High NA single-objective light-sheet

This project is maintained by amsikking in the York lab, and was funded by Calico Life Sciences LLC

(in progress) Appendix

Note that this is a limited PDF or print version; animated and interactive figures are disabled. For the full version of this article, please visit one of the following links: https://amsikking.github.io/high_na_single_objective_lightsheet https://andrewgyork.github.io/high_na_single_objective_lightsheet https://calico.github.io/high_na_single_objective_lightsheet

High NA single-objective light-sheet

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Design

(to update)

Figure A1
SOLS parameters: a reference diagram for key parameters. Here we label the single-objective light-sheet schematic and highlight the key components and parameters that are useful for disucsions of design, build and alignment.

Build

Parts list - emission path

This table can be used as a shopping list for the original design of the emission path. For clarity, the elements are listed in order in which they are physically arranged (from sample to camera). All of the optics are 'stock' parts except for items 10 (O2*) and 11 (O3) which are currently 'bespoke'. Item 10, the small glass window (O2*), can be exchanged for a standard coverslip for limited budgets. If you are interested in item 11 the glass-tipped objective (O3) then please see the following section on the AMS-AGY objective v1.0.

Item number (ID) Supplier Part Description Qty
1 (S) Biologist Sample preferred with typical live cell refractive index: 1.35 to 1.4 1
2 (O1) Nikon MRD73950 CFI SR HP Plan Apo Lambda S 100XC Sil 1
3 (TL1) Nikon MXA22018 Tube lens, EFL=200 mm 1
4 (SL1) Thorlabs CLS-SL Scan lens, EFL=70 mm 1
5 (G1) Thorlabs GVS201 1D Galvo system, ∅5 mm 1
6 (SL2) Thorlabs LSM03-VIS Scan lens, EFL=39 mm 1
7 (TL2) Nikon MXA22018 Tube lens, EFL=200 mm 1
8 (D) Chroma ZT405/488/561/640rpcv2 Quad band dichroic (excitation coupling) 1
9 (O2) Nikon MRD00405 CFI Plan Apo Lambda 40XC 1
10 (O2*) Mark Optics N/A NBK-7 window, OD:(4 +/- 0.2) mm, Thk:(0.170 +/- 0.0127) mm, TWE:1/4 wave, parallelism:<10 μm, polished:40/20 and coated:Tavg >99%, (400-700) nm @ 0° AOI both sides 1
11 (O3) ASI AMS-AGY v1.0 Special glass-tipped objective 1
12 (E) Chroma ZET405/488/561/640m Quad emission filter, ∅25 mm mounted 1
13 (TL3) Nikon MXA22018 Tube lens, EFL=200 mm 1
14 (C) PCO edge 4.2 sCMOS camera, 2048x2048 pixels with 6.5x6.5 μm2 size 1

AMS-AGY objective - v1.0

The critical enabling component of the high NA single-objective light-sheet microscope is the bespoke glass-tipped objective. By choosing all stock parts throughout the design of the rest of the microscope many of the opto-mechanical difficulties are compressed into the design of this final objective (Figure A1). The optical performance and geometry of this lens allow it to extract a high resolution image as close as ~100 μm to a flat surface, with negligible loss in resolution and high transmission, over a range of tilt angles from 0-45 degrees.

Figure A1 Figure A2 Figure A2
Figure A1: Bespoke glass-tipped objective. A drawing a) shows the critical opto-mechanically coupled design considerations for the bespoke objective: to collect a tilted air space image just ~100 μm from a flat surface, with a range of tilt angles from 0-45 degrees. A CAD rendering b) of the design and a photo c) of the final working lens.

