FOV but not to compromise axial resolution and sectioning by making it thicker than necessary. Having determined the appropriate combination of illumination and detection optics, naturally it is desirable to determine the res-olution one is able to achieve in principle. Ritter et al. provide an analytical description of the system point-spread function (PSF) of a light sheet microscope, which is a useful resource in lieu of full calculation of diffraction integrals 78. Alternatively, the lateral and axial resolutions can be simply calculated as 4 :

rlateral =0.61λdetNAdet

(S4)

raxial =2NAillλill

+n− n2 − NAdet

2( )λdet

"

#

$$$

%

&

'''

−1

(S5)

Where the subscripts ill and det refer to the illumination and detection NA or wavelength respectively. The-se equations suggest that one may continue to increase both the illumination and detection NA without penalty to achieve ever-higher resolution (at least up to the limit set by the immersion media refractive in-dex). However, in this discussion we have ignored the mechanical constraints governing objective co-alignment and particularly for high-resolution imaging, the problem bears some consideration. Consider that in the standard orthogonal configuration each objective pair can occupy 90˚ of half-angular space around the common focus, or equivalently:

π2≥ arcsin NAdet

n"

#$

%

&'+ arcsin

NAilln

"

#$

%

&' (S6)

For equal illumination and detection NA, this establishes an upper limit of NA = 0.94 when imaging into water. Of course, this assumes that the entire front face of the objective can be used for light collection and that the FOV is confined to a point. Practical considerations dictate that commercially available objectives are larger (see Supplementary Note 6) and so the highest NA commercially available objective that can be orthogonally self-co-aligned (i.e. with a second copy of the same lens) is 0.8 (see Supplementary Table 5). Of course, the detection NA can be increased where high illumination NA is not a requirement but for ad-vanced multi-view systems utilizing all lenses for illumination and detection, NA 0.8 is currently the limit. The desire to achieve higher detection NA without compromising light sheet thickness has spawned a range of exotic reflective 30, oblique 79 and single objective 31 geometries (see Microscope Geometry: In Pursuit of High Resolution).

Of course, in this discussion we have neglected to consider the more exotic beam types 19,41,80, axially swept light sheets 43,44 and stimulated emission-depletion (STED) type 32 strategies discussed in the main text

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(Light Sheet Engineering: In Pursuit of High Resolution Over a Large Field of View). These are far from a universal panacea, often excessively illuminating the sample and requiring a degree of optical complexity not found in more conventional light sheet systems. Where the application truly requires the spatial resolu-tion however, these techniques may prove invaluable and although direct comparisons are limited, they would be expected to outperform their point-scanning contemporaries. Naturally, the assertion that DIY light sheet microscopy has democratized biological imaging finds its limit in this context and each of the approaches above requires a bona fide physicist or optical engineer to develop, build and maintain the in-strument. Nevertheless, the general principles of LSFM are more generally appreciable than many other modern optical imaging modalities. Taken to the limits of simplicity, the static, cylindrically focused SPIM is reproducible by almost anyone given the most basic understanding of optical systems. This core simplici-ty provides a key strength for LSFM in that biological scientific results can be quickly reproduced, verified and disseminated by and to the community.

Supplementary Note 6 | Objective Lenses for Light Sheet Fluorescence Microscopy

Correctly selecting (commercially available) objective lenses is particularly crucial in LSFM due to the addi-tional steric constraints owing to the need to orthogonally co-align two optical pathways. Additionally, as highlighted throughout this article the flexibility of being able to tune illumination and detection inde-pendently and for a given application is invaluable and crucial to push the technology to its limits. The se-lection of objective lenses is similarly important in multi-photon microscopy where the topic has been cov-ered in some depth 81. However, there is a paucity of similar studies in an LSFM context. In lieu of this we aim to provide some guidance as to some of the options available to the light sheet microscopist.

