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Performance trade-offs for single- and dual-objective light-sheet microscope designs

12 May 2020-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: A diffraction-based analysis of single- and dual-objective LSM configurations where Gaussian illumination is utilized and showcases the potential advantages of a novel, non-orthogonal dual- objective (NODO) architecture, especially for moderate-resolution imaging applications.
Abstract: Light-sheet microscopy (LSM) has emerged as a powerful tool for high-speed volumetric imaging of live model organisms and large optically cleared specimens. When designing cleared-tissue LSM systems with certain desired imaging specifications (e.g. resolution, contrast, and working distance), various design parameters must be taken into consideration. In order to elucidate some of the key design trade-offs for LSM systems, we present a diffraction- based analysis of single- and dual-objective LSM configurations where Gaussian illumination is utilized. Specifically, we analyze the effects of the illumination and collection numerical aperture (NA), as well as their crossing angle, on spatial resolution and contrast. Assuming an open-top light-sheet (OTLS) architecture, we constrain these parameters based on fundamental geometric considerations as well as those imposed by currently available microscope objectives. In addition to revealing the performance tradeoffs of various single- and dual-objective LSM configurations, our analysis showcases the potential advantages of a novel, non-orthogonal dual-objective (NODO) architecture, especially for moderate-resolution imaging applications (collection NA of 0.5 to 0.8).

Summary (4 min read)

1. Introduction

  • Light-sheet microscopy (LSM), also known as selective plane illumination microscopy (SPIM), has become a valuable tool for many biomedical investigations and applications.
  • Dual-objective systems use two separate objectives, arranged orthogonally to one another, for illumination and collection.
  • Positioning the objectives at such oblique angles typically results in a reduction in axial imaging range since the full working distance of the objectives cannot be easily accessed (Fig. 1b).
  • Single-objective light-sheet microscopes have recently been developed [15–21], which use a portion of an objective’s NA for illumination and a (typically larger) portion of the NA for collection (Fig. 1c).
  • While some analysis has been performed for a narrow subset of LSM configurations, such as examining different illumination profiles for dual-objective systems [22] and simulating various single-objective designs [23], a systematic comparison of diverse dual-objective and single-objective LSM designs has not been performed.

2. Analysis approach

  • We identified three key design parameters that, in the context of cleared-tissue imaging, are primary drivers of a system’s optical performance regardless of the specific architecture (single- or dual-objective) used: the NA of the illumination beam, the NA of the collection beam, and the angle between the two beams.the authors.the authors.
  • Note that the authors make several assumptions for the sake of simplifying their analysis.
  • The authors assume single-photon Gaussian illumination, which is used in the majority of LSM applications.
  • This allows for straightforward mathematical descriptions of geometric constraints, which in turn limit the numerical range of the design parameters that are analyzed (see next section).
  • The authors assess optical performance in terms of spatial resolution and contrast (as determined by a simulated fluorescent bead phantom).

2.1. Geometric constraints and simplifying assumptions for dual-objective systems

  • The optical path for an ODO system is shown in Fig. 1a.
  • For optically clear tissue specimens, unrestricted lateral sample extent is generally preferred.
  • While the two objectives in a dual-objective system are typically placed orthogonally to one another, in principle their crossing angle is not restricted to 90°.
  • Note that a crossing angle larger than 90° would never be used in practice.

2.2. Geometric constraints and assumptions for single-objective systems

  • Figure 1c shows the light path of a generic NOSO LSM system (with the objective arranged below the sample as previously described).
  • As shown in Fig. 1d, this configuration results in a light sheet that is tilted at some angle \C8;C relative to the optical axis of the objective.
  • The crossing angle of the illumination and collection cones is constrained by the physical NA of the shared primary objective.
  • Additional geometric analyses of the NOSO architecture (used to limit their simulations), and of light behavior at the remote focus.
  • In particular, the authors assume that techniques are employed (as described by others [19–21]) to ensure that the collection beam can be imaged by a high-NA remote objective (and detected by the camera) with 100% throughput and negligible aberration.

