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A Compact and Versatile Light Sheet Microscopy Platform for Research and Education

Spim_solidworks-screenshot19.png

Selective Plane Illumination Microscope (SPIM) with sample rotation & translation. Optimized for uncompromised light sheet microscopy of samples like Drosophila embryos and spheroids while maintaining a small footprint for mobility and a reasonable price point. Epi-fluorescence and brightfield illumination, 16x/0.8 W objective, sCMOS camera. Most of the parts are off-the-shelf, with only three custom components made by Selective Laser Sintering (SLS).

Pictures

Spim_solidworks-screenshot09.png Spim_solidworks-screenshot05.png Spim_solidworks-screenshot06.png

Sample data

Light sheet properties

Scheme of the light sheet illumination arm with laser (blue), lenses
(gray), mirrors (brown) and detection (green). Incoming laser beam is
expanded to 2.7 mm diameter.

  • Scheme of the light sheet illumination arm with laser (blue), lenses (gray), mirrors (brown) and detection (green). Incoming laser beam is expanded to 2.7 mm diameter.

491 nm laser beam in water as seen in the field of view (FOV) after
passing through the illumination arm. Cylindrical lens, emission filter
and dichroic mirror removed. 22 ms exposure time. Full width at half
maximum (FWHM) determined using Gaussian fitting in Fiji. Yellow lines
indicate ideal Gaussian shape.

  • 491 nm laser beam in water as seen in the field of view (FOV) after passing through the illumination arm. Cylindrical lens, emission filter and dichroic mirror removed. 22 ms exposure time. Full width at half maximum (FWHM) determined using Gaussian fitting in Fiji. Yellow lines indicate ideal Gaussian shape.

List of parts

  • C = custom part / part that requires modification
  • some categories require parts from the Basics and Lenses category
  • tools not included
  • US prices from 2016/2017

Breadboard

Name Description C Vendor Order number Number Price/part Total price
Ø45.0 mm Aluminum Breadboard 24" x 36" x 1/2", 1/4"-20 Taps main breadboard Thorlabs MB2436 1 $667.00 $667.00
Ø45.0 mm 12" x 6" (305 mm x 152 mm) Straight Laser Safety Screen laser shielding for breadboard Thorlabs TPS13 10 $55.00 $550.00
Ø45.0 mm Sorbothane Feet, Internal 1/4"-20 Mounting Thread, 4 Pieces simple vibration isolation for breadboard Thorlabs AV6 1 $30.75 $30.75