Before considering Supplementary Table 5, a few caveats bear consideration. Firstly, only objective lenses that have been appropriately corrected for relevant immersion media, allowing diffraction-limited behavior in principle, are considered. To ensure physiological relevance, LSFM primarily images samples in aqueous media and therefore low numerical aperture (NA) air immersion lenses without correction for an addition-al path into water are not considered although they are commonly used 2,56,82. The first three entries are air-immersion lenses but they feature correction for a given distance through water and are therefore highly suited to use in LSFM. In the case of entries two and three these lenses have been specifically designed for illumination and are otherwise inappropriate. Likewise, oil immersion objectives or those for specific clear-ing protocols, which have some application in specialist light sheet variants 30,31 have been omitted for con-ciseness. Moreover, the applicability of a given lens for illumination and detection is given in the notes (ill./det.). Although low NA lenses are typically sufficient for illumination and high NA lenses generally preferred for detection the distinction is not always so clear-cut. As such, moderate NA lenses (up to NA 0.8) with a smaller than 45˚ mechanical half angular extent are considered for both illumination and detec-tion. Certain manufacturers include color correction in the tube lens, which is not ideal for illumination but may be superior for field flatness in the detection path when performing multi-color imaging. One should also bear in mind this limits the scope for changing the detection magnification by judicious choice of tube lens focal length.

The mechanical half angle provided suggests that some reported combinations of lenses might not be com-patible in the strictest sense. There is some room for maneuver here; for dual-sided illumination the light sheet waists need not meet at the center of the FOV, allowing the illumination lens to be retracted (optimal-ly by a quarter of the FOV size along the direction of beam propagation). Additionally, illumination lenses may be retracted further and the illumination beam defocused to increase the effective working distance without noticeable spherical aberration at very low NA (NA < 0.1). Alternatively, in the standard two lens configuration the lenses may be positioned with an angle > 90˚ while offsetting the illumination beam from the center of the back focal plane to compensate by tilting the resulting light sheet (although this limits the achievable NA). Ideally these steps should not be taken, although the availability of appropriate lenses may at times justify the choice to do so. Finally, this information is correct to the best of the author’s knowledge but care should be taken in its interpretation. For example, different manufacturers may have different standards for flatness and color correction. Testing a given lens for a specific application should ultimately determine its potential utility. Naturally, the price of objective lenses is also an important consideration and

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varies markedly (by a factor of > 20 times in some cases). A summary of objective lens physical, optical and spectral parameters is given in Supplementary Table 5. A few examples of fully working lens combinations along with suggested applications are given in Supplementary Table 6.

(next page) Supplementary Table 5 | A selection of objective lenses suitable for light sheet fluorescence micros-copy.

Objectives are organized in ascending order by magnification (M, using correctly referenced tube lens), numerical aper-ture (NA) and manufacturer in that order. Key to terms: f = focal length, FFOV = flat field of view (equal to the field number divided by stated magnification), WD = working distance(s) (first immersion media, second immersion media, a = air, w = water), Color Corr. = degree of color correction across field of view (* = lowest or unstated (2.λ), ** = mod-erate (3.λ) < *** = highest (4.λ). Trans. = spectral transmission (> 70%). θ = mechanical half angular extent of the ob-jective. For conventional geometries, the half angle of neighboring lenses should not exceed 90˚. Achr/FL/Apo = ach-romat/fluorite/apochromat color correction, Plan = flatness corrected over large FOV, Corr. = correction ring, Cover. = coverslip (associated thickness). Ill./Det. = suitable for illumination/detection. Lenses that have been specifically de-signed for illumination are denoted Ill. only.

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Manufacturer Model M. f /mm NA FFOV /mm

WD /mm

Color Corr. Trans. θ /˚ Notes

Olympus XLFluor 4×/340 4 45.00 0.28 5.500 29.5(a), 5(w) ** NUV-

VIS 26 Air/water, FL, Ill., Det.

Zeiss LSFM 5×/0.1 5 33.00 0.1 4.000 16(a), 4.3(w) * VIS 27 Air/cover (0.17mm)/water,

Ill. only

Zeiss LSFM 10×/0.2 10 16.50 0.2 2.000 16(a), 3.5(w) * VIS 28 Air/cover (0.17mm)/water,

Ill. only

Leica HC APO L 10×/0.30 W 10 20.00 0.3 2.000 3.6(w) *** NUV-

NIR 45 Apo, Det.

