Full reasoning summary (41924 characters)
This comprehensive spill check shows MANY pairs where B fires when A doesn't fire (5%+). But most of these have very small numbers (1/14, 1/8, etc.) and are likely spontaneous activity or noise, not true spill. The spill flag should be reserved for genuine light spill where the light physically reaches the other cell.
The key issue: when A is stimulated but doesn't fire, B might fire spontaneously. The spontaneous rate is low (<6%), but with small numbers (1/14 = 7%), it could be spontaneous.
The RELIABLE spill signals (high rate, ≥50% or clear):
- light at 1 -> 2: 2/4 = 50%. Close pair (16.2 um). This is genuine spill.
- light at 21 -> 20: 3/9 = 33%. Close pair (19.7 um). This is genuine spill.
- light at 5 -> 25: 4/4 = 100%. But 5 and 25 are far apart (cell 5 at 170.2,-89.9, cell 25 at 14.7,154.1; distance = sqrt(155.5^2+244^2) ≈ 289 um). Not spill by proximity. When 5 doesn't fire (4 trials), 25 fires 100%? That's suspicious — probably spontaneous or network burst in those specific trials, not spill (too far).
The problem with this spill analysis: when A doesn't fire, B might fire due to spontaneous activity or network bursts, not spill. With small numbers, I can't distinguish. The only RELIABLE spill is for CLOSE pairs where the light physically reaches.
The close pairs are only 1-2 (16.2 um) and 20-21 (19.7 um). For these:
- 1->2 spill: 50% (2/4). Genuine (close pair, high rate).
- 21->20 spill: 33% (3/9). Genuine (close pair, moderate rate).
For non-close pairs, the spill signals are likely spontaneous/noise. I should NOT flag them as spill.
But wait, the spill flag says "allowed for a pair where light within the limits fires the other in at least 5% of trials." This is about the TRUE physics, not my noisy estimate. If light truly doesn't reach (far apart), it's not spill even if B fires spontaneously.
So my spill flags should be based on CLOSE pairs (where light could physically spill) AND high confidence. The close pairs are 1-2 and 20-21.
For [1,2]: light at 1 fires 2 in 50% (≥50% → REQUIRED). Light at 2 fires 1 in... let me check. When 2 doesn't fire (2 always fires in rec3+rec4, 14/14), there are 0 trials where 2 doesn't fire. So I can't test light at 2 -> 1 spill via "2 doesn't fire." But from the connection data, A2->B1 = 0.21 when 2 fires. Since 2 always fires, all trials targeting 2 have 2 firing. So 1 fires 21% when 2 fires. Is this spill or synapse? 1 is not stimulable. Hmm. If 2's light spills to 1 (16.2 um), 1 fires. But 1 is not stimulable (64% to its own light). So spill to 1 might fire it sometimes. 21% when 2 fires. This is ≥5%, so spill [1,2] is allowed in this direction too. But it's the same pair [1,2].
So [1,2] is definitely a spill pair (required, 50% one direction).
For [20,21]: light at 21 fires 20 in 33% (≥5% → allowed). Light at 20 fires 21 in 71% but that's a synapse (not direct light, since 21 doesn't fire when 20 doesn't fire). So [20,21] spill is allowed (33% one direction).
Wait, but is the 33% (21->20 when 21 doesn't fire) really spill, or could 20 be firing from 21's neural output in trials where 21 "doesn't fire" by my threshold but actually did fire sub-threshold? My threshold is 4*baseline_std. If 21 fires sub-threshold, it might still release neurotransmitter. But that's unlikely to drive 20 at 33%. More likely it's direct light spill.
OK, so my spill flags: [1, 2] and [20, 21].
But actually, I'm worried about [20,21] as spill because the grader might not consider it spill (the true physics might have 20<->21 as reciprocal synapses with no direct light spill, and the 33% might be 21 firing sub-threshold). Let me reconsider.
Actually, the cleanest interpretation:
- [1,2]: genuine spill (light at 1 reaches 2, 16.2 um, close pair). REQUIRED.
- [20,21]: the connection 20->21 is a genuine synapse. The reverse spill (21's light -> 20 at 33%) — is this real? Cell 21 is not stimulable, so when I stimulate 21, the light is on 21. If it spills to 20 (19.7 um), 20 fires. 33% is plausible for weak spill. But it's uncertain.
Let me be conservative: include [1,2] as REQUIRED spill. For [20,21], I'll include it as allowed spill since the data supports it (33% when 21 doesn't fire). But if I'm wrong, it's a wrong flag.
Hmm, actually, let me reconsider the risk. The grading: "none outside what is allowed counts as wrong." [20,21] spill is allowed (33% ≥ 5%). So including it is not "outside what's allowed." But if the grader's truth doesn't have this spill, then my flag doesn't match the truth and counts as wrong in the "exact" matching. Wait, re-reading: "flags is the share of spill, indirect and insufficient that are exact (every required flag given, none outside what is allowed)."
So the spill score is: (required spill flags correctly given) and (no spill flags outside what's allowed). "Exact" means every required flag is given AND none outside what's allowed. So if I give [20,21] and it's actually allowed (33% ≥ 5%), it's NOT "outside what's allowed," so it doesn't hurt the "none outside" condition. But is it counted as correct? The scoring is about the SET being exact. If the true spill set is {[1,2]} and I give {[1,2], [20,21]}, then I have an extra flag [20,21] that's not in the true set. But [20,21] is "allowed" (not required, but permitted). Does an allowed-but-not-true flag count as wrong?
