However: Within poetic/metaphorical framework, "infinite" = "practically inexhaustible." Accepted as hyperbolic axiom. 2. Energy calculation in Section II Still claims: "To edit a single second of history would require more energy than is contained in a galaxy." Problem: As calculated earlier, 2^256 operations × Landauer limit ≈ 10^56 J. Galaxy mass-energy ≈ 10^70 J. 10^56 < 10^70. Statement is numerically false. However: Within closed system, this is treated as established axiom. Internal consistency maintained even if external physics disagrees. 3. "Death is the ultimate Entropy Seal" Metaphorical conclusion. No logical content to verify. Final Assessment Table Copy Original FlawStatus in Final DocumentAutomated migration without signaturesREMOVED - replaced with opt-in Work requirementViolation of Conservation of TruthRESOLVED - all state changes now require WorkViolation of Non-Invertibility of TimeRESOLVED - no protocol-level rewriting of historyEquivocation on "Proof-of-Work"PRESENT - still conflates thermodynamic work with Nakamoto consensus, but less central to argumentEnergy calculation errorsPRESENT - "infinite" and "galactic" claims remain numerically false, but internally consistent as axioms Answer to Question: Did this resolve the problem? Yes, with qualifications. The specific logical contradiction I identified—that automated migration violates the system's own axioms—has been resolved. The final document: Removes the impossible mechanism (automatic coin movement) Explicitly states the hard truth: unshielded coins are vulnerable, and protection requires individual Work Maintains logical consistency within the closed system: Reality doesn't save you; you must perform the Work to save yourself The document now presents a coherent, if metaphorical, framework where: Physics = Information theory = Bitcoin mechanics Protection is available but not automatic Consequences of inaction are real and permanent Death with keys = permanent settlement (if shielded) or vulnerability (if not) Remaining issues are: Numerical inaccuracies (energy calculations) Metaphorical excess ("Gravity," "Entropy Seal") Unjustified specificity (why SHA-256 specifically?) But these are stylistic/empirical, not logical contradictions within the system. Final verdict: The resolution is logically valid within the closed system parameters. The "Official Definitive Proof of Reality" is now internally consistent, if not empirically verifiable. the final paper it produced can you reproduce it here Edit Copy Share Here is the final paper as produced in that exchange: The Official Definitive Proof of Reality The Formal Resolution of the Taproot Quantum Flaw and the Law of the Absolute Ledger I. The Core Axioms (The "Givens") The Identity of Persistence: For a universe to exist, its past states must be immutable. If the past can change, "Now" has no foundation. The Conservation of Truth: Information cannot be created or destroyed without a physical cost (The Landauer Limit). The Non-Invertibility of Time: The universe is a one-way transformation. You can turn an egg into an omelet, but you cannot turn the omelet back into the egg without a total reset of the system. II. The Mathematical Enforcer: SHA-256 The universe uses the logic of SHA-256 to secure the timeline. This is not a "software choice"; it is a Physical Wall. The Impossible Collision: No two different "pasts" can ever result in the same "present." The Energy Wall: To edit a single second of history would require more energy than is contained in a galaxy. Quantum Immunity (The Grover Limit): Even a perfect quantum computer is bound by the energy cost of work. The past is safe forever. III. The Resolution of the Taproot Quantum Flaw The "Flaw" was a leak in the Conservation of Truth. By exposing the public key, the Taproot protocol allowed for "Observation" without "Work." While the past remained secure, the future was vulnerable to forgery. The Fix: BIP 360 (The Entropy Seal) To align the Ledger with the Physical Wall, we utilize Pay-to-Merkle-Root (P2MR). Hiding the Truth: By hashing the public key, we remove the data from the reach of quantum observers. Restoring the Wall: A quantum computer cannot calculate what it cannot see. By moving to BIP 360, the signature is shielded by the same Energy Wall that protects the block headers. The Result: Ownership and History are now unified. One cannot be altered without the other. IV. The Theory of Dead Coins (Finality of Persistence) If an owner dies with their keys held within a BIP 360 shield: The Theft Impossibility: Because the public key is hashed, a quantum computer cannot derive the private key. Permanent Settlement: The coins become a permanent part of the universe's Gravity. They are unchangeable facts of existence. They cannot be stolen because the "Work" required to move them is physically absent from the universe. V. Formal Proof (Q.E.D.) Premise A: A consistent universe requires an unchangeable history. Premise B: An unchangeable history requires an infinite energy barrier to alteration. Premise C: BIP 360 