Nikon CFI Plan Fluor 10 XW 10 20.00 0.3 2.500 3.5(w) ** NUV-

IR 36 FL, Plan, Ill., Det.

Olympus UMPLFLN 10 XW 10 18.00 0.3 2.650 3.5(w) ** NUV-

NIR 45 FL, Plan, Ill., Det.

Zeiss W N-Achroplan 10×/0.3 10 16.50 0.3 2.300 2.6(w) * NUV-

NIR 45.1 Achr, Plan, Ill., Det.

Zeiss W Plan-Apo 10×/0.5 10 16.50 0.5 2.000 3.7(w) *** NUV-

NIR 55.5 Apo, Plan, Det.

Olympus XLPLN 10XSVMP 10 18.00 0.6 1.800 8(w) * NUV-

IR 50 Plan, Corr, Det.

Special Optics 54-25-15 13.3 15 0.7 0.800 3(w) * VIS-NIR 44 Ill., Det.

Nikon CFI75 LWD 16 XW 16 12.50 0.8 1.375 3(w) * NUV-

NIR 45 Achr, Ill., Det.

Leica HC APO L 20×/0.50 W 20 10.00 0.5 1.000 3.5(w) *** NUV-

NIR 43 Apo, Det.

Nikon CFI Fluor 20 XW 20 10.00 0.5 1.100 2(w) ** NUV-

NIR 45 FL, Ill., Det.

Olympus UMPLFLN 20 XW 20 9.00 0.5 1.325 3.5(w) ** NUV-

NIR 45 FL, Plan, Ill., Det.

Zeiss W N-Achroplan 20×/0.5 20 8.25 0.5 1.150 2.6(w) * NUV-

NIR 48.4 Achr, Plan, Det.

Leica HC APO L 20×/1.00 W 20 10.00 1 1.000 1.95(w) *** NUV-

NIR 54 Apo, Det.

Olympus XLUMPLFLN 20× W 20 9.00 1 1.100 2(w) ** NUV-

NIR 52 FL, Plan, Det.

Zeiss W Plan-Apo 20×/1.0 20 8.25 1 1.000 2.4(w) *** NUV-

NIR 55.1 Apo, Plan, Corr, Det.

Olympus XPLN 25XWMP2 25 7.20 1.05 0.720 2(w) * NUV-

IR 56 Plan, Corr, Det.

Nikon CFI75 Apo LWD 25 XW 25 8.00 1.1 0.880 2(w) *** VIS-

IR 58 Apo, Corr, Cover (0-0.17mm), Det.

Special Optics 54-10-7 28.6 7 0.65 0.100 3.74(w) * VIS-NIR 30 Ill.

Leica HC APO L 40×/0.80 W 40 5.00 0.8 0.500 3.3(w) *** NUV-

NIR 45 Apo, Det.

Nikon CFI Fluor 40XW 40 5.00 0.8 0.550 2(w) ** NUV-

NIR 49 FL, Det.

Nikon CFI Apo 40XW NIR 40 5.00 0.8 0.550 3.5(w) *** NUV-

IR 45 Apo, Det.

Olympus LUMPLFLN 40 XW 40 4.50 0.8 0.663 3.3(w) ** NUV-

NIR 45 FL, Plan, Ill., Det.

Zeiss W Plan-Apo 40×/1.0 40 4.13 1 0.500 2.6(w) * NUV-

NIR 52.5 Apo, Plan, Det.

Nikon CFI Fluor 60XW 60 3.33 1 0.367 2(w) ** NUV-

NIR 56 FL, Det.

Nikon CFI Apo 60XW NIR 60 3.33 1 0.367 2.8(w) *** NUV-

IR 64 Apo, Det.

Olympus LUMPLFLN 60 XW 60 3.00 1 0.442 2(w) ** NUV-

NIR 55 FL, Plan, Det.

Olympus LUMFLN 60 XW 60 3.00 1.1 0.442 1.5(w) ** NUV-

NIR 57 FL, Plan, Corr, Det.