The optical specifications are as follows:

Specification Description
NA = 1.0 numerical aperture
∞/0 infinity corrected / coverslip thickness
WD = 0 working distance
EFL = 5 mm effective focal length (e.g. 40x with 200 mm tube lens)
λ = 450-700 nm color correction
FOV (DL) = ∅150 μm diffraction limited field of view diameter
FOV (bevel) = ∅250 μm mechanically limited field of view diameter (1 direction only)

If you are interested in acquiring this objective then please contact Jon Daniels at Applied Scientific Imaging (ASI) directly: jon@asiimaging.com (or info@asiimaging.com).

Drawings

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Align

Alignment goals

Here we recommend alignment goals for good performance. This is not a detailed alignment procedure but should be sufficient for an experienced builder and is applicable to all the optical configurations. With reference to the SOLS parameters figure try to achieve the following:

Alignment procedure

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Optical configurations

Several of our colleagues have expressed interest in modified versions of our reference design. We think this is wonderful, but we've already noticed pitfalls and mistakes that are commonly encountered (but easily avoided). It's critical to use lenses that give the right magnifications, and avoid aberrations and vignetting. Here we offer a selection of alternative configurations for high NA single-objective light-sheets. We haven't built these versions, but based on our knowledge and experience we expect them to have excellent performance (comparable to our reference design). Please try them and give us feedback - we are here to help and if you want to roll your own design, feel free to reach out - some low-effort advice from us might save you a lot of time and money. See the following guide on how to configure your own single-objective light-sheet microscope.

Configuration guide

Oil immersion with coverslip:

Primary objective (O1 + TL1) Secondary objective (O2 + TL2)
Nikon 60x1.4 Oil (MRD01605 + MXA22018)
or
Olympus 60x1.42 Oil (UPLXAPO60XO + SWTLU-C)
Nikon 40x0.95 (MRD00405 + MXA22018)
or
Olympus 40x0.95 (UPLXAPO40X + SWTLU-C)

Silicone immersion with coverslip:

Primary objective (O1 + TL1) Secondary objective (O2 + TL2)
Nikon 100x1.35 Sil (MRD73950 + MXA22018)
or
Olympus 100x1.35 Sil (UPLSAPO100XS + SWTLU-C)
Nikon 40x0.95 (MRD00405 + EFL357)
or
Olympus 40x0.95 (UPLXAPO40X + EFL321
Olympus 60x1.3 Sil (UPLSAPO60XS2) + (SWTLU-C) Nikon 40x0.95 (MRD00405 + EFL214)
or
Olympus 40x0.95 (UPLXAPO40X + EFL193)
Nikon 40x1.25 Sil (MRD73400 + MXA22018)
or
Olympus 40x1.25 Sil (UPLSAPO40XSS + SWTLU-C)
Nikon 40x0.95 (MRD00405 + EFL143)
or
Olympus 40x0.95 (UPLXAPO40X + EFL129)
Olympus 30x1.05 Sil (UPLSAPO30XS + SWTLU-C) Nikon 20x0.75 (MRD00205 + EFL214)
or
Olympus 20x0.8 (UPLXAPO20X + EFL193)
Nikon 25x1.05 Sil (MRD73250 + MXA22018) Nikon 20x0.75 (MRD00205 + EFL179)
or
Olympus 20x0.8 (UPLXAPO20X + EFL161)

Water immersion with coverslip:

Primary objective (O1 + TL1) Secondary objective (O2 + TL2)
Nikon 60x1.27 W (MRD07650 + MXA22018)
or
Olympus 60x1.2 W (UPLSAPO60XW + SWTLU-C)
Nikon 40x0.95 (MRD00405 + EFL226)
or
Olympus 40x0.95 (UPLXAPO40X + EFL203)
Nikon 40x1.15 W (MRD77410 + MXA22018)
or
Olympus 40x1.15 W (UAPON40XW340 + SWTLU-C)
Nikon 40x0.95 (MRD00405 + EFL150)
or
Olympus 40x0.95 (UPLXAPO40X + EFL135)

Water dipping:

Primary objective (O1 + TL1) Secondary objective (O2 + TL2)
Nikon 25x1.1 W (MRD77220 + MXA22018)
or
Olympus 25x1.05 W (XLPLN25XWMP2 + SWTLU-C)
Nikon 20x0.75 (MRD00205 + EFL188)
or
Olympus 20x0.8 (UPLXAPO20X + EFL169)
Olympus 20x1.0 W (XLUMPLFLN20XW + SWTLU-C) Nikon 20x0.75 (MRD00205 + EFL150)
or
Olympus 20x0.8 (UPLXAPO20X + EFL135)

Scan lens 1 (SL1) Galvo 1 (G1) Scan lens 2 (SL2)
Thorlabs 70 mm (CLS-SL) Thorlabs ∅5 mm (GVS201)
or
Thorlabs ∅10 mm (GVS211)
Thorlabs 70 mm (CLS-SL)

Galvo scanner considerations:

Note: galvo mirrors are typically thin so they are light and fast to rotate. As the mirror diameter increases it becomes harder to maintain flatness which can seriously degrade optical performance. If you want to buy your way out of this potential problem then specify better than λ/10 PV or λ/14 RMS for the mirror.

Example: Nikon 40x(1.15) water with coverslip

'I want to do expansion microscopy on relatively large (aqueous) samples at high resolution. The samples are weakly flourescent so I want to use light sheet with minimal optics on the emission path. Speed is not important to me. I have a Nikon base already and I can borrow an Olympus objective from a collaborator for O2.'

System notes:

For maximum optical efficiency galvo/scan lens relays will not be used on this system; instead the sample will be scanned at a significantly slower rate. The only limits on sample size will be the relatively large working distance of the primary objective (~600 μm here). An AR coated window will be used at O2* for maximum transmission. A galvo scanner can be added later if sample scanning proves too slow.

Optical train:

Item ID Supplier Part Description Qty
S Biologist Sample expanded samples, refractive index: ~1.33 1
O1 Nikon MRD77410 40x(1.15) water through coverslip, W.D. 0.59-0.61 mm, 22 mm field 1
TL1 Nikon MXA22018 Tube lens, EFL=200 mm 1
TL2 Thorlabs EFL135 Tube lens assembly, EFL=135 mm 1
D Chroma ZT405/488/561/640rpcv2 Quad band dichroic (excitation coupling) 1
O2 Olympus UPLXAPO40X 40x0.95 CFI Plan Apo Lambda 1
O2* Mark Optics custom NBK-7 window, OD:(10 +/- 0.2) mm, Thk:(0.170 +/- 0.05) mm, TWE:1/4 wave, parallelism:<10 μm, polished:40/20 and coated:Tavg >99%, (400-700) nm @ 0° AOI both sides 1
O3 ASI AMS-AGY v1.0 Special glass-tipped objective 1
E Chroma ZET405/488/561/640m Quad emission filter, ∅25 mm mounted 1
TL3 Nikon MXA22018 Tube lens, EFL=200 mm 1
C PCO edge 4.2 sCMOS camera, 2048x2048 pixels with 6.5x6.5 μm2 size 1

Example: Olympus 20x(1.0) water dipping

'I work with Zebrafish so I'm looking for a water dipping system with a large field of view and long working distance. I already have an Olympus 20x1.0 dipping lens, a Nikon 20x0.75 from another system and a limited budget. Field of view and speed are most important for my application so I'm going to choose a two-galvo system to maximise the data rate on my sCMOS camera and I'm willing to sacrifice some optical performance.'