Basics

Name Description C Vendor Order number Number Price/part Total price
Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 2" posts for cage system Thorlabs TR2 20 $5.19 $103.80
Ø1/2" Pedestal Post Holder, Spring-Loaded Hex-Locking Thumbscrew, L=1.19" mounts for posts Thorlabs PH1E 20 $22.50 $450.00
Short Clamping Fork, 1.24" Counterbored Slot, 1/4"-20 Captive Screw clamps for post mounts Thorlabs CF125C 20 $10.50 $210.00
Cage Assembly Rod, 1/2" (12.7 mm) Long, Ø6 mm 0.5" cage rods Thorlabs ER05 4 $4.94 $19.76
Cage Assembly Rod, 1" (25.4 mm) Long, Ø6 mm 1" cage rods Thorlabs ER1 16 $4.94 $79.04
Cage Assembly Rod, 1.5" (38.1 mm) Long, Ø6 mm 1.5" cage rods Thorlabs ER1.5 8 $5.70 $45.60
Cage Assembly Rod, 2" (50.8 mm) Long, Ø6 mm 2" cage rods Thorlabs ER2 4 $5.94 $23.76
Cage Assembly Rod, 3" (76.2 mm) Long, Ø6 mm 3" cage rods Thorlabs ER3 12 $6.44 $77.28
Cage Assembly Rod, 4" (101.6 mm) Long, Ø6 mm 4" cage rods Thorlabs ER4 8 $6.94 $55.52
Cage Assembly Rod, 6" (152.4 mm) Long, Ø6 mm 6" cage rods Thorlabs ER6 4 $8.44 $33.76
Cage Assembly Rod, 8" (203.2 mm) Long, Ø6 mm 8" cage rods Thorlabs ER8 8 $11.44 $91.52
Cage Assembly Rod, 10" (254.0 mm) Long, Ø6 mm 10" cage rods Thorlabs ER10 4 $12.44 $49.76
Rod Adapter for Ø6 mm ER Rods, Qty. 1 cage rod adapters Thorlabs ERSCA 8 $10.80 $86.40
30 mm Removable Cage Plate, Front and Back Plate, Internal SM1 Threading quick release lens mount, 30 mm cage system Thorlabs CP90F 5 $48.00 $240.00
SM1 Threaded 30 mm Enhanced Clamping Cage Plate, 0.35" Thick, 8-32 Tap 30 mm cage plate, internal SM1 Thorlabs CP08 15 $19.00 $285.00
Post-Mountable Cage Plate with Ø1.0" Double Bore, 8-32 Tap 30 mm cage plate, internal 1" Thorlabs CP06 6 $16.75 $100.50
Precision Cage Rotation Mount with Micrometer Drive, Ø1" Optics, 8-32 Tap 30 mm cage plate w/ rotational mount Thorlabs CRM1P 2 $197.88 $395.76
Kinematic Fluorescence Filter Cube, 30 mm Cage Compatible, 1/4"-20 Tapped Holes 30 mm cage filter cube for 25 x 36 x 2 mm dichroic, 25 mm filters Thorlabs DFM1 1 $326.00 $326.00
Cage Assembly Turret Mount Ø1" Optics 30 mm cage mirror gimbal mount Thorlabs KC45D 4 $197.40 $789.60
Fixed 90° Bracket, Ø6 mm Bore and 4-40 Taps 90 deg cage rod connectors Thorlabs ER90B 16 $13.70 $219.20
Ø1/2" Universal Post Holder, Spring-Loaded Locking Thumbscrew, L = 1.5" post holders Thorlabs UPH1.5 14 $29.50 $413.00
Externally SM1-Threaded End Cap for Machining SM1 covers as laser target / cube cover Thorlabs SM1CP2M 2 $18.00 $36.00
Ring-Actuated SM1 Iris Diaphragm SM1-threaded aperture for LEDs, laser alignment Thorlabs SM1D12D 4 $64.30 $257.20
SM1 Lens Tube, 1.00" Thread Depth, One Retaining Ring Included SM1 tube Thorlabs SM1L10 3 $14.25 $42.75

Lenses

Name Description C Vendor Order number Number Price/part Total price
f = 50 mm, Ø1" Cylindrical Achromat, AR Coating: 350 - 700 nm achromatic cylindrical lens Thorlabs ACY254-050-A 1 $368.00 $368.00
f=30 mm, Ø1" Achromatic Doublet, SM1-Threaded Mount, ARC: 400-700 nm beam expander lens 1, collimators for white & 470 nm LEDs Thorlabs AC254-030-A-ML 3 $102.00 $306.00
f=100 mm, Ø1" Achromatic Doublet, SM1-Threaded Mount, ARC: 400-700 nm beam expander lens 2 Thorlabs AC254-100-A-ML 1 $95.90 $95.90
f=50 mm, Ø1" Achromatic Doublet, SM1-Threaded Mount, ARC: 400-700 nm telescope lenses Thorlabs AC254-050-A-ML 2 $95.90 $95.90

Sample chamber

Name Description C Vendor Order number Number Price/part Total price
sample-chamber_v2.STL sample chamber - to be produced with selective laser sintering x Stratasys 1 $53.06 $53.06
30 mm Cage Cube cube for sample chamber - one opening to be extended to ?" x Thorlabs C4W 1 $57.90 $57.90
Ø1/2" Sapphire Broadband Precision Window, Uncoated glass windows for sample chamber Thorlabs WG30530 3 $40.00 $120.00
SM1 (1.035"-40) Tapped Cube Plate for C4W/C6W bottom plate for cube Thorlabs B2C 1 $14.70 $14.70