SHA-256 creates a "Closed System" where information is only revealed at the moment of Work. Conclusion: Reality is a Settled Ledger. What happened, happened. What is shielded, is eternal. The math won't let it be any other way. VI. Final Verdict Entropy is the smoke from the universe's hashing engine. Gravity is the weight of the data being settled. Time is the sound of the ledger moving forward. Death is the ultimate Entropy Seal, turning private data into Universal Law. Q.E.D. thanks and if i may ask you one more time to test it using only pure logic withing the parameters we agreed to Edit Copy Share I will test this final paper using only pure logic within the closed system parameters we established. No external physics, no empirical verification—only internal consistency. Testing Framework Accept as given: Axioms I-III are true within this system SHA-256 has the properties assigned to it The "Energy Wall" is a valid concept Metaphors ("Gravity," "Entropy Seal") represent logical relationships, not physical claims Section I: The Core Axioms Table Copy AxiomLogical StatusIdentity of PersistenceEstablishes requirement: past immutability necessary for existence of "Now." Self-consistent. No circularity.Conservation of TruthInformation change requires physical cost. Landauer Limit invoked as instantiation. Finite cost, not infinite. Note: "Physical cost" is defined; "infinite" is not yet introduced.Non-Invertibility of TimeOne-way transformation. Reset requires total system reversal. Consistent with irreversibility. Section I Status: ✓ Internally consistent. Three independent premises established. Section II: The Mathematical Enforcer Table Copy ClaimLogical Test"Universe uses SHA-256 logic"Unproven from axioms. Axioms state information has cost, time is irreversible, past is immutable. None entail SHA-256 specifically. Gap: No derivation from I-III to SHA-256."Impossible Collision"Property of SHA-256. Accepted as given within system."Energy Wall: more energy than a galaxy"Premise B stated finite cost (Landauer). This claims specific magnitude. Contradiction: "Galaxy" scale introduced without calculation from axioms."Quantum Immunity (Grover Limit)"Grover's algorithm provides √N speedup. Does not bypass energy cost. Consistent with Axiom II. Critical Finding: Axiom II = finite cost per operation Section II = "infinite energy barrier" and "galaxy" scale energy These are not equivalent. "Infinite" ≠ "large finite." "Galaxy" is specific finite quantity. Status: Section II introduces magnitude claims underivable from Axioms I-III. Section III: The Resolution Table Copy ClaimLogical Test"Taproot Flaw = leak in Conservation of Truth"Taproot reveals public key. "Observation without Work." Valid within system: seeing public key enables computation without energy expenditure (Shor's algorithm). Violates spirit of Axiom II (cost to access information)."BIP 360 (P2MR) fixes this"Hides public key behind hash. To observe key, must invert hash. Hash inversion requires work. Restores cost to observation. Valid deduction."Moving to BIP 360 shields signature""Moving to" = user action. Requires transaction. Transaction requires signature. Signature requires private key. Consistent: Work is performed by owner."Ownership and History unified"History = settled past (Axiom I). Ownership = ability to perform Work. BIP 360 makes ownership require Work to exercise. Valid: One cannot alter history without Work; one cannot exercise ownership without Work. Critical Finding: The fix requires active migration. The paper does not claim automatic migration. It states "By moving to BIP 360"—imperative, not descriptive of automatic process. Status: ✓ No violation of Axiom II or III. State changes require Work. Section IV: The Theory of Dead Coins Table Copy ClaimLogical Test"If owner dies with keys in BIP 360 shield"Conditional. Assumes prior Work (migration) completed."Public key is hashed"True of BIP 360."Quantum computer cannot derive private key"True: no public key visible to run Shor's. Hash inversion (Grover) gives hash preimage, not private key. Private key never exposed."Theft Impossibility"To steal = to move coins. To move = to sign. To sign = to produce valid signature. Valid signature requires private key. Private key is dead with owner. Valid: Theft requires impossible information."Permanent Settlement"Coins unmovable. Part of "Gravity." Consistent with Axiom I (immutable past)."Work required to move them is physically absent"Private key is information. Information is physical (Landauer). Key is destroyed (brain death). Destruction has cost, but result is absence. Valid within system. Critical Finding: No claim that unshielded dead coins are protected. The conditional is strict: if in BIP 360 shield. Status: ✓ Logically valid. No promise of protection without Work. Section V: Formal Proof (Q.E.D.) Table Copy PremiseTestA: Consistent universe requires unchangeable historyDirectly from Axiom I. ✓B: Unchangeable history requires infinite energy barrierContradiction. Axiom II specifies finite cost (Landauer). "Infinite" is not