Light-sheet microscopy (Huisken et al.) is a versatile 3D imaging tool with advantages such as fast imaging, minimal phototoxicity and photobleaching, and the ability to view millimeters into tissue and from different angles. As such, it is ideal for live imaging of large samples in 3D and is widely used e.g. to describe early embryonic development in zebrafish (Keller et al., Mongera et al.), to visualize gastrulation events in Drosophila embryos (Amat et al.), and to characterize developing organoids (de Medeiros et al.). ?
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Why I use light-sheet microscopy:?As a postdoc in the UiO:LifeScience funded project "Integrated technologies for tracking organoid morphogenesis" (ITOM), I built my own light-sheet microscope to characterize how mouse embryo models (cardiovascular gastruloids made from MP1 reporter cell line) grow and develop. Especially, we are interested in how the first organs form, and how these model embryos change shape from spherical to elongated (prolate) spheroids.?To fully utilize my volumetric imaging tool, I also study the model organisms fruit fly and zebrafish larvae, in collaboration with various groups at UiO.?
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Workflow:?The workflow described here is developed for 3D imaging of fixed (dead) mouse gastruloids, as well as fruit fly and zebrafish larvae. By controlling the temperature it also works for live imaging of both fruit fly and zebrafish larvae.
- Step 1: Mount the sample inside of a capillary tube. During imaging (Step 2) the capillary tube holding the sample is placed in front of the viewing objective of the microscope, see Figure 1.
- Step?2: Collect stacks of images into the sample in different color channels (such as green and red) and from different angles, typically every 45 or 90 degrees.
- Step 3: Simultaneously fuse and deconvolve the image stacks to obtain a single sharp 3D image for each color channel.
- Step 4: The final step is to render, segment and analyze the 3D data to obtain quantitative measures such as the shape of cells, their medial distance, and their orientation.??
To ease the storytelling, I explain Step 2 - Imaging first, and explain the sample mounting subsequently.
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Step 2 - Imaging: Optics and laser illumination: Our light-sheet microscope (Figure 1) is a stripped-down version of the Open-SPIM platform (Pitrone et al.). The illumination laser?(Cobolt 6 series, Hübner Photonics) has laser heads with four different wavelength (λ = 375, 488 561, 647 nm), corresponding to UV, green, red and far red light. By exciting fluorescently tagged molecules within gastruloids we visualize the?gastruloid nuclei (λ =488 nm) and membrane (λ =375 nm), as well as the mesoderm (T/Bra GFP reporter; excitation: 488 nm, emission: 509 nm) and endoderm germ layers (Sox17 RFP reporter; excitation: 561 nm, emission: 588 nm). Finally, we visualize blood vessels in the zebrafish brain and fat cells in Drosophila by illuminating the sample with green laser light (488 nm), and invading cells in zebrafish and autophagosomes in Drosophila by illuminating the sample with red laser light (561 nm).? ??
A fiber optic cable safely guides the laser light to a beam head with a built-in collimator. To minimize photobleaching of the specimen, the beam passes through a neutral density filter (ND507A, ThorLabs), and to form the light sheet, it passes through a cylindrical lens (ACY254-050-A, f = 50 mm, ThorLabs), two mirrors (POLARIS-K1, ThorLabs) and an adjustable slit (VA100/M, ThorLabs). Two spherical lenses (AC127-050-A-ML, f = 50 mm, ThorLabs; AC127-025-A-ML, f = 25 mm, ThorLabs; spaced 75mm apart) serve to collimate the light, and a water dipping lens (UMPLFLN 10XW, 10X/0.5, Olympus) focuses the light on to the specimen, giving an in-plane spatial resolution of 1.5 μm in x and 2.1 μm in y, and an out-of-plane (z) resolution of 6.7 μm (as measured by the full width half maximum of the point spread function).