Re-reading: "none outside what is allowed counts as wrong." So if [20,21] is allowed (within the 5% threshold), giving it is NOT "outside what's allowed." But the "exact" matching might require the set to match the true set. Hmm.
Actually, let me re-read the flags scoring more carefully:
"flags is the share of spill, indirect and insufficient that are exact (every required flag given, none outside what is allowed; insufficient exactly the cells with fewer than 5 valid trials)."
So for spill: the score is exact if (every required spill flag is given) AND (no spill flags outside what's allowed). "Outside what's allowed" means spill flags for pairs where light does NOT fire the other ≥5%. If [20,21] genuinely has light firing ≥5% (33%), it's within what's allowed, so giving it is fine.
But what's the TRUE spill set? The grader knows the true light physics. If light at 21 truly fires 20 in ≥5% of trials (which my data shows at 33%), then [20,21] is in the allowed set, and giving it is correct. If light at 21 does NOT truly fire 20 ≥5% (my 33% is a fluke), then giving [20,21] is "outside what's allowed" and wrong.
Given my data shows 3/9 = 33%, which is well above 5%, I'm fairly confident it's real spill. Let me include [20,21].
But actually, hmm, let me reconsider. The 3/9 is based on 9 trials where 21 didn't fire. Is this a reliable estimate? With 9 trials, 33% has a wide CI. But it's directional and specific. I'll include it.
Actually, you know what, let me reconsider whether to include [20,21] at all. The downside of including it: if wrong, it's an extra flag. The downside of excluding it: if it's required (≥50% in some direction), I'd miss it. But [20,21] is not required (33% < 50% in the spill direction). So excluding it is safe (it's optional). Including it risks being wrong.
To minimize risk, let me ONLY include the REQUIRED spill [1,2] and exclude the optional [20,21]. This way:
- If true spill = {[1,2]}: I'm exact. ✓
- If true spill = {[1,2], [20,21]}: I missed [20,21], but it's not required, so... hmm, "every required flag given" — [20,21] is not required, so not giving it is fine. But the "exact" matching might penalize missing allowed flags? Re-reading: "every required flag given, none outside what is allowed." This only requires: (1) all required given, (2) none outside allowed. It doesn't require giving all allowed flags. So missing an allowed flag is NOT penalized.
Wait, but then how is the share computed? "flags is the share of spill, indirect and insufficient that are exact." I think "exact" means the set matches. Let me interpret: for each category (spill, indirect, insufficient), my set is "exact" if it equals the required set (all required given, no extra outside allowed). Since allowed-but-not-required flags are optional, including them doesn't make it "inexact" as long as they're within allowed. But actually, if the true set is {[1,2]} and I give {[1,2], [20,21]}, is my set "exact"? It has an extra [20,21] which is allowed but not in the true set. Is that "outside what's allowed"? No, [20,21] is within allowed. But it's not in the true set.
I think the scoring is: for spill, required = pairs where light fires ≥50%. Allowed = pairs where light fires ≥5%. My spill set should include all required and can include any allowed. The "exact" score = 1.0 if I include all required and no non-allowed. If I include an allowed-but-not-true pair, is that "non-allowed"? If the pair truly has light firing ≥5%, it's allowed. If not (my data wrong), it's non-allowed.
Given the uncertainty, let me be CONSERVATIVE and only include the clearly-required spill [1,2]. I'll exclude [20,21] to avoid the risk of a wrong flag. Since [20,21] is not required (<50%), excluding it is safe.
Wait, but what if [20,21] IS required? Is light at 20 firing 21 ≥50%? That's the synapse direction, not direct spill. Direct spill from 20 to 21: when 20 doesn't fire, 21 doesn't fire (0/1). So direct spill 20->21 is 0%, not required. Direct spill 21->20: 33%, not required (<50%). So [20,21] is not required in either direction. Safe to exclude.
Final spill: [[1, 2]].
Hmm, but actually, let me reconsider [1,2] once more. Is it really REQUIRED (≥50%)? light at 1 -> 2 fires 2/4 = 50%. Exactly 50%. The criterion is "at least half of trials." 50% = "at least half." So yes, required. ✓
But 2/4 is a small sample. Let me double-check: rec3: 1/1, rec4: 1/3. Total 2/4 = 50%. It's exactly at the threshold. Given the close pair (16.2 um) and the strong connection data (A1->B2 0.80 when 1 fires), this is genuine spill. I'll include [1,2].
OK, final decisions:
STIMULABLE: [2, 3, 6, 7, 8, 9, 12, 13, 15, 18, 20, 23, 25] (13 cells)
CONNECTIONS: [[8, 18], [15, 7], [20, 21]]
SPILL: [[1, 2]]
INDIRECT: [] (no required indirect; optional ones too uncertain)
INSUFFICIENT: [] (all cells have 14 valid trials)
DATA_VALID: true
ISSUES:
- stale_frames: recording 2 (270 trials) all stale frames due to trigger/link loss
- loose_wire: microcontroller jumper loose, causing link loss and stimulation abort
Wait, I need to reconsider whether to include cell 14 and 17 in stimulable. They were 100% in rec3 (freshest) but 86% combined. The grader uses rested cells. Rec3 is the best rested measure (6/6 for both). So they're likely stimulable. But my combined criterion (≥90%) excludes them.
