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Protocol
Imaging Cellular Spheroids with a Single (Selective) Plane Illumination Microscope Jim Swoger, Francesco Pampaloni, and Ernst H.K. Stelzer
In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems. However, investigation of physiological context requires specimens that display the complex three-dimensional (3D) relationship of cells that occurs in tissue sections and in naturally developing organisms. The imaging of highly scattering multicellular specimens presents a number of challenges, including limited optical penetration depth, phototoxicity, and fluorophore bleaching. Light-sheetbased fluorescence microscopy (LSFM) overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The specimen is illuminated from the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. The principles of LSFM are implemented in the single (or selective) plane illumination microscope (SPIM). Cellular spheroids are spherical aggregations of hundreds to thousands of cells and they provide a useful model system for studies of 3D cell biology. Here we describe a protocol for imaging cellular spheroids by SPIM.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agarose (low-gelling; 1% [w/v] in PBS) Prepare aliquots of 1% agarose in 1.5-mL microfuge tubes.
Cellular spheroids stained or tagged with fluorescent probes Live spheroids can be stained with a variety of fluorescent dyes. Members of the CellTracker family (Life Technologies) are suitable dyes for identifying and tracking a population of cells inside the spheroid. Nuclear morphology can be investigated with 4′ ,6-diamino-2-phenylindole (DAPI) (excitation 345 nm; emission 458 nm) or DRAQ5 (BioStatus Limited; excitation 647 nm; emission 665 nm). Of course, cells tagged with fluorescent proteins are, in many cases, more convenient than using dyes.
Dulbecco’s modified Eagle’s medium (DMEM; 1× without phenol red) Phosphate-buffered saline (PBS; pH 7.4) Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080176
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Imaging Cellular Spheroids with a SPIM
Equipment
Beaker (glass) Dissection stereomicroscope with transmitted light illumination and incident light illumination with fiber-optic cold-light source (e.g., Stemi 2000, Carl Zeiss MicroImaging, Inc.) Electrical wire with diameter 1–1.8 mm (e.g., 10/0.1-mm tinned copper wire 01-1535 or 7/0.2-mm wire 01-2246; Rapid Electronics) Embryo dishes or watch glasses These are molded clear glass dishes for viewing free-floating specimens (e.g., Agar Scientific L4161). Alternatively, glass well/depression slides can be used.
Glass cutter (diamond-tipped) Heating blocks (set at 37˚C and 65˚C) Micropipettes (glass; 100-μL or 200-μL; e.g., BLAUBRAND intraMARK 708744, inner diameter [ID] 1.0 mm, outer diameter [OD] 1.7 mm, or intraMARK 708757, ID 1.45 mm, OD 2.25 mm, BRAND GMBH) Molding dough (e.g., Nakiplast, Pelikan) Nail polish Pipette tips (200-μL yellow tips with cut ends and intact 10-μL tips) Scalpel or razor blade SPIM To image 100-μm spheroids at subcellular resolution, use a SPIM with (1) a 40×/NA 0.75–0.80 water-dipping objective lens (e.g., Zeiss Achroplan 40×/0.8 W) and (2) a CCD camera suitable for fluorescence microscopy (e.g., Hamamatsu ORCA-AG, 1344 × 1024 pixels, cell size 6.45 × 6.45 µm). With this combination of objective lens and camera, the field of view is 217 × 165 µm. A CCD camera with a larger chip (e.g., the PCO.2000 camera [PCO AG imaging] chip size 2112 × 2072 pixels, cell size 7.40 × 7.40 µm) could be used with a 63× objective lens, such as a Zeiss Achroplan 63×/1.0 W. Objective lenses with lower magnification and NA (e.g., Zeiss Achroplan 10×/0.3 W or Achroplan 20×/0.5 W) are recommended when subcellular resolution is not the main issue.
SPIM imaging chamber SPIM sample holder METHOD Mounting Spheroids for Imaging For an image of a cellular spheroid embedded in agarose, see Figure 1.