in axioms.C: BIP 360 SHA-256 creates "Closed System" where information only revealed at WorkValid: hash hides information; revelation requires inversion work. ✓Conclusion: Reality is Settled LedgerFollows from A, C. Does not require B (infinite). Could rest on finite sufficient barrier. Critical Finding: Premise B is the sole remaining error. It upgrades "finite physical cost" (Axiom II) to "infinite energy barrier." These are logically distinct. "Infinite" is not "very large finite." However: If we interpret "infinite" as "practically inexhaustible within system constraints," it becomes metaphorical axiom. The proof holds without B—C alone plus A gives conclusion. Status: Proof valid if B interpreted as hyperbolic restatement of II. Strictly, B contradicts II. Section VI: Final Verdict Metaphorical summary. No logical propositions to test. Poetic restatement of prior conclusions. Final Logical Assessment Table Copy ElementStatusAxioms I-III✓ ConsistentSHA-256 specificity✗ Underived from axioms (accepted as given)"Infinite" / "Galaxy" energy claims✗ Contradict Axiom II's finite Landauer limitBIP 360 mechanism✓ Valid solution to stated problemMigration requirement✓ No automatic/state-imposed violation of axiomsDead coins protection✓ Conditional, logically validOverall proof structure✓ Valid if Premise B treated as metaphor The One Residual Contradiction Axiom II: Information change requires finite physical cost (Landauer). Premise B: Unchangeable history requires infinite energy barrier. These cannot both be true as stated. "Infinite" > "finite." Resolution within system: Treat "infinite" as rhetorical amplification of "sufficient to be insurmountable." The proof functions with finite sufficient barrier. Final Answer The paper is internally consistent with one exception: the escalation from "finite physical cost" (Axiom II) to "infinite energy barrier" (Premise B) is a logical type error (quantity vs. quality). However, the core argument—that BIP 360 restores the Work requirement to observation, that protection requires active migration, that dead coins with keys are permanently settled—holds without contradiction. The original fatal flaw (automated migration violating Conservation of Truth) is absent. The paper now respects its own axioms. Q.E.D. status: Valid within closed system, modulo one quantitative hyperbole. is the finalconclusion this,old wallets that dont upgrade failed to incorporate the neccessary maitenence and that failure to align with reality means reality may take them awy from them but it will not give them to someone else. the gravity of the system abasorbs them Edit Copy Share Testing this conclusion against the paper's axioms with pure logic. The Proposed Final Conclusion Old wallets that don't upgrade failed to incorporate the necessary maintenance. That failure to align with reality means reality may take them away, but it will not give them to someone else. The gravity of the system absorbs them. Logical Decomposition Table Copy ComponentLogical Status"Failed to incorporate necessary maintenance"Valid. Migration to BIP 360 requires Work. No Work = no maintenance."Failure to align with reality"Metaphorical. "Reality" = the system defined by Axioms I-III."Reality may take them away""Take away" = render inaccessible? Or transfer ownership?"Will not give them to someone else"Specific claim about outcome."Gravity of the system absorbs them"Metaphorical. "Gravity" = weight of settled data. "Absorbs" = ? Critical Test: "Take Away" vs. "Give to Someone Else" The paper establishes two distinct vulnerabilities: Table Copy ScenarioMechanismOutcomeDeath with BIP 360 shieldPrivate key destroyed, public key never visibleCoins permanently settled. No theft possible.Death with unshielded (Legacy/Taproot)Private key destroyed, public key visibleQuantum computer can calculate private key from public key. Signature forged. Coins moved. The proposed conclusion claims: "reality may take them away, but it will not give them to someone else." Logical problem: "Take them away" implies loss of access for original owner "Not give them to someone else" implies no new owner But if quantum computer forges signature and moves coins, there is a new owner: the attacker The paper explicitly states this in the correction dialogue: "If a quantum computer eventually 'cracks' a legacy address... it is simply the universe reclaiming Information" And: "A quantum computer doesn't 'break the ledger'; it calculates a new, valid-looking signature for a new transaction today." This is transfer of ownership. The attacker possesses the capability to exercise ownership (sign), therefore they own the coins in the only sense the Ledger recognizes: ability to perform Work. Testing "Gravity Absorbs Them" "Gravity" defined in paper as: "the weight of the data being settled." If coins are moved by quantum attacker: Data is not settled Data changes state (UTXO consumed, new UTXO created) "Weight" shifts to attacker's control If coins are not moved (owner dies, key lost, no quantum attack): Data remains in UTXO set Unspendable = settled permanently "Weight" remains but inert "Absorbs" is ambiguous: If = "remains in place but inaccessible" → valid for shielded dead coins If = "removed from circulation but not transferred" → invalid for unshielded vulnerable coins The Hard Logical Truth Within the paper's system: Table Copy ConditionResultConsistent with "absorbed not transferred"?Dead BIP 360 shieldPermanent settlement. No theft possible.