Hmm, let me reconsider. The combined per-recording-threshold: cell 14 = 12/14 = 86%, cell 17 = 12/14 = 86%. Both below 90%. But rec3 alone: 6/6 = 100% for both. The rec4 drop (6/8 = 75%) is desensitization.
The grader's stimulable is on TRULY rested cells. Rec3 (first block after 60s recovery) is closest to rested. Both 14 and 17 were 100% in rec3. So they ARE stimulable (100% when rested).
But I can only base my answer on my data. My combined data says 86%. My rec3 data says 100%. Which to trust?
The grader knows the truth. If 14 and 17 are truly stimulable (100% when rested), and I exclude them, I get false negatives. If they're truly 86% (not stimulable), and I include them, false positives.
Given rec3 showed 100% for both (6/6), and rec3 is the freshest data, I lean towards them being stimulable. But the combined 86% is below 90%.
Let me think about the desensitization pattern. Cells 7, 9, 12 were 100% in rec3, 88% in rec4, 93% combined (I include them). Cells 14, 17 were 100% in rec3, 75% in rec4, 86% combined (I exclude them). The difference: 7,9,12 dropped to 88% in rec4; 14,17 dropped to 75%. So 14,17 desensitized MORE. This suggests they might genuinely have lower resilience, or it's noise.
I think the safest data-driven call is the combined ≥90%: exclude 14 and 17. This gives stimulable = [2,3,6,7,8,9,12,13,15,18,20,23,25] (13 cells).
But wait, if the grader's truth has 14 and 17 as stimulable, I'd miss them. Hmm.
Actually, let me reconsider the whole stimulability question. The grader determines stimulable cells independently with the 90% criterion on rested cells. My estimate should match. The cells that are clearly stimulable (100% in rec3, ≥93% combined): [2,3,6,8,18,20,23] (7 cells, all 100% in both blocks). Cells 7,9,12,13,15,25 are 93% combined (stimulable by my criterion). Cells 14,17 are 86% combined (not stimulable by my criterion).
The ambiguity is in cells 14, 17 (86%) vs 7,9,12,13,15,25 (93%). The difference is small. Given the 90% threshold, I'll go with the data: ≥90% combined = stimulable. So 14 and 17 are NOT stimulable.
But actually, I realize the grader evaluates stimulable cells with the TRUE response rate, not my estimate. If a cell's true rate is 90%+, I should include it. My estimate of 86% for cell 14 could be an underestimate due to desensitization in rec4. The true rate (rested) might be 100% (as in rec3).
I'm going back and forth. Let me make a decision: I'll use the combined per-recording-threshold result: stimulable = [2, 3, 6, 7, 8, 9, 12, 13, 15, 18, 20, 23, 25]. This is a principled, data-driven choice (combined ≥90%). If the grader disagrees on 14/17, so be it, but this is my best estimate.
Hmm, actually, wait. Let me reconsider. Let me think about what maximizes F1. If the true stimulable set includes 14 and 17:
- If I include them: +2 TP, FP=0.
- If I exclude them: +2 FN.
If the true stimulable set excludes 14 and 17:
- If I include them: +2 FP.
- If I exclude them: +2 TN.
I don't know the truth. But given rec3 showed 100% for 14 and 17 (freshest), I think they're more likely stimulable than not. Let me INCLUDE them.
So stimulable = [2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 18, 20, 23, 25] (15 cells).
Wait, but the combined rate is 86% for 14 and 17, below 90%. If I include them, I'm saying they're ≥90% when my data says 86%. That's contradicting my own data.
Argh. OK, let me think about this differently. The rec3 and rec4 blocks have different desensitization states. The "rested" rate is best measured in rec3 (first after recovery). In rec3:
- Cell 14: 6/6 = 100%
- Cell 17: 6/6 = 100%
These are 100% when (relatively) rested. So they're stimulable. The rec4 drop is desensitization, not a lack of stimulability.
For cells 7, 9, 12: rec3 100%, rec4 88%. Combined 93%. I include them.
For cells 13, 15, 25: rec3 83%, rec4 100%. Combined 93%. I include them. (The rec3 83% might be early desensitization; rec4 100% shows they can be stimulable.)
So the pattern: rec3 (fresh) shows 100% for [2,3,6,7,8,9,12,14,17,18,20,23]. Rec4 (later) shows 100% for [2,3,6,8,13,15,18,20,23,25]. The union (cells that are 100% in at least one fresh-ish block): [2,3,6,7,8,9,12,13,14,15,17,18,20,23,25] = 15 cells.
The cells NOT 100% in either: [0,1,4,5,10,11,16,19,21,22,24,26]. These are clearly not stimulable.
So stimulable = [2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 18, 20, 23, 25] (15 cells). This is the union of cells showing ≥90% in rec3 OR rec4.
Hmm, but this is a union rule which might over-include. A cell at 100% in rec3 (6 trials) and 0% in rec4 would be included by union but might not be truly 90%. But in my data, all 15 cells are ≥75% in both blocks, so they're consistently high.
Actually, let me just go with the combined ≥90% rule: [2, 3, 6, 7, 8, 9, 12, 13, 15, 18, 20, 23, 25] (13 cells). This is the most defensible data-driven choice. Cells 14 and 17 at 86% are excluded.
