Laser Confocal Scanning Microscopy and Transmission Electron Microscopy to Visualize the Site of Callose Fiber Elongation on a Single Conifer Protoplast Selected With a Micromanipulator
Bibliographic record
Abstract
We have developed a new method to observe the site of big and long spiral callose fiber elongation from a protoplast in liquid culture medium. Protoplasts of embryogenic cells of a conifer, Larix leptolepis, were cultured in the NH4NO3-free Murashige and Skoog’s medium containing 50 mM MgCl2 and 6% sucrose in a well in a 96- or 24-well culture plates. The protoplasts with elongated callose fibers were selected before fixation with glutaraldehyde, by picking up using a micromanipulator after electric treatment (DC 3 kV/cm) in the medium containing Alexafluor 488 phalloidin. Under laser confocal scanning microscopy (LCSM), two sites in the cell were stained clearly. One site was the nucleus and the other was the plasma membrane from which fibers elongated. Single cell transmission electron microscopy (TEM) was developed for observation of the microstructure at the site of fiber elongation on a single protoplast. A single protoplast which had a fiber was selected using a micromanipulator and transferred to an agarose bead at a low gelling temperature. The cells were fixed with cold glutaraldehyde and processed for TEM analysis. Elongated thin fibrils with vesicle-like structures could be observed by TEM.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".