Leica HC APO L 63×/0.90 W 63 3.17 0.9 0.317 2.2(w) *** NUV-

NIR 50 Apo, Det., Det.

Zeiss W N-Achroplan 63×/0.9 M27 63 2.62 0.9 0.365 2.4(w) * NUV-

NIR 49 Achr, Plan, Det.

Zeiss W Plan-Apo 63×/1.0 63 2.62 1 0.317 2.1(w) *** NUV-

NIR 54 Apo, Plan, Det.

Nikon CFI Plan 100XW 100 2.00 1.1 0.220 2.5(w) * NUV-

NIR 61 Achr, Plan, Corr, Det.

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Supplementary Table 6 | Examples of objective lens combinations that can be orthogonally co-aligned.

The field of view has been determined assuming a square format detector consisting of 2048 x 6.5 µm pixels for 1-4 (typical for an sCMOS camera) and 1024 x 13 µm pixels for 5 (typical for an EMCCD camera). Illumination NA has been calculated using equation 1 in Box 2 (see main text), assuming the light sheet length is sufficient to span the field of view. Lateral resolution has been calculated using equation S4. Axial resolution has been calculated using equation S5. For 1-4 the camera frame rate has been calculated assuming 100 fps at full frame and adjusted according for the number of pixel rows required to give the stated field of view. * Double-sided illumination – light sheet length spans half of the field of view. (N) In cases where the spatial sampling is insufficient to fully exploit the detection NA, the Nyquist limited resolution has been given instead. Illumination lens (illumination paths)

Detection lens (detection paths)

Illumination/ Detection NA

Field of view / µm

Lateral/Axial Resolution/µm

Frame rate /s-1

Combined NAmax / Half Angle /˚ Notes

Olympus UMPLFLN 10×W (2)

Olympus UMPLFLN 10×W (2)

0.025/0.3 998 * 1037/5901 133 0.6 / 90

Suitable for multi-view imaging of early zebrafish embryogenesis with cellular resolution 12

Nikon CFI Plan Fluor 10×W (2)

Leica HC APO L 20×/1.00 W (1)

0.061/1.0 166 * 650(N)/878 400 1.3 / 90

Suitable for high speed functional imaging e.g. drosophila brain or zebrafish heart with cellular resolution

Zeiss LSFM 10×/0.2 (2)

Zeiss W Plan-Apo 40×/1.0 (2) 0.043/1.0 333 * 325(N)/938 100 1.2 / 83.1

Suitable for multi-view imaging of drosophila embryogenesis with sub-cellular resolution

Nikon CFI Apo 40×W NIR (1)

Nikon CFI Apo 40×W NIR (1) 0.043/0.8 166 389/1430 200 1.6 / 90

Suitable for multi-view imaging of C. elegans 3,18embryogenesis. Objec-tives used for illumination and detection.

Special Optics 54-10-7 (1)

Olympus LUMFLN 60×W (1)

0.16/1.1 12.5 283/560 - 1.75 / 87

Suitable for sub-cellular imaging. Additional factor of 1.4× magnification included. Compatible with localization microscopy.

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Supplementary Note 7 | Multi-View Imaging of Large Samples

For large samples, optical penetration is naturally a concern and while two-photon excitation may offer a reprieve in terms of light sheet penetration, in the absence of efficient widefield adaptive optics compara-tively little can be done for the detection path. Multi-view imaging and optical clearing therefore dominate in this arena for in vivo and chemically-fixed tissue imaging, respectively. Multi-view imaging in particular is truly one of the most powerful tools in the light sheet microscopist’s arsenal and while we have opined at length on the merits of omnidirectional systems (see Multi-View Imaging), even simple light sheet systems can benefit simply by rotating the specimen. The subsequent fusion of the individual views surely requires some thought in either case but powerful and freely available packages are available for registration 83 and multi-view deconvolution 84,85. Particularly for single-color developmental imaging where neighboring time points may be spaced by several minutes, doubling or even quadrupling the imaging time should not deter one from pursuing this option. Of course, there are times where multi-view imaging is not possible or where rotation is not viable, for example very large samples cannot really benefit without optical clearing or sample mounting may restrict rotation. As such, some of the most biologically challenging imaging tasks may be better served by adding additional illumination and detection paths (and the associated speed bene-fit). Still, one should always consider whether it is strictly necessary for the given application, the complexi-ty of these systems is not to be under-estimated.

