We characterized the MORSOM imaging performance in terms of resolution, SNR, and overall image quality using a phantom consisting of 10-μm spheres that were randomly dispersed in agar gel and juvenile Zebrafish at 21 dpf. At 21 dpf, Zebrafish have a length of ~10 mm and diameters ranging from 1 to 2 mm (Supplementary Fig. S4 ). These dimensions are not accessible by optical or optoacoustic microscopy techniques, such as SPIM18 (link) or multiview optical resolution photoacoustic microscopy6 (link), 19 (link), 20 (link), and correspond to dimensions for which optoacoustic mesoscopy may be ideally suited. Zebrafish were embedded in low melting agar (Sigma-Aldrich, St Louis, MO, USA) and imaged ex vivo, consistent with the current regulations and processes allowed by the Government of Upper Bavaria for adult Zebrafish. The agar was mixed with ~10 μm diameter black microspheres (black polystyrene microspheres, Polybead, Polysciences Inc., Warrington, PA, USA), which we use as fiducial markers to confirm the exact alignment of the different RSOM projections onto the common MORSOM image reconstruction scheme. The Zebrafish/agar specimen was mounted on a syringe, as previously described for SPIM21 , and placed in a water bath for acoustic coupling.
Low melting agar
Manufactured by Merck Group
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Low melting agar is a type of agar with a low gelling temperature, typically in the range of 25-30°C. It is a polysaccharide extracted from certain red algae and is commonly used as a solidifying agent in microbiological and cell culture applications.
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6 protocols using low melting agar
1
Multimodal Optoacoustic Mesoscopy of Zebrafish
2
Soft Agar Assay for Tumor Formation
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To assess tumor formation in vitro, soft agar clonogenic assays were performed. Each well of a 6-well plate was coated with 2 ml of 0.5% (w/v) low-melting agar (Sigma-Aldrich; Merck KGaA) in DMEM with 10% FBS. Cells were mixed and 5×103 cells in 2 ml 0.3% low-melting agar with 10% FBS were added above the polymerized base solution. Plates were incubated (37°C; 5% CO2) for 14 days before colony number and diameter were quantified microscopically using a X71 (U-RFL-T) fluorescence microscope (Olympus Corporation; magnification, ×40).
3
Clonogenic and Soft Agar Assays
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A total of 2 × 103 cells were seeded on six-well plates and maintained in a medium containing FBS for 10–14 days until visible clones appeared. For staining of colonies, 500 μl of Giemsa solution (Keygen, Nanjing, China) was added into each well and incubated for 30 min and removed followed by three washes using PBS.
For performing colony formation in soft agar, each well of a six-well plate that contained 2 ml of 0.5% (w/v) low-melting agar (Sigma–Aldrich, St. Louis, MO, United States) in DMEM medium with 10% FBS was laid in each well. Suspended cells were mixed equally, and 5 × 103 cells in 2 ml of 0.3% low-melting agar in 10% FBS were added above the polymerized base solution. Plates were incubated (37°C, 5% CO2) for 14 days before colony number, and diameter was quantified microscopically.
For performing colony formation in soft agar, each well of a six-well plate that contained 2 ml of 0.5% (w/v) low-melting agar (Sigma–Aldrich, St. Louis, MO, United States) in DMEM medium with 10% FBS was laid in each well. Suspended cells were mixed equally, and 5 × 103 cells in 2 ml of 0.3% low-melting agar in 10% FBS were added above the polymerized base solution. Plates were incubated (37°C, 5% CO2) for 14 days before colony number, and diameter was quantified microscopically.
4
Brain Slice Preparation for Electrophysiology
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Hatchlings were deeply anesthetized by an intramuscular injection of a 3:1 mixture of Ketamine (50 mg/kg) and Rompun (20 mg/kg). Following decapitation, the brain was swiftly extracted from the skull and transferred into oxygenated ACSF (120 mM NaCl, 3 mM KCl, 3 mM MgCl2 · 6H2O, 23 mM NaHCO3, 1.2 mM Na2HPO4 · H2O, 2 mM CaCl2 · 2H2O, 11 mM D+-glucose) cooled to 4°C. The telencephalon and cerebellum were removed, the two mesencephalic hemispheres were separated midsagittally and embedded in low-melting agar (15 g/l, Sigma-Aldrich, St. Louis, MO, USA) in HEPES buffer (200 mM Saccharose, 3 mM KCl, 3 mM MgCl2 · 6H2O, 5 mM HEPES). Hemispheres were orientated at an angle differing from the classical transversal plane (Figure 1A ) and sliced into 500 μm or 2,000 μm slices using a vibratome (VF-200 Microtome, Precisionary Instruments Inc., Greenville, NC, USA), and collected in oxygenated ACSF. The slices were kept in an interface chamber bubbled with carbogen for recovery for 45–60 min and subsequently kept submerged in continuously oxygenated ACSF at room temperature.
5
3D Colony Forming Assay
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We performed assays with minor modifications from a published protocol (81 ). Briefly, we made a base layer of 0.7% agar (Sigma-Aldrich) dissolved in DMEM medium, adding 0.2% serum after the agar solution cooled to ∼37 °C before transferring 1 mL per well to 6-well plates. After the base layer solidified at room temperature, we prepared 0.35% low melting agar (Sigma) in DMEM medium, adding the same concentration of serum as in the base layer and 5 × 104 cells per mL when the solution cooled to ∼37 °C (n = 3 wells per cell type and serum condition). We immediately transferred 1 mL per well of the low melting point agar/cell solution to each well and cooled the plate briefly at 4 °C before placing it in a cell culture incubator. We added 0.5 mL fresh DMEM medium with 0.2% serum every 2 days. After one week, we obtained nine bright-field images per well on an inverted microscope (Olympus IX73 with 20× objective). Immediately after microscopy, we added 150 µg/mL luciferin (Promega) to each well; incubated in a cell culture incubator for 10 min; and then acquired a bioluminescence image of total viable cells on an IVIS Lumina (30 s image, large field of view) (Perkin Elmer). A person blinded to experimental conditions enumerated colonies and quantified imaging data (Living Image, Perkin Elmer).
6
Immunocytochemical Evaluation of Apoptosis Markers
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The assessment of BAX, BCL2, and active caspase-3 proteins expression was performed by immunocytochemistry method. The free and embedded islets in the scaffolds were xed in 4% paraformaldehyde for 15 min. The islets and the scaffolds were both embedded in low melting agar (Sigma, Germany) and para n (18). The cell blocks were sectioned at 5µm thickness.
Immunocytochemistry was performed using primary antibodies against BAX, BCL2, and active caspase-3 (abcam, USA). Incubation in primary antibody was done overnight. After incubation in secondary antibody (abcam, USA) for 1h, the positive cells were detected by 3,3′-Diaminobenzidine (DAB).
Counterstain was carried out with Hematoxylin staining. The samples were evaluated and the H-score was calculated as follows: H score=1 × (% light staining) + 2 × (% moderate staining) + 3 × (% strong staining) (19) .
Immunocytochemistry was performed using primary antibodies against BAX, BCL2, and active caspase-3 (abcam, USA). Incubation in primary antibody was done overnight. After incubation in secondary antibody (abcam, USA) for 1h, the positive cells were detected by 3,3′-Diaminobenzidine (DAB).
Counterstain was carried out with Hematoxylin staining. The samples were evaluated and the H-score was calculated as follows: H score=1 × (% light staining) + 2 × (% moderate staining) + 3 × (% strong staining) (19) .
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