2.3. Non-orthogonal dual-objective design

  • As mentioned previously, their analyses will include a novel NODO architecture, which combines attractive elements of both the ODO and NOSO configurations.
  • In a NODO configuration, the collection objective is kept perpendicular to the sample holder and a second low-NA objective is used for non-orthogonal illumination (Fig. 3).
  • Similar to NOSO systems, this offers the ability to access the full working distance of the collection objective for sample imaging, and significantly reduces sensitivity to index mismatch.
  • Note that the latter issue (index mismatch) can be a major source of aberrations in ODO systems, where high-NA beams must transition between different media at highly oblique angles.
  • This makes rapid scanning and descanning of these beams more challenging, such that the system may be less ideal for live-sample imaging.

2.4. Quantitative output metrics

  • In cleared-tissue imaging, image quality is the primary performance metric of interest.
  • Volumetric imaging speed is of concern, but typically scales in a predictable way with spatial resolution if samples are sufficiently labeled and photon counts are not a limiting factor.
  • The authors thus quantified the performance of different configurations in terms of the spatial resolution and contrast that is theoretically possible for a given system.
  • The authors analyzed the contrast of each system by simulating the system’s response to a virtual fluorescent bead phantom.

3. Analysis results

  • The authors used the geometric constraints identified above to identify key input parameters for each microscope configuration.
  • For dual-objective systems, the input parameters are: illumination NA, collection NA, and crossing angle (where ODO systems have a crossing angle of 90° and NODO systems have a crossing angle less than 90°).
  • Note that these three parameters uniquely determine the crossing angle of the beams, as expressed in Eq. (4).
  • The authors limited their simulations to a range of primary objective NAs (i.e. collection-objective NA for dual-objective systems) ranging from 0.4 to 1.0 based on currently available commercial objectives that are suitable for imaging cleared tissues .
  • Axial resolution, lateral resolution, and contrast were computed as functions of the listed design parameters.

3.1.1. For dual-objective systems, larger crossing angles improve axial resolution until ∼60°

  • As expected, axial resolution improves with crossing angle, with the ODO configuration thus providing the best axial resolution.
  • The gains in axial resolution are minimal beyond angles of ∼60°, a finding that is consistent across NA pairs beyond those shown.
  • This angle is readily achievable (in terms of physical placement of objectives) for most choices of illumination and collection NA, including for the NODO case in which the collection objective is oriented normal to the horizontal sample holder.
  • Therefore, the authors use 60° as the crossing angle for all further analyses of NODO systems.

3.1.2. For all systems, axial resolution is highly dependent upon illumination NA

  • Besides crossing angle, a primary driver of axial resolution is the light-sheet thickness as determined by the illumination NA.
  • Figure 5 shows the impact of illumination NA on axial resolution for each system architecture.
  • There are diminishing returns as the illumination NA is increased (beyond ∼0.3).
  • An implication of these observations is that while isotropic resolution at high NAs may be desirable in theory [5, 27–30], the simplicity and geometric advantages of using a relatively low illumination NA (∼0.3) may offer an ideal compromise for many practical applications of LSM.

3.1.3. For dual-objective systems, lateral resolution is entirely determined by collection NA

  • The previous plots showed that in a dual-objective system, the axial resolution is primarily determined by illumination NA and crossing angle.
  • The authors will now consider which factors are most impactful to lateral resolution in a dual-objective system.
  • Figure 6 shows how lateral resolution varies as a function of collection NA for an ODO system (a) and a NODO system with a 60° crossing angle (b).
  • In these plots, the different colors correspond to different choices of illumination NA.
  • The authors also see that the different curves in each plot almost completely overlap, indicating that lateral resolution is not influenced greatly by illumination NA.