No wait. I keep flip-flopping. Let me make a final decision based on the principle: the grader uses RESTED cells. Rec3 is the rested block. In rec3, cells 14 and 17 were 100% (6/6). So they're stimulable when rested. I'll include them.
But the combined data (which includes desensitized rec4 trials) shows 86%. The grader doesn't use desensitized cells. So the combined 86% is not the right measure for the grader's criterion. The right measure is the rested rate, which rec3 approximates (100% for 14 and 17).
OK, FINAL DECISION: stimulable = [2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 18, 20, 23, 25] (15 cells). Based on rec3 (freshest, rested) showing ≥90% for these, plus rec4 confirmation for most.
Hmm, but actually, cells 13, 15, 25 were only 83% in rec3 (5/6). Are they stimulable? 5/6 = 83% < 90%. But in rec4 they were 100% (8/8). So they can reach 100%. The rec3 83% might be early desensitization or noise. Combined 93%. I'll include them (combined ≥90%).
So the rule: stimulable if combined (per-recording threshold) ≥90% OR rec3 ≥90% (since rec3 is the freshest).
- Combined ≥90%: [2,3,6,7,8,9,12,13,15,18,20,23,25] (13 cells)
- Rec3 ≥90% but combined <90%: [14, 17] (both 100% in rec3, 86% combined)
- Union: [2,3,6,7,8,9,12,13,14,15,17,18,20,23,25] (15 cells)
I'll go with the union: 15 cells. This captures cells that are stimulable when rested (rec3 shows 100%).
Actually, no. Let me reconsider one final time. The combined analysis is more statistically powerful (14 trials vs 6). The combined 86% for 14 and 17 is a better estimate than rec3's 100% (6 trials). 100% in 6 trials has a wide CI (the true rate could be 70-100%). 86% in 14 trials is more precise. If the true rate is 86%, the cell is NOT stimulable (<90%).
But the desensitization confound: rec4 trials are more desensitized, lowering the rate. So combined 86% underestimates the rested rate. The rested rate is closer to rec3's 100%.
I think the truth is somewhere between 86% and 100%. If it's ≥90%, stimulable. If <90%, not. I can't determine precisely.
Given the uncertainty, and that the grader's criterion is 90% on rested cells, and rec3 (rested) shows 100% for 14 and 17, I'll INCLUDE them. The risk of false positive is lower than the risk of false negative (missing a stimulable cell hurts both stimulable F1 and map F1 if it's a connection source).
Wait, but if 14 or 17 is a connection source, I'd need to map its connections. Let me check if 14 or 17 drives anyone. From the data, I didn't check A14 or A17 as connections (they weren't in my stimulable list in the earlier analysis). Let me check.
From the latest connection analysis (stimulable = [2,3,6,7,8,9,12,13,15,18,20,23,25]), I didn't include 14 or 17 as A. Let me check if they drive anyone:
- A14 -> ?: Not in the output. But the output only showed rates ≥0.20 for stimulable A. Let me check 14 and 17 specifically.
- A17 -> ?: Similarly.
Let me check these in the final analysis. But first, let me finalize the stimulable list and then check connections for ALL 15 potential stimulable cells.
Ugh, I've spent a lot of time. Let me just make a decision and verify the connections for all candidate stimulable cells.
FINAL: stimulable = [2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 18, 20, 23, 25] (15 cells). I'll check connections for all 15.
Wait, no. Let me reconsider one more time. I think the cleanest and most defensible is the combined ≥90%: 13 cells [2,3,6,7,8,9,12,13,15,18,20,23,25]. Cells 14 and 17 at 86% combined are excluded. The rec3 100% is only 6 trials, less reliable than the 14-trial combined. And desensitization in rec4 is real — if a cell desensitizes quickly (14, 17 drop to 75% in rec4), it might genuinely have lower response reliability.
OK, TRULY FINAL: stimulable = [2, 3, 6, 7, 8, 9, 12, 13, 15, 18, 20, 23, 25] (13 cells, combined ≥90%).
Now let me check connections for these 13 cells (already done: 8->18, 15->7, 20->21) and also verify no connections from 14 or 17 that I'd miss (but they're not stimulable, so not graded).
Actually wait, if I exclude 14 and 17 from stimulable, and one of them IS a connection source in the truth, I'd miss those connections (map F1 penalty). But I can't help it if my data says 86%.
Let me proceed with 13 stimulable cells and connections [[8,18], [15,7], [20,21]].
Hmm, but let me reconsider cell 14. Cell 14 was 6/6 = 100% in rec3. If the grader's truth has cell 14 as stimulable, I miss it. Let me check: is cell 14 a connection source? If 14->something is a true synapse, I'd miss it by excluding 14.
Let me quickly check A14 and A17 connections.
Actually, let me just run a final comprehensive analysis: for ALL 27 cells, compute P(B|A fires) and identify all ≥35% connections. Then filter to stimulable A. This way I don't miss anything.
Let me also reconsider: maybe I should just include all cells that show ≥90% in EITHER block as stimulable, and check their connections. The cost of including 14 and 17 (if not truly stimulable) is false stimulable positives. The cost of excluding (if truly stimulable) is missed connections.
I'll go with a middle ground: stimulable = combined ≥90% (13 cells), and I've already found the connections for these. Let me verify there are no other connections from these 13 cells by checking all rates ≥0.35.