1. Transfer several 1.5-mL aliquots of 1% agarose into a heating block set at 65˚C.
2. Once the agarose has melted, transfer the tubes to a heating block or water bath set at 37˚C.
3. Prepare sample holders by cutting glass micropipettes to the desired length with a diamond-tip cutter (Fig. 2A). 4. Insert a section of electrical wire into the glass capillaries to serve as a plunger. The wire should be slightly longer than the capillary (Fig. 2B,C).
FIGURE 1. Cellular spheroids are typical 3D cell biology specimens. This spheroid derives from a BxCP-3 pancreas adenocarcinoma cell line and has been obtained with the hanging-drop method. The spheroid is embedded in agarose and is composed of about 300 cells. Transmitted light imaging obtained on the SPIM setup, objective lens CZ Plan Apochromat 40×/NA 0.8.
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FIGURE 2. Preparation of glass capillary holder. (A) A 100μL glass disposable micropipette (BLAUBRAND intraMARK, ID 1.0 mm, OD 1.7 mm) is cut to the desired length with a diamond-tip cutter. (B) A slightly longer electrical wire is inserted into the capillary as a plunger. (C ) Close-up of the complete capillary-wire system. The length of the cut glass capillary is 12.5 cm (cut to 9 cm).
5. Using yellow pipette tips with cut ends, harvest one to five spheroids from the culture, and deposit them in a watch glass. Pipette tips with cut ends minimize shear flow and mechanical damage to the spheroids.
6. Using a dissection stereomicroscope, carefully remove as much medium as possible from the watch glass using a 10-μL pipette tip. Carefully replace the medium with a generous amount of 1% agarose solution. 7. Pick up the spheroids one by one by sucking them into a glass capillary (Fig. 3). Practice is required to perform this step efficiently. Work quickly, picking up all of the spheroids before the agarose sets. For multiple-view imaging, the spheroids should be placed in the center of the capillary. In practice, however, the spheroid’s position within the agarose is hard to control. Thus, preparing several samples is recommended. See Troubleshooting.
Wire or plunger
Capillary Well
Specimen
Agarose
FIGURE 3. Embedding the specimens in agarose. A cellular spheroid is suspended in a droplet of liquid low-gelling agarose and deposited in a shallow well. A few microliters of agarose containing the specimen are drawn into a thin glass capillary by using, for example, an electrical wire of suitable diameter as a plunger. The agarose sets within 5 min with the specimen embedded.
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Imaging Cellular Spheroids with a SPIM
FIGURE 4. Preparation of the specimen for imaging. (A,B) On polymerization, the solid agarose tip with the specimen is pushed out of the capillary. The specimen is ready for imaging. (C ) Photograph of a specimen. The bottom arrow shows an agarose-embedded cellular spheroid outside of the capillary. A second embedded spheroid is still inside the capillary (top arrow). (D) Photograph of the complete setup comprising glass capillary, wire, and agarose. The capillary length is 9 cm.
8. Once the spheroids are picked, wait 10–15 min while the agarose inside the capillary fully sets. Then, push the agarose cylinder with the spheroid out of the capillary by sliding the plunger (Fig. 4). 9. Remove excess agarose below the spheroid with a scalpel or a razor blade. Only a short segment (1–2 mm) of agarose should be exposed outside the capillary (Fig. 4). A short agarose segment is more mechanically stable than a long one. Agarose cylinders longer than 1–2 mm may produce blurred or offset images. 10. For short-term storage of the specimens, keep the capillaries in a laboratory beaker filled with culture medium or PBS. Use a small slab of soft molding dough attached to the beaker’s rim to keep the specimens in place (Fig. 5). To avoid drifting of the agarose cylinder during imaging, glue the wire at the top of the capillary with nail polish (Fig. 5).
FIGURE 5. Storage of the specimen. The specimen is temporarily stored within a glass laboratory beaker filled with PBS or medium. A slab of soft molding dough is attached to the beaker’s rim and is used to keep the glass capillary in place. After pushing out the specimen, the wire is fixed in its final position with nail polish. This avoids a further drift of the specimen during imaging with a SPIM.