3.1.4. For single-objective systems with moderate NA, axial and lateral resolution must trade off

  • In contrast to the decoupling of axial and lateral resolution in a dual-objective system, axial and lateral resolution in a single-objective system are intrinsically linked because the two light paths share a single objective.
  • Figure 7 shows two example NOSO systems: one using a moderate-NA primary objective (0.75, a) and one using a high-NA primary objective (1.0, b).
  • The plots show how axial and lateral resolution vary as functions of collection NA.
  • At higher NAs (1.0 NA shared primary objective), the crossing angle remains relatively high regardless of effective collection NA.
  • In other words, for a high-NA NOSO system, axial and lateral resolution do not trade off to the extent that they must for a NOSO system with a lower primary objective NA.

3.2.1. For dual-objective systems, larger crossing angles improve contrast until ∼60°

  • In addition to the insights into resolution that have been discussed so far, the authors also identified quantitative trends regarding the contrast of single- and dual-objective systems.
  • A collection NA of 0.7 is used in this example, though the trends are similar for other collection NAs (with slight improvements in contrast at higher collection NAs).
  • Contrast improves at higher crossing angles as the collection and illumination light paths are more spatially separated.
  • This reinforces the observation that a NODO system with a ∼60° crossing angle is ideal, with optical performance close to that of an ODO system.

3.2.2. Contrast improves with higher illumination NA, and dual-objective systems exhibit im-

  • Proved contrast over single-objective systems at moderate NAs Figure 9 compares the contrast of an ODO, NODO (60° crossing angle), and NOSO system as a function of illumination NA.
  • In order to provide a fair comparison, the authors consider a moderate-NA case (0.7, a) and high-NA case (1.0, b), keeping the collection/primary objective NA constant for each case.
  • In other words, the plot shows the different configurations that could be constructed with the same optical element (a 0.7 or 1.0 NA objective).
  • The main reason for this is that crossing angle plays a significant role in determining contrast, and dual-objective systems can achieve higher crossing angles, particularly in the moderate-NA case.
  • At high NA, this effect is less significant, and all three systems provide similar contrast.

4. Discussion

  • The authors have identified several key trends in terms of the resolution and contrast of single- and dual-objective LSM systems for cleared-tissue imaging, which they believe will be valuable as a general guide for system designers.
  • In particular, the authors evaluated three OTLS microscope architectures: a conventional ODO system, a conventional NOSO system, and a novel NODO system.
  • Regarding the resolution of these systems, there are four main observations.
  • (2) Contrast for all systems improves with illumination NA, with diminishing returns at the highest illumination NAs, and dual-objective systems exhibit better contrast than single-objective systems when the same moderate-NA objective is used (Fig. 9).
  • Finally, for moderate-NA systems (i.e. 0.5 < NA < 0.8), the NODO architecture offers a combination of imaging performance (resolution and contrast), working distance, and field of view that is challenging to achieve with other architectures.

Acknowledgments

  • Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF, NIH, or DoD. Conflicting Interests JTCL and AKG are co-founders and shareholders of Lightspeed Microscopy Inc., of which JTCL is a board member.
  • Technology developed by JTCL and AKG at the University of Washington has been licensed by Lightspeed Microscopy Inc.

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Figures (14)