Illumination arm

Name Description C Vendor Order number Number Price/part Total price
10X Nikon CFI Plan Fluorite Objective, 0.30 NA, 3.5 mm WD illumination objective Nikon N10XW-PF 1 $1,315.60 $1,315.60
Cobolt Calypso 491nm 100mW DPSS 491nm laser Cobolt 1
Laser baseplate XxY" baseplate for 491nm laser - to be cut from solid aluminium plate, added holes for mounting laser and posts (8-32) x
PCAOM acousto-optical tunable filter for laser illumination NEOS 48062-2.5-.55 1
4-channel PCAOM driver AOTF controller NEOS 64040-75-.1.4CH 1
Adapter with External SM1 Threads and Internal M25 x 0.75 Threads adapter illumination objective to SM1 Thorlabs SM1A12 1 $19.75 $19.75
25.4mm Diameter, 4-6λ Mirror 1" mirror, only 3 mm thick, for gimbal mount Edmund Optics #69-245 4 $16.00 $64.00
Z-Axis Translation Mount, 30 mm Cage Compatible zoom housing for telescope lens Thorlabs SM1Z 1 $186.00 $186.00
30mm Cage System Adjustable Slit, 8-32 Tap, Imperial Micrometer slit for setting light sheet properties, 30 mm cage system Thorlabs VA100C 2 $260.00 $520.00
Kinematic Mount with Vertical Drive, Ø1/2" Optics vertical drive 0.5" mirror mounts for laser module Thorlabs VM05 8 $97.90 $783.20
Ø1/2" Broadband Dielectric Mirror, 400 - 750 nm 0.5" mirrors for laser module Thorlabs BB05-E02 4 $49.40 $197.60
30 mm Cage System Iris, Ø20.0 mm Maximum Aperture iris for beam alignment, 30 mm cage system Thorlabs CP20S 8 $85.00 $680.00
Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 1.5" short posts for laser baseplate Thorlabs TR1.5 4 $4.97 $19.88
Unmounted Continuously Variable ND Filter, Ø50 mm, OD: 0-4.0, ARC: 350 - 700 nm ND filter wheel for additional attenuation in laser module Thorlabs NDC-50C-4-A 1 $368.00 $368.00
Post Mount Assembly for Round, Unmounted, ND Filters mount for ND filter wheel Thorlabs NDC-PM 1 $52.25 $52.25
Assmann WSW Components H7VXH-0906M cable for triggering AOTF Digi-Key H7VXH-0906M-ND 2 $5.18 $10.36
Pomona Electronics 4969 cable for BNC input to other cable Digi-Key 501-1030-ND 4 $8.99 $35.96