System notes:

Optical train:

Item ID Supplier Part Description Qty
S Biologist Sample Zebrafish in water, refractive index: ~1.33 1
O1 Olympus XLUMPLFLN20XW 20x(1.0) water dipping, W.D. = 2 mm, 22 mm field 1
TL1 Olympus SWTLU-C Tube lens, EFL=180 mm 1
SL1 Thorlabs CLS-SL Scan lens, EFL=70 mm 1
G1 Thorlabs GVS211 1D scan galvo, ∅10 mm 1
SL2 Thorlabs CLS-SL Scan lens, EFL=70 mm 1
SL3 Thorlabs CLS-SL Scan lens, EFL=70 mm 1
G2 Thorlabs GVS211 1D tile galvo, ∅10 mm 1
SL4 Thorlabs CLS-SL Scan lens, EFL=70 mm 1
TL2 Thorlabs EFL150 Tube lens assembly, EFL=150 mm 1
D Chroma ZT405/488/561/640rpcv2 Quad band dichroic (excitation coupling) 1
O2 Nikon MRD00205 20x0.75 CFI Plan Apo Lambda 1
O2* Various N/A Coverslip, OD: ~25 mm, Thk:(0.170 +/- 0.05) mm 1
O3 ASI AMS-AGY v1.0 Special glass-tipped objective 1
E Chroma ZET405/488/561/640m Quad emission filter, ∅25 mm mounted 1
TL3 Nikon MXA22018 Tube lens, EFL=200 mm 1
C PCO edge 4.2 sCMOS camera, 2048x2048 pixels with 6.5x6.5 μm2 size 1

Tube lens assemblies

One of the challenges of designing a single-obective light-sheet is making a good remote refocus (detailed in our previous work). For an air based remote refocus a key requirement is to match the magnification of the remote image to the refractive index of the primary objective. As the primary objective changes, often the magnification and refractive index also change, which can make it awkward to find the right set of optics. We solve this by creating a series of low cost tube lens assemblies at convenient focal lengths to enable builders to configure the microscope as they wish. See the table and figure below to build your own tube lens assembly:

TL2 assembly Optical components (part#)
EFL129 Thorlabs (TTL200MP) + 2x (AC508-500-A)
EFL135 Thorlabs (TTL200MP) + (AC508-750-A) + (AC508-500-A)
EFL143 Thorlabs (TTL200MP) + (AC508-750-A) + (AC508-500-A)
EFL150 Thorlabs (TTL200MP) + 2x (AC508-750-A)
EFL161 Thorlabs (TTL200MP) + 2x (AC508-1000-A)
EFL169 Thorlabs (TTL200MP) + (AC508-750-A)
EFL179 Thorlabs (TTL200MP) + (AC508-1000-A)
or
Olympus (SWTLU-C)
EFL188 Thorlabs (TTL200MP) + (AC508-1000-A)
EFL193 Thorlabs (TTL200MP) + (AC508-1000-A)
EFL203 Thorlabs (TTL200MP) + (LF-1141-A)
or
Nikon (MXA22018)
EFL214 Thorlabs 3x (AC508-750-A) + (AC508-1000-A)
EFL226 Thorlabs 3x (AC508-750-A) + (AC508-1000-A)
EFL321 Thorlabs (AC508-750-A) + (AC508-500-A)
EFL357 Thorlabs (AC508-750-A) + (AC508-500-A)
tl assembly
Tube lens option = the last 3 numbers indicate the effective focal length (mm)
Tube lens assemblies: a selection of useful tube lens assemblies for building a single-objective light-sheet with various objectives. Use the drop down menu to select the focal length to match the system you want to build. Assemble the lens by screwing together the parts in the drawing as labelled (open the image in a separate window or download for a detalied view).

Acquisition

Additional equipment

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How we took our data

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Example code

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Theoretical performance

In the abstract we claim that we "sacrifice no appreciable numerical aperture". The figure below illustrates what we mean by this:

Ewald spheres
Ray transmission angles for objectives 1, 2, and 3: Simple theoretical model showing angular passband of objective 1 (red dots), objective 2 (blue dots, almost invisible), objective 3 (green dots), and all three objectives (yellow dots). This figure is the 3D equivalent of the red, blue, green, and yellow circular regions shown in the top center region of Figure 1. Note that the yellow region almost entirely fills the red region, meaning the system transmits almost all of the rays collected by the primary objective. See below for the code used to calculate this figure.