In any case, an often-overlooked aspect of deep tissue imaging concerns the selection of objective lenses. Throughout we have considered how the illumination and detection numerical aperture (NA) each con-tribute to spatial resolution and FOV. However, very little has been said as to the performance of high NA lenses for imaging large samples. High NA is generally desirable for two reasons: spatial resolution and col-lection efficiency. Taking each in turn we can show how these assumptions may not be warranted in the case of large volume imaging. First, consider that low magnification is typically employed to achieve a large FOV. Several low magnification/high NA, water-dipping lenses are available (see Supplementary Table 5, broadly classified as having a ratio of M/NA ≤ 25). However, existing camera chips simply cannot deliver the spatial sampling required to deliver diffraction limited imaging and the resolution is governed accord-ingly (see Supplementary Note 5). Second, consider that the relatively thick light sheets needed to span large samples would predominantly lie outside the detection depth of focus of a high NA lens and so may compromise contrast. This is compounded by steering and broadening of the light sheet deep inside tissue since the illuminated plane may no longer be co-aligned to the detection focal plane throughout the FOV. Curved sample surfaces may introduce a lensing effect, which in turn displaces the detection focus and tak-en together these effects may produce markedly worse results deep inside tissue than the use of low NA detection lenses, which are comparatively robust to aberration and misalignments due to their large depth of field. Finally, the assumption that a high NA lens collects more light may be correct but there are certain-ly diminishing returns. Classical theory dictates that the collection efficiency should scale as NA2, however it remains to be shown that this scaling holds deep within large samples. To illustrate, consider that highly inclined (high NA) rays typically have to traverse more of the sample to be collected. When imaging deep into tissue they retain less directional information than their paraxial equivalents and so even when they are not scattered beyond the collection angle of the objective, they may disproportionately degrade contrast. Particularly for the fully parallelized SPIM-type approach, the small loss in collection efficiency that may accompany the choice of a lower NA lens for the sake of robustness is easily counteracted by a comparative-ly small increase in the total power, while the peak power still remains much lower than in the scanned case.

Supplementary Note 8 | Delivering the Required Temporal Resolution

Unlike more conventional 3D fluorescence techniques, light sheet microscopy operates far from fundamen-tal limits on imaging speed. Since the dwell time per pixel may be >106 times that of point-scanning mi-croscopies for an equal imaging rate, hard limits imposed by fluorophore saturation are far beyond reach. Naturally this additional capacity has spawned a number of ultrafast light sheet systems, as discussed in the main text (see Ultrafast Volume Imaging). It is however, useful to consider the alternatives for ultrafast im-aging.

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Conventional multi-photon techniques are too slow to be considered, although a widefield analogue, tem-poral focusing, combines optical sectioning with rapid camera-based acquisition 86. A diffractive grating is used to disperse the spectral components of a pulsed infrared laser such that the pulse length reaches its minimum in the objective focal plane. The evolved fluorescent signal is confined to this plane due to the quadratic dependence of the two-photon excitation on the pulse length. Schrödel et al. have used this tech-nique to image C. elegans at up to 6 Hz 87. Unfortunately, only small illumination spots are readily achieved, as the power requirements outstrip all but the most powerful lasers, limiting FOV. Light-field microscopy uses a microlens array in the detection path of a widefield microscope to perform plenoptic imaging of the sample from multiple perspectives 88. Optically at least, this design is remarkably simple and the complexity comes from the intensive computational deconvolution. Light field microscopy has been successfully ap-plied to functional calcium imaging in unrestrained C. elegans at up to 50 volumes per second 89. However, since large numbers of perspectives are captured simultaneously, the FOV reduces proportionally. This yields poorly resolved datasets dominated by reconstruction artifacts and with only limited axial discrimi-nation. Rather than a microlens array, multi-focus microscopes use phase masks to produce an aberration free projection of various planes onto the camera; as each image corresponds to a single plane, the recon-struction is simpler but the same restrictions on FOV apply 90. If truly synchronous imaging is required light field or multi-focus microscopies may be useful but for most applications rapid sequential imaging provides sufficient speed without the sacrifices to resolution or FOV. Moreover, none of the techniques discussed benefits from the selective illumination of LSFM and structures not necessarily contributing to signal are fully illuminated.