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A
B Wire
Metal holder
Plastic holder
Holder type II
Holder type I
O-ring
Glass capillary Agarose
A
FIGURE 6. Capillary holders for SPIM. Two types of glass capillary holders developed at European Molecular Biology Laboratory. (A) Aluminum holder. A metal spring keeps the glass capillary firmly in place. (B) Plastic holder. In this simplified holder type, two rubber O-rings keep a tight hold on the capillary.
B
Holder Glass capillary Objective lens Metal chamber
Specimen
C
D
1 mm
FIGURE 7. SPIM imaging setup. (A,B) Schematic of the SPIM imaging setup. The specimen mounted in the glass capillary is immersed in a liquid medium (e.g., DMEM without phenol red or PBS). The medium is contained in a chamber with glass windows on the side and on the front. The glass windows are glued to the chamber by using nail polish or a silicon-based glue and can be easily replaced. The detection objective lens is directly inserted into the chamber. A spring-loaded radial shaft seal ensures that the junction of the objective lens and the chamber is watertight. (C ) Photograph of the real system. On the left, the illumination lens is visible. The light sheet illuminates the sample from the side as indicated by the blue arrow. (D) A close-up of the specimen, a cellular spheroid with a diameter of 100 µm.
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Imaging Cellular Spheroids with a SPIM
TABLE 1. Typical SPIM recording parameters Cellular spheroid (diameter 100 µm) Zeiss Plan Apochromat 63 ↔ /NA 1.0 CyTRAK dye (nuclei + cytoplasm) 488 nm 2.6 µm 0.540 mW (30 nW/μm2)a 100 msec Hamamatsu ORCA-AG 6.45 µm 1340 × 1024 pixels 130 µm 102 × 102 × 819 nm 159 18 sec
Specimen Objective lens Fluorescence labeling Excitation wavelength Light-sheet thickness Measured laser power on the specimen Exposure time CCD camera type CCD chip cell size CCD chip total size z-stack (total travel) Voxel size Number of x − y planes Total recording time
Specimen, cellular spheroid with a diameter of 150 µm. The laser power was measured by placing the detector at the position of the specimen.
a
11. Insert the capillary into the SPIM specimen holder (examples of holders are shown in Fig. 6), and place it into the imaging chamber by firmly connecting it with the x−y−z-Φ motorized stage (Fig. 7). Imaging Cellular Spheroids with an SPIM
12. Image small spheroids (diameter of 100 µm) at subcellular resolution using a 40× waterdipping objective lens (NA 0.75–0.80) and a CCD camera suitable for fluorescence microscopy. 13. Adjust the illuminating laser light sheet to obtain the best compromise between light-sheet thickness and homogeneity of axial resolution. For a 40× objective, this corresponds to a lightsheet thickness of 3.5 µm full width at half-maximum. 14. Acquire stacks of lateral (x − y) images using a step size of 1.8 µm along the z-axis to obtain a reasonable axial (z) sampling. Typical recording parameters for a spheroid with a diameter of 100 µm are listed in Table 1. Given the nearly perfect spherical shape of the spheroids, multiple-view imaging can be performed quite easily (Figs. 8 and 9).
0°
30°
60°
90°
120°
150°
180°
210°
240°
270°
300°
330°
150 μm
FIGURE 8. Multiple-view angular imaging series of a spheroid derived from BxCP-3 pancreas adenocarcinoma cells. The figure shows 12 maximum-intensity projections of SPIM stacks recorded along 12 different directions. Each stack is composed of 278 planes with 0.5-μm spacing. The cell nuclei are stained with DRAQ5 (excitation 633 nm; emission 665 nm). Objective lens water-dipping Zeiss Achroplan 40×/NA 0.8. The CCD camera used is a Hamamatsu ORCA-AG. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080176
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FIGURE 9. Images of a spheroid derived from BxCP-3 pancreas adenocarcinoma cells recorded with a SPIM. Selected single frames from a single-view SPIM stack. The frames are spaced 10 µm along the z-axis.