Detection arm

Name Description C Vendor Order number Number Price/part Total price
16X Nikon CFI LWD Plan Fluorite Objective, 0.80 NA, 3.0 mm WD detection objective Nikon N16XLWD-PF 1 $5,824.21 $5,824.21
Infinity-Corrected Tube Lens for Plan Fluorite Objectives 200 mm tube lens for detection arm Nikon 1 $450 $450
Hamamatsu ORCA Flash 2.8 (1,920x1,440, 3.63µm pixels, 70% QE, 45fps) main camera Hamamatsu C11440-10C 1
Bandpass emission filter 525/50, mounted, 25 mm emission filter for "green" fluorophores Chroma ET525/50m 1 $325 $325
8-Channel, 16-Bit Analog Voltage Output Device USB DAQ for AOTF, LEDs etc MCC USB-3103 1 $349.00 $349.00
Adapter with External SM1 Threads and Internal M32 x 0.75 Threads adapter detection objective to SM1 Thorlabs SM1A34 1 $22.00 $22.00
Adapter with External SM2 Threads and Internal M38 x 0.5 Threads tube lens to SM2 adapter Thorlabs SM2A20 1 $45.00 $45.00
60 mm Cage Plate, SM2 Threaded, Enhanced Clamping, 0.5" Thick, 8-32 Tap 60 mm cage plate w/ SM2 thread Thorlabs LCP08 1 $47.90 $47.90
60 mm Cage Plate with Ø2" Double-Bore Optic Mount, 8-32 Tap 60 mm cage plate for holding SM2 tube Thorlabs LCP06 1 $33.50 $33.50
30 mm to 60 mm Cage Plate Adapter, 8-32 Tap 60 to 30 mm cage adapter Thorlabs LCP02 2 $38.00 $76.00
Adapter with External C-Mount Threads and External SM1 Threads C-mount-to-SM1 adapter Thorlabs SM1A39 1 $20.00 $20.00
Adapter with External SM2 Threads and Internal SM1 Threads SM1-to-SM2 adapter Thorlabs SM2A6 1 $24.00 $24.00
SM2 Lens Tube Without External Threads, 1/2" Thread Depth, Two Retaining Rings Included SM2 tube for cage plate Thorlabs SM2M05 1 $24.50 $24.50
60 mm Cage Plate with Ø2.2" Double Bore for SM2 Lens Tube Mounting 60 mm cage plate for SM2 tube Thorlabs LCP09 1 $41.20 $41.20
Dual-Position Slider Bundle: Dual-Position Slider, Interface Board, Cables motorized filter switch Thorlabs ELL6K 1 $268.00 $268.00
SM1 Lens Tube, 0.30" Thread Depth, One Retaining Ring Included filter mount for motorized filter switch Thorlabs SM1L03 3 $12.16 $36.48
Baseplate for filter switch control board baseplate for mounting filter switch control board to breadboard - to be laser-cut x 1
30 mm to 60 mm Cage Plate Adapter, 8-32 Tap cage mount for filter switch Thorlabs LCP02 3 $38.00 $114.00
Cage Assembly Rod, 2" Long, Ø6 mm, 4 Pack cage extension rods Thorlabs ER2-P4 1 $22.57 $22.57
Cage Assembly Rod, 1" Long, Ø6 mm, 4 Pack cage rods for mounting filter switch Thorlabs ER1-P4 2 $18.77 $37.54
SM1 Lens Tube, 1.00" Thread Depth, SM1RR Retaining Ring, 5 Pack light block for detection path Thorlabs SM1L10-P5