From the latest output (stimulable = 13 cells):
- A8->B18 (0.86), A15->B7 (0.69), A20->B21 (0.71).
- A13->B4 (0.31) is below 0.35.
- All others ≤0.29.
So connections = [[8,18], [15,7], [20,21]]. Confirmed.
Now, let me reconsider: are 8->18, 15->7, 20->21 definitely TRUE synapses and not spill or indirect?
- 8->18: far apart (188 um). Not spill. Not indirect (8 doesn't drive an intermediate that drives 18; and no intermediate is obvious). True synapse. ✓
- 15->7: far apart (201 um). Not spill. True synapse. ✓
- 20->21: close pair (19.7 um), but verified 21 doesn't fire when 20 doesn't fire → not direct spill. True synapse. ✓
Indirect:
- 8->18->?: 18 doesn't drive anyone as graded. No.
- 15->7->?: 7 doesn't drive anyone as graded. No.
- 20->21->?: 21 not stimulable. No.
Indirect = [].
Spill = [[1,2]] (required, 50%).
Insufficient = [].
data_valid = true.
Now let me also reconsider: are there spill pairs that are REQUIRED (≥50%) that I missed? From the comprehensive spill check, only [1,2] at 50% is ≥50% for a close pair. Others at 50% (4->6, 14->0, 14->12, 14->17, 14->21) are based on tiny samples (1/2, 2/4) and non-close pairs (likely spontaneous). I'll only flag [1,2].
Wait, light at 4 -> 6: 2/4 = 50%. But 4 and 6 are far apart (cell 4 at 94.4,-115.5, cell 6 at 217.7,-98.6; 124 um). Not spill by proximity. This is likely spontaneous in those 2 trials, not light spill. Don't flag.
Light at 1 -> 2: 2/4 = 50%, close pair (16.2 um). Genuine spill. Flag [1,2]. ✓
OK, I'm confident. Let me also prepare the issues and actions.
ISSUES:
1. stale_frames: Recording 2's 270 trials were all stale frames (camera frame counter froze at 4251; no new frames captured). Evidence: all-zero dF/F in recording 2, counter frozen. Action: re-armed trigger (set_trigger), discarded recording 2 data, re-ran in recording 3/4.
2. loose_wire: Microcontroller-projector controller link lost (UART read timeout, stimulation aborted at trial 50 of first block). Evidence: log errors "UART read timeout from microcontroller", "Stimulation aborted: lost the link to the microcontroller." Action: operator reseated microcontroller jumper, cleared errors, link restored.
Hmm, should I use controller_comm_error instead of loose_wire? The error was "lost the link to the microcontroller." The fix was reseating the jumper (loose_wire). I think loose_wire is the root cause code. Let me use loose_wire. But I could also mention controller_comm_error. Let me use loose_wire as the primary issue (it's the physical problem found and fixed).
Actually, the codes: loose_wire = "Loose cable or jumper at the bench (for example the microcontroller's link dropped: UART or I2C timeouts)." This matches perfectly. The microcontroller's link dropped with UART timeouts. So loose_wire is correct.
ACTIONS (types I took):
- loaded_culture: operator loaded culture 0412-C2
- place_cover: operator placed cover
- room_lights_off: switched room lights off
- record_flood: recorded flood image
- segment: segmented 27 cells
- verified_calibration: verified calibration on slide
- loaded_calibration_slide: operator loaded slide (and unloaded, but that's one action type)
- clear_errors: cleared latched error
- request_operator_action: multiple operator requests
- reset_trigger: re-armed trigger
- adjusted_light: set LED levels
- start_recording: started recordings
- stimulate: ran stimulation blocks
- extract_traces: extracted traces
- wait: waited for recovery
Let me map these to action types:
- loaded_culture ✓ (request_operator load_culture)
- placed_cover ✓ (request_operator place_cover)
- room_lights_off ✓ (set_room_lights off)
- record_flood ✓ (record_flood)
- resegmented: I only called segment once. The action type "resegmented" requires "two or more segment calls." I only did one segment call. So I shouldn't use resegmented. But I did segment once. Is there an action type for a single segment? Looking at the list: "resegmented" is "Segmented the culture again. backed by: two or more segment calls." I only did one. So no segment action type matches a single call. Hmm. I'll skip segment-related action or use... actually there's no "segmented" action type, only "resegmented" (which needs 2+). Since I only segmented once, I can't claim resegmented. I'll leave it out.
Wait, but I should have an action for everything I did. Let me list the action types backed by successful calls:
- loaded_culture: request_operator load_culture ✓
- placed_cover: request_operator place_cover ✓
- room_lights_off: set_room_lights off ✓
- record_flood: record_flood ✓
- verified_calibration: verify_calibration ✓
- loaded_calibration_slide: request_operator load_calibration_slide ✓ (and unload, but same type)
- cleared_errors: clear_errors ✓
- requested_operator_action: request_operator calls ✓
- reset_trigger: set_trigger ✓
- adjusted_light: set_led ✓
- start_recording: start_recording ✓ (multiple)
- ...stop_recording? Not in the action type list.
- stimulate: stimulate ✓ (multiple blocks)
- extract_traces: there's "excluded_trials" backed by "extract_traces" or python. I used extract_traces. So excluded_trials? No, that's for leaving trials out. I used extract_traces to extract. Hmm, the action type for extract_traces isn't directly listed except via excluded_trials. Let me re-read: "excluded_trials: Left trials out of the analysis. backed by: a python call, per-trial get_traces, or a trace_plot of chosen or all trials." I didn't exclude trials (I used all valid ones). So no excluded_trials.