TROUBLESHOOTING Problem (Step 7): Spheroids stick to the watch glass bottom. Solution: Clean the glass with ethanol before adding the spheroids.
DISCUSSION
Cellular spheroids are spherical aggregations of hundreds to thousands of cells with diameters of 100– 800 µm (Friedrich et al. 2009), and they were probably the first 3D cell system used in clinical pharmacology (Mueller-Klieser 1997). Many common cell lines form spheroids, including MCF10a, Caco-2, and HepG2 (Kelm et al. 2003). Spheroids from the human teratocarcinoma cell line Ntera-2 are a useful model system for biomedical studies and toxicity assays of the nervous system (Podrygajlo et al. 2009). Cellular spheroids obtained by the aggregation of primary cells, tumor, or nontumor cell lines are often used in tissue engineering, regenerative medicine, oncology, and the development of more reliable drug toxicity assays (Pampaloni et al. 2009). Cell aggregation is induced by physical forces (buoyancy or stirring) and mostly occurs without having to add exogenous scaffolds or extracellular matrix proteins. Buoyancy is exploited in the hanging-drop method, in which droplets of culture medium containing trypsinated cells are suspended on the surface of a Petri dish lid. Compact spheroids are harvested from the droplets within 3–20 d of growth (Kelm et al. 2003; Timmins et al. 2004). Spheroids can also be obtained by seeding cells on nonadhesive surfaces such as 3D alginate porous scaffolds (Glicklis et al. 2000) or from cell cultures in rotating well vessels (Bilodeau and Mantovani 2006). Standardized protocols for the reproducible and reliable culture, handling, and screening of cellular spheroids for pharmacology and toxicology have been published (Friedrich et al. 2009). For further information about the principles of LSFM and SPIM, see Light-Sheet-Based Fluorescence Microscopy for Three-Dimensional Imaging of Biological Samples (Swoger et al. 2014). 112
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Imaging Cellular Spheroids with a SPIM
RECIPE PBS(P)
Reagent NaH2PO4 Na2HPO4 NaCl
Quantity (for 1 L)
Final concentration (10×)
2.56 g 11.94 g 102.2 g
18.6 mM 84.1 mM 1.75 M
Adjust the pH to 7.4 using NaOH or HCl as necessary. This recipe produces a 10× stock solution; prepare 1× PBS(P) by diluting with H2O. Both 1× and 10× PBS(P) can be kept indefinitely at room temperature.
REFERENCES Bilodeau K, Mantovani D. 2006. Bioreactors for tissue engineering: Focus on mechanical constraints. A comparative review. Tissue Eng 12: 2367– 2383. Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. 2009. Spheroid-based drug screen: Considerations and practical approach. Nat Protoc 4: 309–324. Glicklis R, Shapiro L, Agbaria R, Merchuk JC, Cohen S. 2000. Hepatocyte behavior within three-dimensional porous alginate scaffolds. Biotechnol Bioeng 67: 344–353. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK. 2003. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83: 173–180. Mueller-Klieser W. 1997. Three-dimensional cell cultures: From molecular mechanisms to clinical applications. Am J Physiol 273: C1109– C1123.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080176
Pampaloni F, Stelzer EH, Masotti A. 2009. Three-dimensional tissue models for drug discovery and toxicology. Recent Pat Biotechnol 3: 103–117. Podrygajlo G, Tegenge MA, Gierse A, Paquet-Durand F, Tan S, Bicker G, Stern M. 2009. Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture. Cell Tissue Res 336: 439–452. Swoger J, Pampaloni F, Stelzer EHK. 2014. Light-sheet-based fluorescence microscopy for three-dimensional imaging of biological samples. Cold Spring Harb Protoc doi: 10.1101/pdb.top080168. Timmins NE, Dietmair S, Nielsen LK. 2004. Hanging-drop multicellular spheroids as a model of tumour angiogenesis. Angiogenesis 7: 97–103.