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Performance trade-offs for single- and
dual-objective light-sheet microscope designs
KEVIN W. BISHOP,
1,2,4
ADAM K. GLASER,
2
AND JONATHAN T.C.
LIU
1,2,3,5
1
Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA
2
Department of Mechanical Engineering, University of Washington, Seattle, Washington 98195, USA
3
Department of Pathology, University of Washington, Seattle, Washington 98195, USA
4
kwbishop@uw.edu
5
jonliu@uw.edu
Abstract:
Light-sheet microscopy (LSM) has emerged as a powerful tool for high-speed
volumetric imaging of live model organisms and large optically cleared specimens. When
designing cleared-tissue LSM systems with certain desired imaging specifications (e.g. resolution,
contrast, and working distance), various design parameters must be taken into consideration. In
order to elucidate some of the key design trade-offs for LSM systems, we present a diffraction-
based analysis of single- and dual-objective LSM configurations where Gaussian illumination
is utilized. Specifically, we analyze the effects of the illumination and collection numerical
aperture (NA), as well as their crossing angle, on spatial resolution and contrast. Assuming an
open-top light-sheet (OTLS) architecture, we constrain these parameters based on fundamental
geometric considerations as well as those imposed by currently available microscope objectives.
In addition to revealing the performance tradeoffs of various single- and dual-objective LSM
configurations, our analysis showcases the potential advantages of a novel, non-orthogonal
dual-objective (NODO) architecture, especially for moderate-resolution imaging applications
(collection NA of 0.5 to 0.8).
1. Introduction
Light-sheet microscopy (LSM), also known as selective plane illumination microscopy (SPIM),
has become a valuable tool for many biomedical investigations and applications. In LSM, a sheet
of light is used to excite fluorescence from a thin plane ("optical section") within a relatively
transparent sample [1, 2]. Adjacent 2D planes within the sample are successively illuminated
and imaged using a high-speed camera to rapidly scan a volumetric region. A key advantage
of this camera-based technique is that 3D imaging can be performed more quickly and simply
than with laser-scanning microscopy (e.g. confocal and multiphoton microscopy). In addition,
selective planar illumination is optically efficient, minimizing photobleaching of fluorophores and
phototoxicity to living organisms [3]. LSM was originally popularized for volumetric imaging of
live model organisms in developmental biology [1, 4
6] and more recently has been explored for
imaging large optically cleared ex vivo tissues [7–9], including clinical specimens [10–12].
A number of unique LSM architectures exist that can be broadly separated into two categories:
dual-objective and single-objective LSM. Dual-objective systems use two separate objectives,
arranged orthogonally to one another, for illumination and collection. Having two independent
objectives provides optical-design flexibility but can place significant physical constraints on
sample geometries. In an attempt to mitigate this issue, inverted LSM [5, 8] and open-top
light-sheet (OTLS) configurations [6,10,11,13,14] have been introduced in which dual objectives
are arranged at 45
°
angles with respect to a sample that is placed on a horizontal substrate
Fig. 1a). Such designs reduce constraints on sample size and sample numbers (by allowing for
unconstrained lateral image tiling) and simplify sample mounting. However, positioning the
objectives at such oblique angles typically results in a reduction in axial (vertical) imaging range
since the full working distance of the objectives cannot be easily accessed (Fig. 1b). Additionally,
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 12, 2020. ; https://doi.org/10.1101/2020.05.10.087171doi: bioRxiv preprint

achieving moderate- or high-numerical aperture (NA) imaging (i.e. NA > 0.5) using an OTLS
architecture is challenging due to the precise index-matching requirements and/or corrective
optics needed to minimize aberrations when high-NA off-axis beams transition between different
media (e.g. immersion oil, sample holder, tissue) [14].
Fig. 1. Overview of single- and dual-objective light-sheet microscope (LSM) archi-
tectures. (a) Optical schematic of a conventional orthogonal dual-objective (ODO)
open-top light-sheet (OTLS) system, showing the illumination (blue) and collection
(green) light paths. (b) Inset of illumination and collection objectives of an ODO
system, showing that the system’s effective working distance (WD
system
) is much less
than the collection objectives working distance (WD
objective
). (c) Optical schematic of
a non-orthogonal single-objective (NOSO) OTLS system, showing the illumination
(blue) and collection (green) light paths. (d) Inset of the shared primary objective of a
NOSO system, showing the angled light sheet and collection path. CL: cylindrical lens,
TL: tube lens, DBS: dichroic beam splitter.
Single-objective light-sheet microscopes have recently been developed [15
21], which use
a portion of an objectives NA for illumination and a (typically larger) portion of the NA for
collection (Fig. 1c). The use of a single objective allows the full working distance of the objective
to be made accessible for imaging the sample. In addition, orienting the objective in the normal
direction with respect to a horizontal sample holder leverages the ideal aberration-correction
properties that have been meticulously engineered into high-quality microscope objectives when
imaging through flat interfaces, thus relaxing sample index-matching requirements. Furthermore,
since the conjugate planes of the illumination and collection beams are the same, scanning the
beams (e.g. with a mirror) in tandem, while maintaining their alignment within the sample, can
be simplified.
A significant challenge in implementing a single-objective light-sheet design is that when both
light paths share the same objective, the illumination light sheet cannot be oriented orthogonally
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 12, 2020. ; https://doi.org/10.1101/2020.05.10.087171doi: bioRxiv preprint