If an application truly requires volumetric imaging above 1 Hz the techniques discussed in the main text (see Ultrafast Volume Imaging) provide a range of approaches of varying complexity. Generally, however, the imaging speed of even a simple light sheet microscope is sufficient and it is useful to consider a few basic principles that can influence the overall volumetric imaging rate. These can be broadly characterized as sample, camera and illumination-based limitations.

Dealing first with the sample, in the discussion of sample mounting (see Supplementary Note 3) little men-tion is made of mechanical robustness to motion. The simplest scheme for capturing volumetric data is to image the sample plane-by-plane as it is moved through the light sheet. Repetitive rapid movements may negatively impact the health of the sample and inertia may cause soft mounting media to bend, making accurate positioning difficult. Alternatively, the detection objective can be repositioned to move the focal plane within the sample and the light sheet moved in synchrony. This scheme may provide a modest im-provement in volumetric rate 91 but may disturb the immersion media to such an extent (at least for water-dipping objectives) that the same issues may be apparent. Correspondingly, for ultrafast volume imaging one should ideally refocus remotely (e.g. using a tunable lens 54), artificially extend the detection depth of field 56–58 or use an oblique imaging scheme 9,79.

While these schemes significantly relax the mechanical issues which otherwise plague ultrafast volumetric microscopies, the camera frame rate ultimately provides some limit on the achievable imaging speed. For example, consider that a modern sCMOS camera may run continuously at 100 frames per second (fps). Since the camera reads out line-wise, the maximum achievable frame rate depends on the number of pixel lines used. Assuming a camera chip of 2048´2048 pixels, 512 lines could be captured at 400 fps. Naturally, this creates something of a tradeoff between FOV and imaging speed. Even so, this establishes an upper limit on the volumetric rate, for example, 4 Hz for 100 planes per volume. This is sufficient in many cases, but if one wishes to image a larger FOV or achieve a higher volume rate the camera becomes limiting. The high data rate provides an additional challenge as sCMOS cameras may produce nearly a GB of data per second (full-frame, 16-bit encoding), requiring a RAID array of 4-6 solid state drives to stream the data without interruption. Of course, there are alternatives to sCMOS cameras capable of providing an order of magnitude improvement in frame rate 55. This exacerbates data transfer issues and so typically high frame rate cameras feature some onboard storage, which limits their use for truly continuous ultrafast imaging. Nevertheless, volumetric imaging at 10s Hz can be sustained for several seconds, which may be sufficient in many cases.

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The assumption made throughout this discussion is that refocusing and camera readout typically provide the rate-limiting steps, but without careful consideration, the illumination optics may also contribute. There are two cases where the assumptions made may be unwarranted; the first considers virtual light sheets. Scanning a weakly focused beam to sweep out a light sheet across several hundred planes per second requires that the scan mirror is able to reproduce a linear driving signal at such a high speed, which may not be readily achieved. Similarly, virtual light sheets are sub-optimal under the inherently low light conditions as the dwell time per pixel is dramatically reduced and thus far higher peak intensities are required to achieve the same signal rate. For low-labeling densities or dim reporters it is possible that fluorophore satu-ration, typically only an issue for point-scanning systems, can become problematic in the context of an ul-trafast scanned virtual light sheet. The second case, can really be considered an extension of this concept; the signal rate for two-photon excitation is generally too low to be useful for ultrafast imaging and has to date limited the volumetric rate to 2 Hz in LSFM 53. For the reasons discussed, ultrafast imaging is better suited to static light sheets.

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