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Imaging Cellular Spheroids with a Single (Selective) Plane Illumination Microscope Jim Swoger, Francesco Pampaloni and Ernst H.K. Stelzer Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080176 Email Alerting Service Subject Categories
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Protocol
Imaging Cellular Spheroids with a Single (Selective) Plane Illumination Microscope Jim Swoger, Francesco Pampaloni, and Ernst H.K. Stelzer
In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems. However, investigation of physiological context requires specimens that display the complex three-dimensional (3D) relationship of cells that occurs in tissue sections and in naturally developing organisms. The imaging of highly scattering multicellular specimens presents a number of challenges, including limited optical penetration depth, phototoxicity, and fluorophore bleaching. Light-sheetbased fluorescence microscopy (LSFM) overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The specimen is illuminated from the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. The principles of LSFM are implemented in the single (or selective) plane illumination microscope (SPIM). Cellular spheroids are spherical aggregations of hundreds to thousands of cells and they provide a useful model system for studies of 3D cell biology. Here we describe a protocol for imaging cellular spheroids by SPIM.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agarose (low-gelling; 1% [w/v] in PBS) Prepare aliquots of 1% agarose in 1.5-mL microfuge tubes.
Cellular spheroids stained or tagged with fluorescent probes Live spheroids can be stained with a variety of fluorescent dyes. Members of the CellTracker family (Life Technologies) are suitable dyes for identifying and tracking a population of cells inside the spheroid. Nuclear morphology can be investigated with 4′ ,6-diamino-2-phenylindole (DAPI) (excitation 345 nm; emission 458 nm) or DRAQ5 (BioStatus Limited; excitation 647 nm; emission 665 nm). Of course, cells tagged with fluorescent proteins are, in many cases, more convenient than using dyes.
Dulbecco’s modified Eagle’s medium (DMEM; 1× without phenol red) Phosphate-buffered saline (PBS; pH 7.4) Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080176
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Imaging Cellular Spheroids with a SPIM
Equipment
Beaker (glass) Dissection stereomicroscope with transmitted light illumination and incident light illumination with fiber-optic cold-light source (e.g., Stemi 2000, Carl Zeiss MicroImaging, Inc.) Electrical wire with diameter 1–1.8 mm (e.g., 10/0.1-mm tinned copper wire 01-1535 or 7/0.2-mm wire 01-2246; Rapid Electronics) Embryo dishes or watch glasses These are molded clear glass dishes for viewing free-floating specimens (e.g., Agar Scientific L4161). Alternatively, glass well/depression slides can be used.
Glass cutter (diamond-tipped) Heating blocks (set at 37˚C and 65˚C) Micropipettes (glass; 100-μL or 200-μL; e.g., BLAUBRAND intraMARK 708744, inner diameter [ID] 1.0 mm, outer diameter [OD] 1.7 mm, or intraMARK 708757, ID 1.45 mm, OD 2.25 mm, BRAND GMBH) Molding dough (e.g., Nakiplast, Pelikan) Nail polish Pipette tips (200-μL yellow tips with cut ends and intact 10-μL tips) Scalpel or razor blade SPIM To image 100-μm spheroids at subcellular resolution, use a SPIM with (1) a 40×/NA 0.75–0.80 water-dipping objective lens (e.g., Zeiss Achroplan 40×/0.8 W) and (2) a CCD camera suitable for fluorescence microscopy (e.g., Hamamatsu ORCA-AG, 1344 × 1024 pixels, cell size 6.45 × 6.45 µm). With this combination of objective lens and camera, the field of view is 217 × 165 µm. A CCD camera with a larger chip (e.g., the PCO.2000 camera [PCO AG imaging] chip size 2112 × 2072 pixels, cell size 7.40 × 7.40 µm) could be used with a 63× objective lens, such as a Zeiss Achroplan 63×/1.0 W. Objective lenses with lower magnification and NA (e.g., Zeiss Achroplan 10×/0.3 W or Achroplan 20×/0.5 W) are recommended when subcellular resolution is not the main issue.
SPIM imaging chamber SPIM sample holder METHOD Mounting Spheroids for Imaging For an image of a cellular spheroid embedded in agarose, see Figure 1.