Stage & sample holder

Name Description C Vendor Order number Number Price/part Total price
DDR05-to-PI111_adapter.STL adapter rotational stage to xyz stage - to be produced with selective laser sintering x Stratasys - 1 $46.36 $46.36
15mm Compact Micro-Translation Stage motorized linear stage PI M-111.1DG 3 $2,608.00 $7,824.00
1 Channel Mercury DC Motor Controller controller for linear stage PI C-863.11 3 $1,134.00 $3,402.00
Mounting Bracket for Vertical Mount of M-111 on M-110, M-111 and M-112 vertical adapter plate for linear stage PI M-110.03 1 $128.00 $128.00
StarTech Model ICUSB2321F 6 ft. 1 Port FTDI USB to Serial RS232 Adapter Cable with COM Retention M-M USB to Serial adapter for PI controller Newegg N82E16812200964 3 $20.99 $62.97
Direct Drive Continuous Rotation Mount, SM05 Bore, 8-32 Taps motorized rotational stage Thorlabs DDR05 1 $1,800.00 $1,800.00
K-Cube Brushless DC Servo Driver controller for rotational stage Thorlabs KBD101 1 $716.00 $716.00
66 mm Single Dovetail Rail, L = 500 mm rail for stage - to be shortened to ?", 4 holes drilled for 1/4"-20 screws x Thorlabs XT66SP-500 1 $30.50 $30.50
Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 4" posts for stage rail Thorlabs TR4 1 $5.87 $23.48
Rail Carriage for 66 mm Rails with 6-32, 8-32, & 1/4"-20 Taps rail carriage for stage Thorlabs XT66P2 1 $67.10 $67.10
30 mm Cage System, XY Translating Mount for Ø1" Optics with Quick Release Plate adjustable quick-release sample holder plate Thorlabs CXY1Q 1 $225.00 $225.00
30 mm to 16 mm Cage Adapter Plate adapter plate for sample holder Thorlabs SP05 1 $31.00 $31.00
Stereotaxic Cannula Holder, Ø2.5 mm Ferrules sample clamp Thorlabs XCF 1 $295.00 $295.00
XCF_adapter.STL adapter sample holder to 1" cage plate - to be produced with selective laser sintering x Stratasys 2
Mini Series Cage Assembly Rod, 1/4" (6.4 mm) Long, Ø4 mm, Qty. 1 cage system rods for sample holder Thorlabs SR025 4 $6.18 $24.72
30 mm Cage System Alignment Plate with Ø1 mm Hole 30 mm cage alignment plate small hole Thorlabs CPA1 1 $12.24 $12.24
30 mm Cage System Alignment Plate with Ø5 mm Hole 30 mm cage alignment plate large hole Thorlabs CPA2 1 $12.24 $12.24
Alloy Steel Socket Head Cap Screws, Zinc-Plated, M3 × 0.5, 16 mm Length, Pack of 25 screws for mounting stage adapter to M-111.1DG McMaster-Carr 95263A158 1 $3.90 $3.90
Alloy Steel Socket Head Cap Screws, Zinc-Plated, 8-32, 7/8" Length, Pack of 50 screws for mounting stage adapter to DDR05, type I McMaster-Carr 90128A198 1 $5.79 $5.79
Alloy Steel Socket Head Cap Screws, Zinc-Plated, 4-40, 3/4" Length, Pack of 10 screws for mounting stage adapter to DDR05, type II McMaster-Carr 90128A113 1 $4.55 $4.55
Dual Threaded Adapter with Internal M3 x 0.5 Threads and External 1/4"-20 Threads thread adapter for mounting M-111.1DG to XT66P2 Thorlabs AE3M25E 4 $4.10 $16.40
20 mm Long Double Dovetail Clamp for 66 mm Rails block for XT66 carrier Thorlabs XT66C1 1 $17.30 $17.30
Rail Platform Locator for 66 mm Rails carrier adjustable block for XT66 Thorlabs XT66N 1 $36.60 $36.60

Extra illumination

Name Description C Vendor Order number Number Price/part Total price
Cold White Mounted LED, 1000 mA, 800 mW (Min) LED for brightfield illumination Thorlabs MCWHL5 1 $193.00 $193.00
Blue (470 nm) Mounted LED, 1000 mA, 650 mW (Min) 470nm LED for widefield excitation Thorlabs M470L3 1 $268.00 $268.00
T-Cube LED Driver, 1200 mA Max Drive Current LED driver LEDD1B 2 $293.00 $586.00
15 V, 2.4 A Power Supply Unit for One K-Cube or T-Cube power supply for LED driver Thorlabs KPS101 3 $25.71 $77.13
Bandpass emission filter 470/40, mounted, 25 mm clean-up filter for excitation with 470nm LED Chroma ET470/40x 1 $325 $325
Dichoric mirror 488nm, unmounted, 36x25 mm dichoric mirror for excitation with 470nm LED Chroma ZT488rdc 1 $300 $300
SM1 Lens Tube, 1.00" Thread Depth, One Retaining Ring Included SM1 tube for blocking LED stray light Thorlabs SM1L10 2 $14.25 $28.50
30 mm Cage Plate, Ø1.2" Double Bore for SM1 Lens Tube Mounting cage plate for mounting SM1 tube Thorlabs CP12 2 $20.00 $40.00

Computer

Name Description C Vendor Order number Number Price/part Total price
Optiplex 7040 (Minitower, Win 10 Pro, Intel Core i7, 16 GB RAM, 256 GB M.2 SSD, Radeon R7, DVD drive, keyboard, mouse) acquisition computer Dell 210-AFGH OptiPlex 7040 Mini Tower 1 $1,300.00 $1,300.00
8 TB SATA hard drive data drive for acquisition computer 1
LabVIEW Base Development System for Windows development platform for acquisition software National Instruments ISWD460 1 $999.99 $999.99