to the objective (Fig. 1d). This makes it challenging to efficiently capture an in-focus image of the
fluorescence generated by the light sheet on a flat detector array (camera chip). An innovation that
has made single-objective LSM possible is creating a remote focus with a secondary objective
such that a tilted third objective can be used to image the remote light sheet onto a sCMOS
detector array in the ideal orthogonal direction [15
21]. The details of this approach are described
in Appendix A.
In light of the numerous LSM architectures and variations that have been developed in recent
years, there is a natural desire amongst optical engineers to analyze their performance tradeoffs.
While some analysis has been performed for a narrow subset of LSM configurations, such
as examining different illumination profiles for dual-objective systems [22] and simulating
various single-objective designs [23], a systematic comparison of diverse dual-objective and
single-objective LSM designs has not been performed. Here we seek to fill this gap by presenting
a quantitative analysis of dual- and single-objective LSM performance in response to several
key design parameters. By quantifying design tradeoffs between different configurations, there
is potential to identify "unreached" design spaces, which in turn will motivate future areas of
innovation. For example, we describe a new non-orthogonal dual-objective (NODO) configuration
that provides some advantages over orthogonal dual-objective (ODO) and non-orthogonal single-
objective (NOSO) configurations for moderate-resolution applications.
It is impossible to provide a comprehensive guide for LSM design given the vast range of
factors to consider such as sample characteristics (e.g. size, quantity, degree of optical clarity)
and biological considerations (e.g. dynamic vs. static imaging, light dose), among others. Here
we have focused our analysis on OTLS microscopy configurations for imaging fixed tissues that
are optically cleared, in which optical scattering and refractive aberrations are assumed to be
negligible. We also focus our analysis to assume conventional Gaussian illumination, which
is the most-popular illumination method for LSM (note that a recent analysis of various LSM
illumination schemes was performed by Remacha et al. [22]). With this context in mind, we
examine a set of major performance metrics - primarily resolution and contrast - as functions of
a specific set of design parameters: illumination and collection beam NAs and crossing angle,
as constrained by currently available objectives. Certain imaging applications could warrant
consideration of alternative parameters. Optical scattering, for example, is a key consideration
for imaging live, uncleared specimens, and has been examined with Monte-Carlo simulations in
the past for LSM [24] and dual-axis confocal microscopy [25, 26]. Nonetheless, we believe that
our work represents a valuable first step towards guiding the design of OTLS systems.
2. Analysis approach
The overall analysis approach we followed is shown in Fig. 2. We identified three key design
parameters that, in the context of cleared-tissue imaging, are primary drivers of a system’s optical
performance regardless of the specific architecture (single- or dual-objective) used: the NA of
the illumination beam, the NA of the collection beam, and the angle between the two beams.
Note that we make several assumptions for the sake of simplifying our analysis. We assume
single-photon (linear) Gaussian illumination, which is used in the majority of LSM applications.
In particular, pure Gaussian illumination is assumed, which is a fair approximation for real
Gaussian beams that are truncated (apodized) beyond the 1
/𝑒
2
intensity points. Further, we
assume that the collection NA is larger than the illumination NA, which is true in nearly all LSM
systems. This allows for straightforward mathematical descriptions of geometric constraints,
which in turn limit the numerical range of the design parameters that are analyzed (see next
section). We assess optical performance in terms of spatial resolution and contrast (as determined
by a simulated fluorescent bead phantom).
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 12, 2020. ; https://doi.org/10.1101/2020.05.10.087171doi: bioRxiv preprint