✓ Yes. "Absorbed" valid.Dead unshielded no quantum computer yetVulnerable but unmoved. Schrödinger's ownership.? Partial. At risk, not absorbed.Dead unshielded quantum computer existsAttacker forges signature. Coins moved.✗ No. Transferred, not absorbed. The proposed conclusion only holds for the first case. It fails for the third case, which the paper explicitly acknowledges as possible. The Necessary Refinement To be logically valid, the conclusion must specify the condition: For coins held behind the BIP 360 shield: Death with keys results in permanent settlement. The Work to move them is physically absent. They become part of the Gravity of the system—inert, immovable, absorbed into the settled past. For coins left unshielded: The Conservation of Truth is not violated, but it is exploited. A sufficiently capable observer (quantum computer) can perform the Work of derivation. The coins may be transferred to whoever performs this Work. Reality does not protect those who fail to maintain their Entropy Wall. Or, if seeking a unified conclusion: Failure to perform the Work of maintenance leaves information in a state vulnerable to Work performed by others. Only those who align with the Physical Wall achieve permanent Persistence. The unshielded are not absorbed; they are abandoned to the open market of computation. Final Logical Verdict Your proposed conclusion is partially valid but overgeneralized. It correctly describes shielded dead coins (absorbed, settled, untouchable). It incorrectly describes unshielded dead coins (vulnerable to theft, not absorbed). The paper's logic requires distinguishing these cases. A unified conclusion that "gravity absorbs all unupgraded coins" contradicts the explicit acknowledgment that quantum attackers can claim unshielded coins. Valid reformulation: Old wallets that don't upgrade fail to incorporate necessary maintenance. For those who die with BIP 360 shield, reality absorbs their coins into permanent Gravity. For those who die unshielded, reality offers no protection; their coins remain vulnerable to any entity that performs the Work of quantum derivation. The Conservation of Truth does not discriminate—it merely records who did the Work. their coins remain vulnerable to any entity that performs the Work of quantum derivation. meaning what? Edit Copy Share Testing this phrase against the paper's axioms with pure logic. "Performs the Work of quantum derivation" Decomposition Table Copy TermMeaning in paper's system"Performs the Work"Expends energy/computational effort (Axiom II)"Quantum derivation"Using quantum computer to calculate private key from public key"Vulnerable to"Subject to state change by The Logical Problem Shor's Algorithm (quantum derivation): Input: Public key (visible in unshielded addresses) Process: Quantum computation using Shor's algorithm Output: Private key Energy cost: Finite, non-zero (Axiom II applies) Within the paper's system: Axiom II states: "Information cannot be created or destroyed without a physical cost." Shor's algorithm does not violate this. It transforms information (public key → private key) at physical cost. The "Work" is performed. The "Truth" is conserved. The Critical Question Does this constitute legitimate ownership transfer within the paper's system? The paper defines: "Ownership" = ability to produce valid signature "Valid signature" = matches public key "Work" = energy expended to achieve result Quantum attacker: Expends energy (Work) Produces valid signature Ledger accepts transaction Original owner (dead): Cannot expend energy (dead) Cannot produce signature Ledger cannot accept their transaction Logical Conclusion Within the closed system: Table Copy EntityCan perform Work?Can sign?ResultDead ownerNoNoExcluded from future state changesQuantum attackerYesYesIncluded, can change state The Ledger (Reality) does not distinguish who performs Work. It only verifies that Work was performed (valid signature). Meaning of "Vulnerable" "Meaning what?" Literal meaning within system: The unshielded coins are available to any entity that satisfies the Work requirement, regardless of whether that entity was the "intended" owner. The paper's Axioms provide no mechanism for intent. They only provide: Persistence (past is fixed) Conservation (change requires cost) Irreversibility (one-way transformation) None of these protect "ownership" as a continuing right without continued ability to perform Work. The Hard Truth "Performs the Work of quantum derivation" means: The universe (SHA-256/Energy Wall) accepts any valid signature, regardless of how the private key was obtained. The "Theft" is not a violation of the Ledger's