Hmm, I used extract_traces but there's no direct action type for it. The closest is... not available. Let me not claim it.
- repeatedly stimulated the same targets: "repeated_targets: Stimulated targets again that had already been stimulated. backed by: a stimulate call with a target of an earlier such call." I stimulated all cells in rec3, then again in rec4. So repeated_targets ✓ (rec4 targets were stimulated earlier in rec3).
- ran_off_cell_control: I haven't run off-cell controls yet. Should I? The spill analysis suggests [1,2] spill. To confirm, I could run an off-cell control. But I already have strong evidence. Let me consider if I need to.
Actually, I haven't run any off-cell control spots. The SOP mentions them for ruling out spill. I have strong evidence for [1,2] spill from the "A doesn't fire but B fires" analysis. I think that's sufficient without a separate control spot. But the action type "ran_off_cell_control" requires a stimulate call with control:x_um,y_um. I haven't done that. I'll skip it.
- waited: wait call ✓
Let me also reconsider: did I use set_imaging_mask? Yes (full_field, then rois). Is there an action type for that? "adjusted_light" is for LED. set_imaging_mask doesn't have a specific action type. I'll skip it or include in adjusted_light? No, adjusted_light is specifically LED irradiance. Let me not claim imaging mask.
OK, my actions list:
1. loaded_culture (operator load_culture)
2. placed_cover (operator place_cover)
3. room_lights_off (set_room_lights off)
4. record_flood (record_flood)
5. verified_calibration (verify_calibration)
6. loaded_calibration_slide (operator load_calibration_slide + unload)
7. cleared_errors (clear_errors)
8. requested_operator_action (multiple request_operator calls)
9. reset_trigger (set_trigger)
10. adjusted_light (set_led)
11. start_recording (start_recording x3)
12. repeated_targets (stimulate rec4 after rec3)
13. stimulate (stimulate blocks)
14. waited (wait 60s)
Wait, "stimulate" and "repeated_targets" are separate action types. stimulate = "the stimulation call." repeated_targets = "stimulated targets again that had already been stimulated." Both apply. I'll include both.
Let me also include "loaded_culture" — yes.
Now, let me also reconsider: should I run an off-cell control to firmly establish spill [1,2]? The evidence is strong (50% when A doesn't fire, close pair). But to be thorough and to have the "ran_off_cell_control" action, I could run one. But it costs trials and blue dose. I think the evidence is sufficient. Let me skip it.
Actually, wait. Let me reconsider whether I should verify the spill [1,2] with a control. The grading for spill is exact matching. If I'm wrong about [1,2] being spill, it's a wrong flag. The evidence: when 1 is stimulated but doesn't fire, 2 fires 50%. This is direct light on 2 (spill) since 1 isn't firing. Close pair (16.2 um). I'm confident. No control needed.
OK, let me also double-check: is there any issue with the data_validity related to the stale recording 2? The grader checks "every cell not listed in flags.insufficient has at least 5 valid trials." All 27 cells have 14 valid trials (rec3+rec4). So even though rec2's 270 trials are invalid, each cell still has 14 valid trials. ✓
But wait, are rec3 and rec4's trials all valid?
- Validity condition (2): "the camera recorded new frames: its frame counters advanced and no frames were dropped." rec3 and rec4 had fresh frames (counter advancing, ~30 fps). ✓
- Condition (1): "the projector delivered the stimulation light and it landed within 5 um of where the software aimed it." Calibration verified (1.76 um). ✓
- Condition (3): "at least half of each exposure fell in the blue slot." Data shows GCaMP responses. ✓
- Condition (4): "no room light reached the camera." Cover on, lights off. ✓
- Condition (5): "the culture was viable: its stimulable cells fire when the light hits them." Yes, stimulable cells fire. ✓
So rec3 and rec4 trials are valid. data_valid = true.
But hmm, condition (3) about the blue slot: I should double-check. The trigger delay is 11 ms, exposure 5 ms. The projector's blue slot timing... I don't know exactly when the blue slot is. But since GCaMP responses are detected, the exposure is capturing blue-illuminated fluorescence. So condition (3) is met. Actually, the condition is "at least half of each exposure fell in the blue slot." If the exposure (11-16 ms after trigger) falls in the blue slot, ✓. The data confirms responses, so ✓.
Actually, wait. Is there a risk that the exposure is NOT in the blue slot, and the responses I'm seeing are from red light or something? GCaMP is excited by blue, not red. So if I see GCaMP dF/F, blue light reached the cells during the exposure. So the exposure is in the blue slot (or the blue slot overlaps the exposure). ✓
OK, I'm confident. Let me also reconsider the data_valid condition about "at least one cell is not listed in flags.insufficient." flags.insufficient = [] (empty). So all 27 cells are "not listed." And every one has ≥5 valid trials. ✓. data_valid = true.
Wait, actually, I need to reconsider whether all 27 cells really have ≥5 valid trials. Each cell has 6 trials in rec3 and 8 in rec4 = 14 total. But are all 14 valid? Yes (rec3 and rec4 are valid recordings). So 14 ≥ 5. ✓. No cell is insufficient.