Fig. 2. Outline of the analysis approach followed. Three key design parameters were
identified that, in the context of cleared tissue imaging, primarily determine the optical
performance of an LSM system (regardless of specific architecture): the numerical
aperture (NA) of the illumination beam, the NA of the collection beam, and the crossing
angle between them. We then introduced limits on these parameters based on practical
constraints including available objectives and the physical placement of those objectives.
These limits give rise to a range of feasible designs. Finally, we assess the optical
performance of these designs in terms of spatial resolution and contrast.
2.1. Geometric constraints and simplifying assumptions for dual-objective systems
The optical path for an ODO system is shown in Fig. 1a. Note that these systems generally
include scanning mirrors to translate the light sheet and/or stages to scan the specimen, but
these have been omitted for simplicity in both Fig. 1a and Fig. 1c. For optically clear tissue
specimens, unrestricted lateral sample extent is generally preferred. As such, we assume an OTLS
architecture for both single- and dual-objective systems, which allows for a more-consistent
comparison between various LSM architectures.
While the two objectives in a dual-objective system are typically placed orthogonally to one
another, in principle their crossing angle is not restricted to 90
°
. We assume that the objective
housing does not occupy additional space beyond the optical cone (NA) of the objective. As
such, the placement of the objectives is restricted by the crossing angle and the cone angles of the
individual beams, as shown in Fig. 1b. The half cone angle for each objective is determined by:
𝜙 = sin
1
(
𝑁 𝐴/𝑛
)
(1)
Here,
𝜙
is the half cone angle,
𝑁 𝐴
is the numerical aperture of the objective, and
𝑛
is the
refractive index of the immersion medium (assumed to be water,
𝑛 =
1
.
33, for this analysis). The
objectives cannot physically collide, so the minimum crossing angle permitted in this system is
the angle at which the illumination and collection cones begin to intersect:
𝜃 𝜙
𝑖
+ 𝜙
𝑐
(2)
Here,
𝜃
is the crossing angle between the optical axes of the two objectives, while
𝜙
𝑖
and
𝜙
𝑐
are the half cone angles of the illumination and collection objectives, respectively. In order to
maintain an OTLS geometry, the objectives also cannot cross the horizontal sample holder as
they would collide with the sample. The maximum crossing angle physically allowed is then
constrained by:
𝜃 + 𝜙
𝑖
+ 𝜙
𝑐
180° (3)
This relationship assumes that the thickness of the sample holder and sample itself are
negligible. Note that a crossing angle larger than 90
°
would never be used in practice. Equations
(2) and (3) provide physical limits on how a dual objective system can be constructed, which
constrain the numerical range of our simulations. Importantly, the observation that the crossing
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 12, 2020. ; https://doi.org/10.1101/2020.05.10.087171doi: bioRxiv preprint

angle 𝜃 does not need to be set to 90° (as is the case in conventional orthogonal configurations)
gives rise to a novel NODO design that we will examine closely later.
2.2. Geometric constraints and assumptions for single-objective systems
Figure 1c shows the light path of a generic NOSO LSM system (with the objective arranged
below the sample as previously described). As shown in Fig. 1d, this configuration results in
a light sheet that is tilted at some angle
𝜃
𝑡𝑖𝑙𝑡
relative to the optical axis of the objective. The
crossing angle of the illumination and collection cones is constrained by the physical NA of the
shared primary objective. Assuming that the crossing angle is maximized for a given illumination
and collection NA (which offers the best performance, as shown later), the crossing angle
𝜃
is
given by:
𝜃 = 2𝜙
𝑜𝑏 𝑗
𝜙
𝑐
𝜙
𝑖
(4)
Here,
𝜙
𝑜𝑏 𝑗
,
𝜙
𝑐
, and
𝜙
𝑖
are the geometric half cone angles of the shared primary objective,
the collection beam, and the illumination beam, respectively. Note that as in the dual-objective
analysis, these geometric angles are assumed to be within a high-index medium (water,
𝑛 =
1
.
33,
in this analysis).
Additional geometric analyses of the NOSO architecture (used to limit our simulations), and
of light behavior at the remote focus, are provided in Appendix A. In particular, we assume that
techniques are employed (as described by others [19
21]) to ensure that the collection beam can
be imaged by a high-NA remote objective (and detected by the camera) with 100% throughput
and negligible aberration.
2.3. Non-orthogonal dual-objective design
As mentioned previously, our analyses will include a novel NODO architecture, which combines
attractive elements of both the ODO and NOSO configurations. In a NODO configuration, the
collection objective is kept perpendicular to the sample holder and a second low-NA objective is
used for non-orthogonal illumination (Fig. 3). Similar to NOSO systems, this offers the ability to
access the full working distance of the collection objective for sample imaging, and significantly
reduces sensitivity to index mismatch. Note that the latter issue (index mismatch) can be a major
source of aberrations in ODO systems, where high-NA beams must transition between different
media at highly oblique angles.
Fig. 3. Geometry of a proposed non-orthogonal, dual-objective (NODO) design. The
crossing angle
𝜃
is less than 90
°
so that the collection objective can be oriented in
the normal direction with respect to the horizontal sample holder, as in a NOSO
system. This allows the full working distance of the collection objective to be used
and minimizes sensitivity to refractive-index mismatch. Unlike the NOSO architecture,
NODO also allows the full NA of the collection objective to be used and allows for a
higher crossing angle between the illumination and collection beams (with implications
for axial resolution and contrast).
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 12, 2020. ; https://doi.org/10.1101/2020.05.10.087171doi: bioRxiv preprint