1. Transfer several 1.5-mL aliquots of 1% agarose into a heating block set at 65˚C.
2. Once the agarose has melted, transfer the tubes to a heating block or water bath set at 37˚C.
3. Prepare sample holders by cutting glass micropipettes to the desired length with a diamond-tip cutter (Fig. 2A). 4. Insert a section of electrical wire into the glass capillaries to serve as a plunger. The wire should be slightly longer than the capillary (Fig. 2B,C).
FIGURE 1. Cellular spheroids are typical 3D cell biology specimens. This spheroid derives from a BxCP-3 pancreas adenocarcinoma cell line and has been obtained with the hanging-drop method. The spheroid is embedded in agarose and is composed of about 300 cells. Transmitted light imaging obtained on the SPIM setup, objective lens CZ Plan Apochromat 40×/NA 0.8.
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FIGURE 2. Preparation of glass capillary holder. (A) A 100μL glass disposable micropipette (BLAUBRAND intraMARK, ID 1.0 mm, OD 1.7 mm) is cut to the desired length with a diamond-tip cutter. (B) A slightly longer electrical wire is inserted into the capillary as a plunger. (C ) Close-up of the complete capillary-wire system. The length of the cut glass capillary is 12.5 cm (cut to 9 cm).
5. Using yellow pipette tips with cut ends, harvest one to five spheroids from the culture, and deposit them in a watch glass. Pipette tips with cut ends minimize shear flow and mechanical damage to the spheroids.
6. Using a dissection stereomicroscope, carefully remove as much medium as possible from the watch glass using a 10-μL pipette tip. Carefully replace the medium with a generous amount of 1% agarose solution. 7. Pick up the spheroids one by one by sucking them into a glass capillary (Fig. 3). Practice is required to perform this step efficiently. Work quickly, picking up all of the spheroids before the agarose sets. For multiple-view imaging, the spheroids should be placed in the center of the capillary. In practice, however, the spheroid’s position within the agarose is hard to control. Thus, preparing several samples is recommended. See Troubleshooting.
Wire or plunger
Capillary Well
Specimen
Agarose
FIGURE 3. Embedding the specimens in agarose. A cellular spheroid is suspended in a droplet of liquid low-gelling agarose and deposited in a shallow well. A few microliters of agarose containing the specimen are drawn into a thin glass capillary by using, for example, an electrical wire of suitable diameter as a plunger. The agarose sets within 5 min with the specimen embedded.
108
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Imaging Cellular Spheroids with a SPIM
FIGURE 4. Preparation of the specimen for imaging. (A,B) On polymerization, the solid agarose tip with the specimen is pushed out of the capillary. The specimen is ready for imaging. (C ) Photograph of a specimen. The bottom arrow shows an agarose-embedded cellular spheroid outside of the capillary. A second embedded spheroid is still inside the capillary (top arrow). (D) Photograph of the complete setup comprising glass capillary, wire, and agarose. The capillary length is 9 cm.
8. Once the spheroids are picked, wait 10–15 min while the agarose inside the capillary fully sets. Then, push the agarose cylinder with the spheroid out of the capillary by sliding the plunger (Fig. 4). 9. Remove excess agarose below the spheroid with a scalpel or a razor blade. Only a short segment (1–2 mm) of agarose should be exposed outside the capillary (Fig. 4). A short agarose segment is more mechanically stable than a long one. Agarose cylinders longer than 1–2 mm may produce blurred or offset images. 10. For short-term storage of the specimens, keep the capillaries in a laboratory beaker filled with culture medium or PBS. Use a small slab of soft molding dough attached to the beaker’s rim to keep the specimens in place (Fig. 5). To avoid drifting of the agarose cylinder during imaging, glue the wire at the top of the capillary with nail polish (Fig. 5).
FIGURE 5. Storage of the specimen. The specimen is temporarily stored within a glass laboratory beaker filled with PBS or medium. A slab of soft molding dough is attached to the beaker’s rim and is used to keep the glass capillary in place. After pushing out the specimen, the wire is fixed in its final position with nail polish. This avoids a further drift of the specimen during imaging with a SPIM.