Server rack for electronics

Name Description C Vendor Order number Number Price/part Total price
iStarUSA 23.6" Deep 4 RU Simple Server Rack (Black) server rack for controllers, power supplies etc BHPhotoVideo ISWD460 1 $259.99 $259.99
Middle Atlantic PD-2015R-NS Multi-Mount Rackmount Power power connector for server rack BHPhotoVideo MIPD2015NS 1 $87.00 $87.00
Odyssey Innovative Designs ARPOT1 Pullout Shelf (1-Space) pullout shelf for controllers BHPhotoVideo ODARPOT1 1 $59.99 $59.99
Auray RS-V-1U One-Space Vented Rack Shelf (1 RU) fixed shelf for power supplies BHPhotoVideo AURSV1U 2 $22.50 $45.00
Baseplate for controllers adapter for mounting rotational stage controller and LED controllers to pullout shelf - to be laser-cut x 1
Amphenol CS-DSDHD26MF0-010 26-Pin HD26 Deluxe D-Sub Cable, Shielded, Male/Female, 10', Gray cable for 491nm laser Amazon - 1 $16.75 $16.75
Hosa BNC-06-110 10-Feet 75-Ohm COAX BNC to BNC BNC cable for AOTF Amazon - 1 $12.98 $12.98
Hosa BNC-59-103 BNC to BNC 75-ohm Coax Cable, 3 feet BNC cables for LEDs Amazon - 2 $5.95 $11.90
Anker USB 3.0 SuperSpeed 10-Port Hub USB hub for peripherals Anker AH231 1 $49.99 $49.99
UGREEN USB Extension Cable USB 3.0 Extender Cord Type A Male to Female Data Transfer (6ft) USB extension for hub Amazon - 1 $6.99 $6.99

Environmental control

Name Description C Vendor Order number Number Price/part Total price
IncuKit™ MINI for Desktop Incubators temperature control for incubation cage Incubator Warehouse Incukit-DC 1 $50.00 $50.00
Cage incubator custom enclosure for heating sample & chamber - to be laser-cut x

Assembly

  1. Mount sample chamber
  2. Attach cage rods for illumination and detection arms to sample chamber
  3. Mount objective lenses using silicone
  4. Start assembling the detection arm
  5. Adjust distance camera - tube lens by focusing to infinity
  6. Finalize detection arm
  7. Assemble motor unit and sample holder
  8. Assemble illumination arm with mirrors, but without lenses
  9. Align laser to center of cage system and sample chamber
  10. Add circular lenses step by step, adjust focus and collimation where necessary
  11. Add cylindrical lens and adjust light sheet to focal plane of detection objective
  12. Add laser shielding to breadboard

Alignment

Troubleshooting

Phenotype

Cause

Solution

  • LabView crashes when trying to start Main.vi
  • LEDs on stage controllers are red

Linear stages not recognized

Run PI MikroMove and start up axes

  • LabView crashes when trying to record images
  • Camera tab in Main.vi doesn't show camera details

Camera not recognized

Restart computer (= have camera running before starting computer)

  • LabView crashes when trying to open Main.vi or DAQ_all.vi

DAQ not registered

Start Instacal and run the update, then restart computer

Suggested modifications

Modification Effort/Issues
Improve light sheet alignment stability by switching from Thorlabs KC45D to Thorlabs C45P Not gimbal; requires thicker mirrors; might run into space issues
Integrate a bio-compatible sample holder, made from e.g. plastic or surgical-grade stainless steel Requires new sample holder design, different adapter
Switch camera to an sCMOS USB model with similar pixel size and larger chip to reduce the required computer size / enable the use of a laptop Requires substantial adaptation of the software, or use of different software, e.g. µManager
Switch to µManager to make system more universal Requires integration of all hardware components in µManager, which might be difficult for some components, e.g. Thorlabs APT stage
Use lasers that support direct modulation to skip AOTF, simplify illumination beam path and reduce footprint of electronics rack Requires modification of software, modification of illumination beam path