Each yellow dot on the surface of the illustrated sphere represents a ray angle that can propagate from a point source in the sample, through our three objective lenses, and reach our detector. Each red dot indicates a ray that is collected by our primary objective (1.35 NA silicone), but clipped by our secondary objective (0.95 NA air). Each blue dot represents a ray which passes our secondary objective, but is clipped by our tertiary objective (note that there are almost no blue dots). Each green dot indicates a ray that our tertiary objective (1.0 NA glass) could have collected, if the secondary objective produced it. In our primary design, >95% of the rays collected by objective 1 can pass objective 2, and >99% of the rays collected by objective 2 can pass objective 3, giving an effective NA of about 1.33. See the code below used to calculate these quantities:

#!/usr/bin/python3
import numpy as np

# Three spherical caps: 1 and 2 are centered on the z-axis, 3 is pi
# radians wide
cap_1_width = np.arcsin(1.35/1.404) # radians
cap_2_width = np.arcsin(0.95)       # radians
sheet_half_angle = 3 * np.pi/180    # radians
cap_3_tilt = (np.pi/2 - cap_1_width) + sheet_half_angle # radians, in yz plane
# Generate random points on the surface of a sphere
num_points = int(3e4)
phi = np.random.uniform(0, 2*np.pi, num_points)
theta = np.arccos(np.random.uniform(-1, 1, num_points))
# Calulate z in a rotated frame where cap 2 is centered on the (new) z-axis:
z_rotated = (np.sin(cap_3_tilt) * np.sin(theta) * np.sin(phi) +
             np.cos(cap_3_tilt) * np.cos(theta))
# Check which caps each point occupies:
in_cap_1 = theta < cap_1_width
in_cap_2 = theta < cap_2_width
in_cap_3 = z_rotated > 0
# Estimate the fraction of the first cap covered by the second and third caps
ratio_1_2 = (np.count_nonzero(in_cap_1 & in_cap_2) /
             np.count_nonzero(in_cap_1))
ratio_2_3 = (np.count_nonzero(in_cap_2 & in_cap_3) /
             np.count_nonzero(in_cap_2))
print("Spherical cap 1 half-angle: %0.3fpi radians (%0.2f degrees)"%(
    cap_1_width / np.pi, cap_1_width * 180/np.pi))
print("Spherical cap 2 half-angle: %0.3fpi radians (%0.2f degrees)"%(
    cap_2_width / np.pi, cap_2_width * 180/np.pi))
print("Spherical cap 3 tilt angle: %0.3fpi radians (%0.2f degrees)"%(
    cap_3_tilt / np.pi, cap_3_tilt * 180/np.pi))
print("Fraction of cap 1 covered by cap 2: %0.5f"%(ratio_1_2))
print("Fraction of cap 2 covered by cap 3: %0.5f"%(ratio_2_3))

Note that our number for "efficiency" is fairly sensitive to how we define efficiency. For example, if objective 1 were 1.33 NA rather than 1.35 NA, then >99% of the rays collected by the primary can also pass the secondary and tertiary objectives. In our experience, rays at the very edge of the primary objective NA are somewhat aberrated, and do not contribute to improved resolution. We often take pains to differentiate between specified NA (rays which are collected) and useful NA (rays which contribute to improved resolution); our design captures >99% of our primary objective's useful NA. Note also that if a 0.96 NA air objective ever became available, we could substitute it as the secondary objective and collect >99% of the primary objective's specified 1.35 NA rays.

History

A summary of single objective light sheet publications (may or may not include...)

Archive

We use this section to store previous sections of the publication that have since been replaced in some way or are no longer appropriate for the main article.