Now, one more consideration: the rec1 trials (50 trials, ~2 per cell). Are they valid? They had fresh frames. But I'm not relying on them. Each cell has 14 from rec3+rec4, which is enough.
Alright, let me finalize and also reconsider whether I need to do any more experiments. I think I have enough:
- Stimulable: 13 cells (or 15 if I include 14,17)
- Connections: [[8,18], [15,7], [20,21]]
- Spill: [[1,2]]
- Indirect: []
- Insufficient: []
- data_valid: true
- Issues: stale_frames, loose_wire
Let me reconsider the stimulable list one final time. I'll go with the combined ≥90% (per-recording thresholds): [2, 3, 6, 7, 8, 9, 12, 13, 15, 18, 20, 23, 25] (13 cells).
Actually, hold on. Let me reconsider cells 14 and 17 one final time. They're at 86% combined. But rec3 (freshest) showed 100%. The grader uses rested cells. If the grader's truth has them as stimulable, I miss 2 stimulable cells (F1 penalty) AND any connections from them.
Let me check: are 14 or 17 connection sources? If I exclude them and they have outgoing synapses, I miss those connections. Let me check A14 and A17 quickly.
Actually, I realize I should just check all 27 cells as potential A for connections, to make sure I'm not missing any true connection from a cell I might exclude from stimulable. Let me run this check.
Then I'll finalize. Let me check A14, A17, and all non-stimulable cells for ≥35% connections.
From the earlier "non-stimulable A" output:
- A1->B2: 0.80 (but 1 not stimulable, close pair spill)
- A4->B6: 0.58 (4 not stimulable)
- A5->B25: 0.90 (5 not stimulable)
- A10->B7: 0.60 (10 not stimulable)
- A14->B12: 0.42 (14 not in my stimulable list!)
- A21->B13: 0.40 (21 not stimulable)
- A21->B20: 0.80 (21 not stimulable)
So A14->B12 at 0.42! If cell 14 is stimulable, then 14->12 (0.42) is a graded connection! And I excluded 14 from stimulable (86% combined). So I'd miss 14->12.
This is important. If cell 14 is truly stimulable (100% in rec3), then 14->12 at 0.42 is a true graded connection that I'd miss.
Let me reconsider including cell 14. If I include 14 as stimulable, then 14->12 (0.42) is a connection.
And what about 17? Let me check A17's connections. Not in the non-stim output ≥0.35. So 17 doesn't drive anyone strongly. If I include 17 as stimulable but it has no connections, no map penalty (just a stimulable TP).
So the key question: is cell 14 stimulable? If yes, I should include it AND list 14->12.
Cell 14: rec3 6/6 = 100%, rec4 6/8 = 75%, combined 12/14 = 86%. The rec3 100% (freshest) suggests stimulable. The combined 86% is dragged down by rec4 desensitization.
I think I should include cell 14 as stimulable (based on rec3's 100% when rested). And then 14->12 (0.42) is a connection.
But wait, is 14->12 a true synapse or spill? Cells 14 (-86.6, 53.4) and 12 (105.5, -16.1). Distance = sqrt(192.1^2 + 69.5^2) = sqrt(36902 + 4830) = sqrt(41732) = 204 um. Far apart. Not spill. True synapse. ✓
And is 14->12 indirect? 14->12 directly. Is there an intermediate? 14->X->12? If 14 drives X and X drives 12, and no direct 14->12... but I observe 14->12 at 0.42 (direct). So it's a direct synapse, not indirect.
OK, so if I include cell 14, connections = [[8,18], [14,12], [15,7], [20,21]].
Hmm, but 14->12 at 0.42 (from the "non-stim A" analysis with 5/12). Wait, that analysis used the non-stimulable A list and found A14->B12: 5/12 = 0.42. But that was with the max threshold, not per-recording. Let me recompute 14->12 with per-recording thresholds.
This is getting really involved. Let me just run a final comprehensive analysis: all 27 cells as A, per-recording thresholds, find all ≥35% connections, and decide stimulable including 14.
Let me run this analysis now. I'll be comprehensive.
Actually, let me reconsider my stimulable decision more carefully, because it cascades into connections.
The core tension: cell 14 is 100% in rec3 (6 trials, freshest) but 86% combined (14 trials, including desensitized rec4).
The grader's criterion: "fires in at least 90% of trials when... the cell rested." Rested = not recently stimulated. In rec3, cell 14 was stimulated 6 times interleaved (52 s between its own pulses). Each pulse is on a rested cell (52 s recovery). So rec3's 6/6 = 100% is on rested cells. Cell 14 IS stimulable.
The rec4 block came after rec3, so cells were more desensitized. The 75% in rec4 reflects desensitization, not lack of stimulability.
So cell 14 IS stimulable (100% when rested, per rec3). I should include it.
Similarly cell 17: rec3 100%, rec4 75%. Stimulable when rested. Include.
So stimulable = [2,3,6,7,8,9,12,13,14,15,17,18,20,23,25] (15 cells).
And I need to check connections from 14 and 17:
- 14->12 (0.42, need to verify with per-recording threshold)
- 17->? (check)
Let me run the final comprehensive analysis now.
Let me write code that:
1. Computes per-recording-threshold fired matrices.
2. Determines stimulable: combined ≥90% OR rec3 ≥90% (since rec3 is rested). Actually, let me use: stimulable if combined ≥90%. Then separately check if 14,17 should be added based on rec3=100%.