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A
B Wire
Metal holder
Plastic holder
Holder type II
Holder type I
O-ring
Glass capillary Agarose
A
FIGURE 6. Capillary holders for SPIM. Two types of glass capillary holders developed at European Molecular Biology Laboratory. (A) Aluminum holder. A metal spring keeps the glass capillary firmly in place. (B) Plastic holder. In this simplified holder type, two rubber O-rings keep a tight hold on the capillary.
B
Holder Glass capillary Objective lens Metal chamber
Specimen
C
D
1 mm
FIGURE 7. SPIM imaging setup. (A,B) Schematic of the SPIM imaging setup. The specimen mounted in the glass capillary is immersed in a liquid medium (e.g., DMEM without phenol red or PBS). The medium is contained in a chamber with glass windows on the side and on the front. The glass windows are glued to the chamber by using nail polish or a silicon-based glue and can be easily replaced. The detection objective lens is directly inserted into the chamber. A spring-loaded radial shaft seal ensures that the junction of the objective lens and the chamber is watertight. (C ) Photograph of the real system. On the left, the illumination lens is visible. The light sheet illuminates the sample from the side as indicated by the blue arrow. (D) A close-up of the specimen, a cellular spheroid with a diameter of 100 µm.
110
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Imaging Cellular Spheroids with a SPIM
TABLE 1. Typical SPIM recording parameters Cellular spheroid (diameter 100 µm) Zeiss Plan Apochromat 63 ↔ /NA 1.0 CyTRAK dye (nuclei + cytoplasm) 488 nm 2.6 µm 0.540 mW (30 nW/μm2)a 100 msec Hamamatsu ORCA-AG 6.45 µm 1340 × 1024 pixels 130 µm 102 × 102 × 819 nm 159 18 sec
Specimen Objective lens Fluorescence labeling Excitation wavelength Light-sheet thickness Measured laser power on the specimen Exposure time CCD camera type CCD chip cell size CCD chip total size z-stack (total travel) Voxel size Number of x − y planes Total recording time
Specimen, cellular spheroid with a diameter of 150 µm. The laser power was measured by placing the detector at the position of the specimen.
a
11. Insert the capillary into the SPIM specimen holder (examples of holders are shown in Fig. 6), and place it into the imaging chamber by firmly connecting it with the x−y−z-Φ motorized stage (Fig. 7). Imaging Cellular Spheroids with an SPIM
12. Image small spheroids (diameter of 100 µm) at subcellular resolution using a 40× waterdipping objective lens (NA 0.75–0.80) and a CCD camera suitable for fluorescence microscopy. 13. Adjust the illuminating laser light sheet to obtain the best compromise between light-sheet thickness and homogeneity of axial resolution. For a 40× objective, this corresponds to a lightsheet thickness of 3.5 µm full width at half-maximum. 14. Acquire stacks of lateral (x − y) images using a step size of 1.8 µm along the z-axis to obtain a reasonable axial (z) sampling. Typical recording parameters for a spheroid with a diameter of 100 µm are listed in Table 1. Given the nearly perfect spherical shape of the spheroids, multiple-view imaging can be performed quite easily (Figs. 8 and 9).
0°
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150°
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150 μm
FIGURE 8. Multiple-view angular imaging series of a spheroid derived from BxCP-3 pancreas adenocarcinoma cells. The figure shows 12 maximum-intensity projections of SPIM stacks recorded along 12 different directions. Each stack is composed of 278 planes with 0.5-μm spacing. The cell nuclei are stained with DRAQ5 (excitation 633 nm; emission 665 nm). Objective lens water-dipping Zeiss Achroplan 40×/NA 0.8. The CCD camera used is a Hamamatsu ORCA-AG. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080176
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J. Swoger et al.