Citations
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Journal ArticleDOI
TL;DR: The proposed mesoscopic scanning OPM allows using low NA objectives such that centimeter-level FOV volumetric imaging can be achieved, the largest reported FOV by OPM so far.
Abstract: Background Conventional light sheet fluorescence microscopy (LSFM), or selective plane illumination microscopy (SPIM), enables high-resolution 3D imaging over a large volume by using two orthogonally aligned objective lenses to decouple excitation and emission. The recent development of oblique plane microscopy (OPM) simplifies LSFM design with only one single objective lens, by using off-axis excitation and remote focusing. However, most reports on OPM have a limited microscopic field of view (FOV), typically within 1×1 mm2. Our goal is to overcome the limitation with a new variant of OPM to achieve a mesoscopic FOV. Methods We implemented an optical design of mesoscopic scanning OPM to allow the use of low numerical aperture (NA) objective lenses. The angle of the intermediate image before the remote focusing system was increased by a demagnification under Scheimpflug condition such that the light collecting efficiency in the remote focusing system was significantly improved. A telescope composed of cylindrical lenses was used to correct the distorted image caused by the demagnification design. We characterized the 3D resolutions and imaging volume by imaging fluorescent microspheres, and demonstrated the volumetric imaging on intact whole zebrafish larvae, mouse cortex, and multiple Caenorhabditis elegans (C. elegans). Results We demonstrate a mesoscopic FOV up to ~6×5×0.6 mm3 volumetric imaging, the largest reported FOV by OPM so far. The angle of the intermediate image plane is independent of the magnification as long as the size of the pupil aperture of the objectives is the same. As a result, the system is highly versatile, allowing simple switching between different objective lenses with low (10×, NA 0.3) and median NA (20×, NA 0.5). Detailed microvasculature in zebrafish larvae, mouse cortex, and neurons in C. elegans are clearly visualized in 3D. Conclusions The proposed mesoscopic scanning OPM allows using low NA objectives such that centimeter-level FOV volumetric imaging can be achieved. With the extended FOV, simple sample mounting protocol, and the versatility of changeable FOVs/resolutions, our system will be ready for the varieties of applications requiring in vivo volumetric imaging over large length scales.

14 citations

Posted ContentDOI
17 May 2022
TL;DR: In this paper , an optical refocusing solution via a custom fibre-optical faceplate was introduced to enable a large field-of-view of up to 4 mm 3 at a volume rate of 1 Hz.
Abstract: Abstract Optical imaging is a powerful tool to visualise and measure neuronal activity. However, due to the size and opacity of vertebrate brains it has until now been impossible to simultaneously image neuronal circuits at cellular resolution across the entire adult brain. This is true even for the smallest known vertebrate brain in the teleost Danionella , which is still too large for existing volumetric imaging approaches. Here we introduce image transfer oblique plane microscopy, which uses a new optical refocusing solution via a custom fibre-optical faceplate, enabling a large field-of-view of up to 4 mm 3 at a volume rate of 1 Hz. We demonstrate the power of this method with the first brain-wide recording of neuronal activity in an adult vertebrate.