Image acquisition: We mount the specimen inside of?a pre-rinsed fluorinated ethylene propylene (FEP) tube (BOLA, ID=0.8 mm, OD=1.6 mm) and place the tube holding the sample in a water filled acrylic viewing chamber (22 mm ×22 mm×37 mm). To minimize optical distortions (Weber et al.), especially spherical aberrations, the refractive index of the FEP-tube (n=1.34) closely matches the refractive index of water (n=1.33) inside the viewing chamber.?A movable stage (USB-4D-Stage, Picard Industries; resolution: 1.5μm) holds the FEP tube, and interfaces with a lab computer to allow the operator to rotate and to translate the sample along the x, y and z axes. Finally, the imaging system consists of a water dipping objective (UMPLFLN 20XW, 20X/0.5, Olympus), an emission filter (OD6 ULTRA Quad-Bandpass, Alluxa) which filters out the excitation wavelengths from the laser and a light sensitive camera (Andor Zyla 5.5, Andor). A lab computer controls the stage and camera via Micro-Manager (Edelstein et al.), while Cobolt Monitor interfaces with the same?computer to control the laser.
Step 1 - Sample mounting: We mount our sample inside the FEP-tube (ID=0.8mm, OD=1.6mm, BOLA) by aspirating it.?The protocol depends on the size of the sample. To aspirate large samples, such as fruit fly and zebrafish larvae, we use a syringe with a luer connector (B BRAUN Omnifix-F, 1ml) in combination with a blunt needle (21 Gauge, i.e. inner diameter=0.82mm) which makes a snug fit with the tube (Figure 2, top row). We then seal the tube either by pushing it into a soft, synthetic polymer like play doh, by sticking it into an agarose slab (1% concentration), or by inserting a stainless steel pin (ID=0.78mm) into the tube.
For smaller samples such as gastruloids, the mounting is much more delicate and requires additional control. Especially, it is critical that there is no dead volume in the flow path, and to avoid this, we use a male-female luer lock fitting (Flanged Fittings Kit, IDEX) and use a flange kit (Easy-Flange, VICI) to make flanged tubing (Figure 2, bottom row). We seal the tube by inserting a stainless steel pin (ID=0.78mm) into the tube. Finally, to ensure that the sample does not stick to the wall of the tube, it is useful to pre-rinse it with a surfactant solution (Anti-Adherence Rinsing Solution, Stemcell Technologies).?? ?
Once the sample, big or small, is mounted, we cut the tube using a razor blade and place it inside of a tube holder, which sits in front of the imaging objective.?
Live imaging: Motion capture of cells in 3D?
To describe how cells multiply, specialize and move, we perform live imaging and track them in 3D. We create physiological conditions by controlling the temperature, pH and oxygen level.??
Temperature control
To control the temperature, we circulate pre-heated water at physiological temperature through channels in the viewing/incubation chamber, see Figure 2.
References
Amat, F., Lemon, W., Mossing, D. P., McDole, K., Wan, Y., Branson, K., ... & Keller, P. J. (2014). Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data. Nature methods, 11(9), 951-958.
Edelstein, A., Amodaj, N., Hoover, K., Vale, R., & Stuurman, N. (2010). Computer control of microscopes using ?Manager. Current protocols in molecular biology, 92(1), 14-20.
Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., & Stelzer, E. H. (2004). Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science, 305(5686), 1007-1009.
Keller, P. J., Schmidt, A. D., Wittbrodt, J., & Stelzer, E. H. (2008). Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science, 322(5904), 1065-1069.
de Medeiros, G., Ortiz, R., Strnad, P., Boni, A., Moos, F., Repina, N., ... & Liberali, P. (2022). Multiscale light-sheet organoid imaging framework. Nature Communications, 13(1), 4864.
Mongera, A., Rowghanian, P., Gustafson, H. J., Shelton, E., Kealhofer, D. A., Carn, E. K., ... & Campàs, O. (2018). A fluid-to-solid jamming transition underlies vertebrate body axis elongation. Nature, 561(7723), 401-405.
Pitrone, P. G., Schindelin, J., Stuyvenberg, L., Preibisch, S., Weber, M., Eliceiri, K. W., ... & Tomancak, P. (2013). OpenSPIM: an open-access light-sheet microscopy platform. Nature methods, 10(7), 598-599.
Weber, M., Mickoleit, M., & Huisken, J. (2014). Multilayer mounting for long-term light sheet microscopy of zebrafish. JoVE (Journal of Visualized Experiments), (84), e51119.
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