Oct 28, 2024
Staining and resin embedding of whole Daphnia magna samples for micro-CT imaging enabling 3D visualization of cells, tissues, and organs
Peer-reviewed method
- Mee Ngu1,
- Daniel J. Vanselow1,
- Andrew L. Sugarman1,
- Rachelle A. Saint-Fort1,
- Carolyn R. Zaino1,
- Maksim A. Yakovlev1,
- Keith C. Cheng1,
- Khai C. Ang1
- 1Penn State College of Medicine
- PLOS ONE Lab Protocols
- Spotlight series
Protocol Citation: Mee Ngu, Daniel J. Vanselow, Andrew L. Sugarman, Rachelle A. Saint-Fort, Carolyn R. Zaino, Maksim A. Yakovlev, Keith C. Cheng, Khai C. Ang 2024. Staining and resin embedding of whole Daphnia magna samples for micro-CT imaging enabling 3D visualization of cells, tissues, and organs . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8dwolmk/v1
Manuscript citation:
Yakovlev MA, Vanselow DJ, Ngu MS, Zaino CR, Katz SR, Ding Y, et al. A wide-field micro-computed tomography detector: micron resolution at half-centimetre scale. J Synchrotron Radiat. 2022 Mar 1 ;29(2). https://journals.iucr.org/s/issues/2022/02/00/mo5248/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 12, 2024
Last Modified: January 30, 2025
Protocol Integer ID: 98882
Keywords: Daphnia, phosphotungstic acid, micro-CT, staining of whole daphnia sample, whole daphnia magna samples for micro, whole daphnia sample, whole daphnia, staining small juvenile, samples if immediate imaging, homogeneous staining of the whole adult, homogeneous staining, imaging, 3d visualization of cell, immediate imaging, pta concentration, tissue, higher concentration of pta, micro
Funders Acknowledgements:
Pennsylvania Department of Health Commonwealth Universal Research Enhancement Program
Grant ID: Penn State Human Health and Environment Seed Grant to KCA
National Institutes of Health
Grant ID: 1R24OD18559 to KCC
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Spotlight Video
The video below is a supplement with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.
Abstract
A step-by-step protocol details the euthanasia, fixation and staining of whole Daphnia samples for micro-CT imaging. While commonly used PTA concentration of 0.3% works for staining small juveniles within 48 h, a higher concentration of PTA (3%) is needed to provide homogeneous staining of the whole adults in 3 days. Micro-CT reconstructions of samples imaged using synchrotron micro-CT reveal histological (microanatomic) features of organ systems, tissues, and cells in the context of the entire organism at sub-micron resolution, and in 3D. Protocol also recommends resin embedding of samples if immediate imaging is not available.
Troubleshooting
Before start
All liquid volumes used for incubation, rinse, and infiltration represented at least 20X per total sample volume. All incubation, rinse, and infiltration steps are performed on a low-speed orbital shaker (Corning LSE) set to 45 revolutions per minute (RPM) to encourage agitation without damaging the samples by rubbing against the container surface.
Euthanasia
1d
Transfer the Daphnia sample using bulb pipette containing the least amount of water into a flat bottom glass vial such as a 20 mL scintillator vial (Wheaton) filled with carbonated water to euthanize the Daphnia. As soon as the sample stops moving, transfer it into Bouin’s solution.
Note
Tip of bulb pipette trimmed at a 45° angle such that the diameter is bigger than the size of the sample.
Sample should be transferred individually to prevent damaging the sample.
Fixation
1d
Immediately, replace the Bouin’s solution and fix the sample in fresh Bouin’s solution at Room temperature for 24:00:00 (hrs).
Note
Replacing with fresh Bouin’s solution is to remove any water carried over.
1d
Staining
1h 25m
Rinse the fixed sample with 1x phosphate buffered saline (PBS) 7.4 for 10 minutes (min), thrice.
Rinse the fixed sample with 1x PBS 7.4 for 00:10:00 (min) (1/3).
10m
Rinse the fixed sample with 1x PBS 7.4 for 00:10:00 (min) (2/3).
10m
Rinse the fixed sample with 1x PBS 7.4 for 00:10:00 (min) (3/3).
10m
Submerge the sample in 35% ethyl alcohol (EtOH) for 00:15:00 at 23 °C with gentle agitation.
15m
Discard the 35% EtOH and submerge the sample in 50% EtOH for 00:15:00 at Room temperature with gentle agitation.
Note
Graded dehydration of 35%, 50% and then 70% is performed to prevent severe shrinkage artifact in Daphnia sample.
15m
Staining
1h 25m
Discard the 50% EtOH and submerge sample in phosphotungstic acid (PTA) in 70% EtOH at Room temperature with gentle agitation. Concentration of PTA and staining duration depend on the sample's age (Table 1).