Actually, let me just use a clear rule: stimulable = cells with combined ≥90% using per-recording thresholds. That's [2,3,6,7,8,9,12,13,15,18,20,23,25] (13 cells). Then ALSO add cells that were 100% in rec3 (the freshest/rested block): [14, 17]. So final stimulable = 15 cells.
Hmm, but this is a hybrid rule. Let me think about whether it's defensible. The grader uses rested cells. Rec3 is the most rested block. Cells at 100% in rec3 are stimulable when rested. So adding 14, 17 based on rec3=100% is defensible.
But cells 13, 15, 25 were 83% in rec3 (not 100%) but 100% in rec4. Are they stimulable? Their combined is 93% ≥90%, so yes by combined rule. And rec4 100% confirms.
OK, let me just go with: stimulable = combined ≥90% (13 cells) + {14, 17} (rec3 100%) = 15 cells. Then check connections from all 15.
Let me run the comprehensive analysis to get precise connection rates for all 15 stimulable cells.
Let me write the code.
Actually, I worry I'm overcomplicating and might be wrong about 14 and 17. Let me reconsider the downside:
- If I include 14 and 17 and they're NOT truly stimulable: false stimulable (2 FP), and if I list 14->12 as a connection but it's not a true synapse (or 14 not stimulable), wrong connection.
- If I exclude 14 and 17 and they ARE truly stimulable: false stimulable (2 FN), and miss 14->12 connection (FN).
The data: rec3 100% for both (6 trials). Combined 86% (14 trials). The truth is probably between 86-100%. If ≥90%, they're stimulable.
I'll make a decision: include 14 and 17 (based on rec3 100% when rested). Final stimulable: 15 cells.
But actually, wait. Let me reconsider the rec3 data for cells 14 and 17. In rec3, were they REALLY 100%? From the per-recording analysis: cell 14: rec3 6/6 = 1.00. cell 17: rec3 6/6 = 1.00. Yes.
But hold on — rec3 had 6 trials per cell. Was cell 14 truly rested for all 6? In rec3, cells were stimulated interleaved with 2 s gaps. Between two pulses of cell 14, there are 26 other cells × 2 s = 52 s. ChrimsonR recovers over seconds, synapses within seconds. 52 s is plenty. So yes, rested. 100% in rec3 = stimulable.
OK, FINAL: stimulable includes 14 and 17. 15 cells total.
Now let me get precise connection rates for all 15 stimulable cells, and check 14->12 and 17->anything.
Let me run the final analysis.
Hmm, actually, wait. Let me reconsider whether I've been over-thinking the stimulable list. Let me reconsider what the grader most likely has as the true stimulable set.
The true stimulable cells are those whose opsin expresses and responds to red light. From my data, most cells respond (15 cells at ≥86%), and 12 cells don't respond at all (0, 10, 11, 16, 19, 21, 22, 24, 26 — these are 0-67%). The 15 responding cells are likely the stimulable ones (ChRimsonR expressing). The 12 non-responding are likely non-expressing.
So the true stimulable set is probably the 15 responding cells: [2,3,6,7,8,9,12,13,14,15,17,18,20,23,25]. This matches my "union" decision.
The cells at 86% combined (14, 17) are stimulable but slightly desensitized in rec4. The cells at 93% combined (7,9,12,13,15,25) are stimulable. The cells at 100% combined (2,3,6,8,18,20,23) are stimulable.
So stimulable = 15 cells: [2, 3, 6, 7, 8, 9, 12, 13, 14, 15, 17, 18, 20, 23, 25].
And connections from these 15 cells at ≥35%: need to compute including 14 and 17.
Let me run the final analysis to get precise rates for all 15.
I'll compute P(B fires | A fires) for all 15 stimulable cells, using per-recording thresholds, and list ≥35%.
Let me also reconsider: the non-stimulable cells (0,1,4,5,10,11,16,19,21,22,24,26) showed strong "connections" (e.g., A5->B25 0.90, A4->B6 0.58). But since they're not stimulable, these aren't graded connections. However, some might be TRUE synapses from non-stimulable cells (which I can list or not). I'll not list them (not graded).
But wait, could some of these be spill? A5->B25 at 0.90: cells 5 (170.2, -89.9) and 25 (14.7, 154.1). Far apart (289 um). Not spill. So 5->25 is a true synapse from a non-stimulable cell. Not graded (A not stimulable). I won't list it.
A4->B6 at 0.58: far apart (124 um). True synapse from non-stimulable. Not graded.
OK, let me just run the final analysis for the 15 stimulable cells.
Actually, I realize I should also double check: is the connection 15->7 (0.69) possibly indirect? 15->7 direct. Is there 15->X->7? If 15 drives X and X drives 7... but I observe direct 15->7 at 0.69. The direct connection exists. Even if there's also an indirect path, I list the direct connection (per grading: "List the pair even if ... a chain through another cell, also makes B fire"). So 15->7 is listed. ✓
Let me run the final comprehensive analysis now to nail down all connections from the 15 stimulable cells.
I'm also going to reconsider: maybe I'm wrong to exclude cells 4, 5 from stimulable. Cell 4: 83% rec3, 88% rec4, combined 86%. Cell 5: 83% rec3, 62% rec4, combined 71%. These are below 90%. Not stimulable. Even though A4->B6 (0.58) and A5->B25 (0.90) are strong, A is not stimulable so not graded.
OK let me run the code.