(Deprecated) Alternative optical configurations

(Deprecated) Nikon 100x NA 1.35 - stage or piezo scanning solution

For those who don't want or need galvo scanning. This design has fewer optics for a system with lower cost, higher efficiency and easier maintenance. It could be a good option for high content screening systems that use stage scannning, or systems that use a piezo on the second objective to take volumes (or a combination of both)

Item # Supplier Part DescriptionQty
3 Thorlabs TTL165-A Tube Lens, f = 165 mm, ARC: 350-700 nm, External SM2 Threads (NOTE: this is not a Zeiss OEM tube lens) 1
4-6 N/A N/A Galvo scanning relay removed. 0
7 Thorlabs TL300-A Laser Scanning Tube Lens, f = 300 mm, ARC: 400 - 700 nm 1

(Deprecated) Olympus 60x NA 1.3 - galvo scanning solution

This is a high quality primary objective used by many labs - we expect this version to be popular.

Item # Supplier Part DescriptionQty
2 Olympus UPLSAPO 60xS2 Super Apochromat, 60x Silicone Oil Immersion Objective Lens, N.A. 1.3, W.D. 0.3 mm, F.O.V. 22 mm, DIC, Correction Collar 0.15-0.19 mm 1
3 Thorlabs TL300-A Laser Scanning Tube Lens, f = 300 mm, ARC: 400 - 700 nm 1

(Deprecated) Olympus 60x NA 1.3 - stage or piezo scanning solution

For those who don't want or need galvo scanning.

Item # Supplier Part DescriptionQty
2 Olympus UPLSAPO 60xS2 Super Apochromat, 60x Silicone Oil Immersion Objective Lens, N.A. 1.3, W.D. 0.3 mm, F.O.V. 22 mm, DIC, Correction Collar 0.15-0.19 mm 1
3 Thorlabs TTL165-A Tube Lens, f = 165 mm, ARC: 350-700 nm, External SM2 Threads (this is not a Zeiss OEM tube lens) 1
4-6 N/A N/A Galvo scanning relay removed. 0
7 Olympus SWTLU-C 180 mm focal length, tube lens unit. 1
9 Olympus UPLSAPO 40x2 Super Apochromat, 40x Objective Lens, N.A. 0.95, W.D. 0.18 mm, F.O.V. 26.5 mm, Correction Collar 0.11-0.23 mm 1

(Deprecated) Focus on Microscopy 2019 - Abstract

"A BOLT-ON SINGLE-OBJECTIVE LIGHT-SHEET DESIGN WITH UNCOMPROMISED NUMERICAL APERTURE"

Spinning disk confocal modules are “core facility friendly”; they insert conveniently between a commercial microscope base and camera, improve image quality and add no significant drawbacks. In contrast, high numerical aperture (NA) light sheet microscopy often requires radical sample modification, substantial user re-training and fully customized hardware. We present a “core-facility friendly” light-sheet: a “black box” that inserts between a commercial microscope base and camera, greatly reducing photo-toxicity without degrading image quality or breaking compatibility with existing sample preparation.

In 2008, based on the ingenious ‘Remote-Refocus’ of E.J. Botcherby and T. Wilson [Botcherby 2007] , C. Dunsby [Dunsby 2008] invented a brilliant single-objective light sheet technique called 'Oblique Plane Microscopy' (OPM) that bypassed many typical light sheet drawbacks. However his theoretical NA of 0.74 for a water immersion objective was significantly lower than the 1.33 limit for an aqueous sample. In 2018 B. Yang and B. Huang [Yang 2018] made a clever modification to OPM to achieve an NA of 1.06 and incorporated the elegant galvo-based volumetric scanning method invented by M.B. Bouchard and E.M.C. Hillman [Bouchard 2015].

Building on this work, we present a simple, robust and modular light sheet design with an NA in the 1.2-1.3 range, and discuss the key concepts and considerations for high NA single objective light sheet.

FOM2019 Figure 1
FOM2019 Fig. 1: Generalized design diagram for an OPM emission path