FIGURE 9. Images of a spheroid derived from BxCP-3 pancreas adenocarcinoma cells recorded with a SPIM. Selected single frames from a single-view SPIM stack. The frames are spaced 10 µm along the z-axis.
TROUBLESHOOTING Problem (Step 7): Spheroids stick to the watch glass bottom. Solution: Clean the glass with ethanol before adding the spheroids.
DISCUSSION
Cellular spheroids are spherical aggregations of hundreds to thousands of cells with diameters of 100– 800 µm (Friedrich et al. 2009), and they were probably the first 3D cell system used in clinical pharmacology (Mueller-Klieser 1997). Many common cell lines form spheroids, including MCF10a, Caco-2, and HepG2 (Kelm et al. 2003). Spheroids from the human teratocarcinoma cell line Ntera-2 are a useful model system for biomedical studies and toxicity assays of the nervous system (Podrygajlo et al. 2009). Cellular spheroids obtained by the aggregation of primary cells, tumor, or nontumor cell lines are often used in tissue engineering, regenerative medicine, oncology, and the development of more reliable drug toxicity assays (Pampaloni et al. 2009). Cell aggregation is induced by physical forces (buoyancy or stirring) and mostly occurs without having to add exogenous scaffolds or extracellular matrix proteins. Buoyancy is exploited in the hanging-drop method, in which droplets of culture medium containing trypsinated cells are suspended on the surface of a Petri dish lid. Compact spheroids are harvested from the droplets within 3–20 d of growth (Kelm et al. 2003; Timmins et al. 2004). Spheroids can also be obtained by seeding cells on nonadhesive surfaces such as 3D alginate porous scaffolds (Glicklis et al. 2000) or from cell cultures in rotating well vessels (Bilodeau and Mantovani 2006). Standardized protocols for the reproducible and reliable culture, handling, and screening of cellular spheroids for pharmacology and toxicology have been published (Friedrich et al. 2009). For further information about the principles of LSFM and SPIM, see Light-Sheet-Based Fluorescence Microscopy for Three-Dimensional Imaging of Biological Samples (Swoger et al. 2014). 112
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Imaging Cellular Spheroids with a SPIM
RECIPE PBS(P)
Reagent NaH2PO4 Na2HPO4 NaCl
Quantity (for 1 L)
Final concentration (10×)
2.56 g 11.94 g 102.2 g
18.6 mM 84.1 mM 1.75 M
Adjust the pH to 7.4 using NaOH or HCl as necessary. This recipe produces a 10× stock solution; prepare 1× PBS(P) by diluting with H2O. Both 1× and 10× PBS(P) can be kept indefinitely at room temperature.
REFERENCES Bilodeau K, Mantovani D. 2006. Bioreactors for tissue engineering: Focus on mechanical constraints. A comparative review. Tissue Eng 12: 2367– 2383. Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. 2009. Spheroid-based drug screen: Considerations and practical approach. Nat Protoc 4: 309–324. Glicklis R, Shapiro L, Agbaria R, Merchuk JC, Cohen S. 2000. Hepatocyte behavior within three-dimensional porous alginate scaffolds. Biotechnol Bioeng 67: 344–353. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK. 2003. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83: 173–180. Mueller-Klieser W. 1997. Three-dimensional cell cultures: From molecular mechanisms to clinical applications. Am J Physiol 273: C1109– C1123.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080176
Pampaloni F, Stelzer EH, Masotti A. 2009. Three-dimensional tissue models for drug discovery and toxicology. Recent Pat Biotechnol 3: 103–117. Podrygajlo G, Tegenge MA, Gierse A, Paquet-Durand F, Tan S, Bicker G, Stern M. 2009. Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture. Cell Tissue Res 336: 439–452. Swoger J, Pampaloni F, Stelzer EHK. 2014. Light-sheet-based fluorescence microscopy for three-dimensional imaging of biological samples. Cold Spring Harb Protoc doi: 10.1101/pdb.top080168. Timmins NE, Dietmair S, Nielsen LK. 2004. Hanging-drop multicellular spheroids as a model of tumour angiogenesis. Angiogenesis 7: 97–103.
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