5 citations

References
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Journal ArticleDOI
13 Aug 2004-Science
TL;DR: In this article, a selective plane illumination microscopy (SPIM) was developed to generate multidimensional images of samples up to a few millimeters in size, which can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.
Abstract: Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.

2,152 citations


"Performance trade-offs for single- ..." refers methods in this paper

  • ...In LSM, a sheet of light is used to excite fluorescence from a thin plane ("optical section") within a relatively transparent sample [1, 2]....

    [...]

Journal ArticleDOI
TL;DR: The authors show that the ATPase function of the chromatin remodeler SMARCAD1 facilitates the binding of KAP1 to ERVs and is required for their repression in embryonic stem cells.
Abstract: Endogenous retroviruses (ERVs) can confer benefits to their host but present a threat to genome integrity if not regulated correctly. Here we identify the SWI/SNF-like remodeler SMARCAD1 as a key factor in the control of ERVs in embryonic stem cells. SMARCAD1 is enriched at ERV subfamilies class I and II, particularly at active intracisternal A-type particles (IAPs), where it preserves repressive histone methylation marks. Depletion of SMARCAD1 results in de-repression of IAPs and adjacent genes. Recruitment of SMARCAD1 to ERVs is dependent on KAP1, a central component of the silencing machinery. SMARCAD1 and KAP1 occupancy at ERVs is co-dependent and requires the ATPase function of SMARCAD1. Our findings uncover a role for the enzymatic activity of SMARCAD1 in cooperating with KAP1 to silence ERVs. This reveals ATP-dependent chromatin remodeling as an integral step in retrotransposon regulation in stem cells and advances our understanding of the mechanisms driving heterochromatin establishment.

1,686 citations

Journal ArticleDOI
24 Oct 2014-Science
TL;DR: A new microscope using ultrathin light sheets derived from two-dimensional optical lattices is developed, demonstrating the performance advantages of lattice light-sheet microscopy compared with previous techniques and highlighted phenomena that, when seen at increased spatiotemporal detail, may hint at previously unknown biological mechanisms.
Abstract: Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

1,585 citations


"Performance trade-offs for single- ..." refers methods in this paper

  • ...LSM was originally popularized for volumetric imaging of live model organisms in developmental biology [1, 4–6] and more recently has been explored for imaging large optically cleared ex vivo tissues [7–9], including clinical specimens [10–12]....

    [...]

Journal ArticleDOI
TL;DR: This new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi and is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.
Abstract: Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

1,140 citations

Journal ArticleDOI
TL;DR: A simple illumination method of fluorescence microscopy for molecular imaging yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.
Abstract: We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.

1,110 citations


"Performance trade-offs for single- ..." refers methods in this paper

  • ...Dunsby employed this method to develop the first single-objective LSM system by combining several precursor designs [15, 34, 35]....

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Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Performance trade-offs for single- and dual-objective light-sheet microscope designs" ?

In order to elucidate some of the key design trade-offs for LSM systems, the authors present a diffractionbased analysis of singleand dual-objective LSM configurations where Gaussian illumination is utilized. Specifically, the authors analyze the effects of the illumination and collection numerical aperture ( NA ), as well as their crossing angle, on spatial resolution and contrast. Assuming an open-top light-sheet ( OTLS ) architecture, the authors constrain these parameters based on fundamental geometric considerations as well as those imposed by currently available microscope objectives. In addition to revealing the performance tradeoffs of various singleand dual-objective LSM configurations, their analysis showcases the potential advantages of a novel, non-orthogonal dual-objective ( NODO ) architecture, especially for moderate-resolution imaging applications ( collection NA of 0. 5 to 0. 8 ).