Image Data

Multi-view imaging

Fixed Drosophila embryo mounted in 1.2% agarose. Sytox Green nuclear marker. 491nm excitation, band-pass 525/50nm emission filter, 50ms exposure time. Z-stacks with 1µm spacing. Multi-view reconstruction in Fiji. Depth projection using temporal color coding with ‘Spectrum’ look-up table in Fiji. Scale bar = 50µm. Sample by Jeehae Park, Harvard Medical School.

Fixed Drosophila embryo mounted in 1.5% agarose. Sytox Green nuclear marker. Band-pass 525/50nm emission filter, 50ms exposure time. Epi-fluorescence illumination: 470nm LED, 488nm long-pass dichroic mirror. Light sheet illumination: 491nm laser. Scale bar = 50 µm. Sample by Jeehae Park, Harvard Medical School.

Live Imaging

Live spheroid from patient-derived ovarian carcinoma cells transfected with lentivirus containing EGFP construct. Mounted in 1.2% agarose. Z-stacks with 1µm spacing recorded with brightfield and light sheet illumination. Brightfield illumination: white LED, 22ms exposure time. Flat field correction and Gaussian-based stack focuser in Fiji. Light sheet illumination: 491nm laser, band-pass 525/50nm emission filter, 50ms exposure time. Single plane at 40µm into the sample. Depth projection generated using temporal color code with ‘Spectrum’ look-up table in Fiji. Scale bar = 50µm. Sample by Marcin Iwanicki, Stevens Institute of Technology.

Specifications

• 463 x 327µm field of view

• 325nm lateral resolution at 520nm

• 227nm lateral sampling

• 45fps at full field of view

• Z-stack recording at full camera speed

• Motorized xyz movement and rotation

• Multi-view and/or time-lapse recording

• 3.1 - 7.3µm static Gaussian light sheet across 200µm at 491nm

• Epi-fluorescence & brightfield illumination

specifications vary with choice of detection lens, camera, illumination optics and excitation wavelength

The light sheet

Beam path

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.

Field of View vs. Full Width at Half Max

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

Off-the-shelf components

• C = custom part / part that requires modification

• Some categories also require parts from the Basics and Lenses category

• Tools not included

• US prices from 2016/201

Custom parts

• Sample chamber and adapters need to be produced with selective laser sintering (SLS), e.g. at Stratasys

• Incubation chamber have to be laser-cut

hard- and Software

LabVIEW code

• This zip file contains all the custom VIs. Compatibility depends on the final components used in each setup.

 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

• The 3D model can be viewed using the free eDrawings Viewer.

 Alignment

 Troubleshooting

1. 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

2. 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)

3. 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

1. Improve light sheet alignment stability by switching from Thorlabs KC45D to Thorlabs C45P
   Not gimbal; requires normal/thicker mirrors (compared to KC45D); might run into space issues

2. Integrate a bio-compatible sample holder, made from e.g. plastic or surgical-grade stainless steel
   Requires new sample holder design, different adapter

3. Switch camera to an sCMOS USB camera 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

4. 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

5. 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

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