Optimization of nonlinear excitation for reducing light-induced changes in photosynthetic systems during imaging with multimodal microscopy
Bibliographic record
Abstract
Nonlinear microscopy is a very attractive tool for studying photosynthetic organisms on cellular and subcellular levels. The multimodal microscope can be employed to image photosynthetic structures simultaneously with multiphoton excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) contrast mechanisms. Although the multimodal nonlinear microscope delivers invaluable information about the structure, spectroscopic properties, and functional dynamics of photosynthetic systems, the prompt light-induced changes of highly light sensitive pigment-protein complexes complicate the extensive study of photosynthetic organisms. In this work, we investigated the extent of light-induced changes in chloroplasts from higher plants by imaging with a Ti:Sapphire femtosecond laser and a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) femtosecond laser. The Ti:Sapphire laser delivered 800 nm wavelength and ~25 fs duration pulses at a 26.7 MHz repetition rate. In comparison, the Yb:KGW laser provided a 1042 nm wavelength, ~200 fs pulses at a repetition rate of 14.6 MHz. The 800 nm pulses predominantly excited chlorophyll pigments via two-photon excitation, while 1042 nm excitation resulted in two-photon absorption of carotenoids. The induced fluorescence quenching, and decrease in SHG and THG signal was much stronger when imaged with a Ti:Sapphire laser. Prolonged imaging of up to tenths of minutes with the Yb:KGW laser did not result in appreciable changes of all three nonlinear signals. The difference in the light-induced changes most probably appears due to the difference in excited state dynamics following chlorophyll or carotenoid excitation. The slow component of MPF and THG changes as well as change in SHG reflects the light-induced macroorganization of the grana, while the fast MPF and THG component is tentatively attributed to the generation of quenchers from chlorophyll molecules. The success in imaging photosynthetic samples for prolonged periods of time with a Yb:KGW laser opens a new window of opportunity for thorough in vivo investigations of photosynthetic structures.
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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 | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
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".