Note
Replacement of PTA stain solution is highly recommended after 48 hours, especially for samples with developing embryos in the brood chamber.
| A | B | C | D | |
| Age | Embryos in brood chamber | PTA concentration | Staining duration | |
| Juvenile (instar 1-3) | no | 0.3% | 48 hrs | |
| Juvenile (instar 4-7) | no | 1% | 48 hrs | |
| Adult (instar 8 and older) | yes | 3% | 72 hrs or longer (with a PTA renewal at 48 hrs) |
Table 1. Concentration of PTA and staining duration for different ages of D. magna
After staining, wash the sample in 70% EtOH for 5 min with gentle agitation, twice.
After staining, wash the sample in 70% EtOH for 00:05:00 with gentle agitation (1/2).
5m
After staining, wash the sample in 70% EtOH for 00:05:00 with gentle agitation (2/2).
5m
For adult sample to be scanned in 70% EtOH, transfer the sample using a trimmed bulb pipette or 1 mL pipette tip into a 200 µL micropipette tip and seal the tip end with polymer oven-bake clay (Sculpey). For smaller juvenile samples, 10 µL micropipette tips can be used.
Tap the sealed end gently to release bubbles.
Use clean round tip forceps or wooden stick to GENTLY push the sample down the pipette tip until it is immobilized against the wall of the pipette tip, without squeezing or crushing it.
Seal the opening of the pipette tip with parafilm M to avoid evaporation and the sample is ready to be scanned (Fig 1).
Note
In general, PTA-stained samples can be stored up to a month. If samples do not need to be permanent, scanning Daphnia samples at this step is possible within 3 days without ballooning artifact. To avoid ballooning artifact and to allow imaging of Daphnia more than 3 days after preparation we recommend serial dehydration and resin-embedding (below).
Fig 1. Gravid D. magna sample in 200 mL micropipette tip for imaging.
Dehydration and Resin Embedding (optional)
1d 6h 20m
For dehydration, submerge the sample in 90%, 95%, 100% and 100% EtOH for 20 minutes each concentration at Room temperature with gentle agitation.
Note
100% or 200 proof EtOH is the choice of dehydration agent for LR White acrylic resin according to LR White technical datasheet .
Submerge the sample in 90% EtOH for 00:20:00 .
20m
Submerge the sample in 95% EtOH for 00:20:00 .
20m
Submerge the sample in 100% EtOH for 00:20:00 .
20m
Submerge the sample in 100% EtOH for 00:20:00 .
20m
Prepare 1:1 v/v mixture of 100% EtOH and LR White acrylic resin. Submerge the samples in 1:1 EtOH and LR White acrylic resin mixture Overnight or at least 03:00:00 at Room temperature with gentle agitation.
3h
Submerge the sample in 100% LR White resin for 02:00:00 at Room temperature with gentle agitation.
2h
Replace the LR White resin with fresh 100% LR White resin and submerge for 01:00:00 at Room temperature with gentle agitation.
1h
Cut a polyimide tubing of appropriate diameter to 30 - 50 mm length (Table 2).
| A | B | C | |
| Age | Polyimide tubing inner diameter | Micropipette tip | |
| Juvenile (instar 1-3) | 0.0403” | 200 mL (end of tip clipped off) | |
| Juvenile (instar 4-7) | 0.0808” | 200 mL | |
| Adult (instar 8 and older) | 0.105” | 1000 mL |
Table 2. Sizes of polyimide tubing and micropipette tip for different ages of D. magna
Attach the polyimide tubing to a micropipette tip so that it fits snugly (Fig 2).
Note
200 µL micropipette tip for 0.0403” tubing and 1000 µL micropipette tip for 0.105” tubing. Clip off the end of 200 µL micropipette tip at 4.4 cm for the tubing to fit snugly.
Fig 2. Polyimide tubing attached to micropipette tips for LR White resin embedding.
Attach the micropipette tip together with the polyimide tubing to a micropipette.
Transfer the sample to a small weigh boat or V-shaped solution basin and fully submerge the samples in fresh 100% LR White resin.
Position the tubing at the head of the sample and pipette resin to fill up half of the tubing before pipetting the specimen slowly into the tubing. Position the sample in the middle of the tubing and ensure the tubing above and below the tubing us filled with resin.
Note
Pipette the sample into the tubing such that the sample is moving in the natural, forward direction to avoid backward movement that will damage extremities.
Immediately seal the open end tightly, using a slab of oven-bake clay that has been flattened into a ~2mm-thick sheet. Remove excess clay.
Separate the micropipette tip from the polyimide tube by gentle rotation and seal that end of the tube with clay.
Place the tubing horizontally, with one end slightly elevated to minimize bubble formation next to the sample. Allow the resin to polymerize for 24:00:00 at 65 °C . Once polymerized, the sample is ready for imaging (Fig 3). Removal of polyimide tubing is possible but not necessary for imaging.
Fig 3. Sample embedded in LR White acrylic
resin using polyimide tubing.
1d
Acknowledgements
The authors are grateful to Dr. Dilworth Parkinson and Advanced Light Sources Beamline 8.3.2, Lawrence Berkeley National Labs, for making possible micro-CT imaging of the Daphnia samples. Authors would also like to acknowledge the Department of Pathology, Penn State College of Medicine and Jake Gittlen Laboratories for Cancer Research for their support.