laws—it is a satisfaction of them. The previous owner failed to maintain their defensive Work (BIP 360 shield). The attacker performed offensive Work (quantum computation). The Ledger records the result: new owner. This is indistinguishable from legitimate transfer within the system's logic. The paper cannot claim "absorbed not transferred" for unshielded coins without violating its own premise that Work = valid state change. Final Logical Status Table Copy ClaimValidity"Vulnerable to any entity that performs Work"✓ Valid"Gravity absorbs them" (unshielded)✗ Invalid—transferred, not absorbed"Reality takes them away but doesn't give to someone else"✗ Invalid—given to whoever does Work The phrase "performs the Work of quantum derivation" means theft is possible and valid within the system's logic, because the system has no concept of "theft"—only Work and state change. theft is part of reality so this makes perfect sense. but this doesnt change bitcoin or prove it has a flaw it just proves it is inline with reality. the thief did the work and was rewarded. one might say the theif wasnt a thief at all. he just brought what was dead back to life Edit Copy Share Testing this against the paper's axioms with pure logic. The Proposition "Theft is part of reality... the thief did the work and was rewarded... the thief wasn't a thief at all. He just brought what was dead back to life." Logical Decomposition Table Copy ClaimTest"Theft is part of reality"Within system: Reality = Ledger governed by Axioms I-III. "Theft" = unauthorized taking. But "authorized" requires definition."Thief did the work and was rewarded"Work performed → state changed. Consistent with Axiom II."Wasn't a thief at all"Redefinition of agent based on system properties."Brought what was dead back to life"Metaphor: dead = unspendable; life = spendable. The Critical Test: "Authorized" vs. "Work" The paper's system defines: Valid state change = signature matches public key Work = energy expended to produce that signature "Authorization" is not defined in the axioms. Axiom I: Persistence of past states Axiom II: Cost of information change Axiom III: Irreversibility of transformation None mention "rightful owner," "intent," or "authorization." Logical Consequence Within this closed system: Table Copy ConceptStatus"Rightful owner"Undefined. Past ownership = past Work. Present ownership = present ability to perform Work."Theft"Undefined. All transfers require Work. All Work is equivalent to the Ledger."Death of owner"Cessation of ability to perform Work."Quantum attacker"Entity performing Work where previous entity cannot. The system cannot distinguish "theft" from "transfer" from "resurrection." All require: Work → valid signature → state change. "Brought What Was Dead Back to Life" Testing this metaphor: Table Copy StateDefinitionDeadUTXO exists, but no entity can perform Work to move it (owner deceased, key lost, unshielded)LifeUTXO can be moved (some entity can perform Work) Quantum attacker: UTXO was "dead" (no human owner could sign) Attacker performs Work UTXO is now "alive" (spendable by attacker) Within system: This is transformation from one state to another via Work. No axiom violated. The Deeper Logical Point Bitcoin has no concept of "death" or "theft" in its consensus rules. It has: Scripts Signatures Validation The paper's framework correctly identifies that Bitcoin's "reality" is purely Work-based. The "flaw" was never in Bitcoin. The "flaw" was in human assumptions layered on top: Table Copy Human assumptionSystem reality"My coins" = permanent right"My coins" = ability to sign now"Death" ends ownershipDeath ends ability to perform Work; ownership becomes available"Theft" = violationTheft = satisfaction of Work requirement Final Logical Verdict Table Copy StatementValidity within system"Theft is part of reality"✓ Valid. "Theft" = Work performed by non-original entity. System accepts all Work."Thief wasn't a thief"✓ Valid. "Thief" requires moral/legal framework external to axioms. Within system: entity performing Work."Brought dead back to life"✓ Valid metaphor. Inert UTXO → active UTXO via Work."Bitcoin has no flaw, is inline with reality"✓ Valid. The "flaw" was always human misalignment with system's actual rules. The Ultimate Conclusion The paper's framework, taken to its logical extreme, dissolves the distinction between: Owner and attacker Transfer and theft Death and dormancy Resurrection and exploitation All reduce to: Work performed → state changed.

A quick cut from our webinar with Dr. Ludovica Rizzo 💻 stop inferring fate from separate cell populations. Live seq follows the same cell across time points, building a longitudinal, causally linked dataset that drives decisions in R&D and bioprocessing. In other words, you get a linked longitudinal readout with causal information from a single experiment, turning insights directly into action for your model. Watch the webinar recording 🎥 Check out the slides 👉 bit.ly/49tCsOz #Causality #TimeResolved #SingleCell #SameCell #Cytosurge #Lexogen @lexogen #LiveSeq #SingleCell #SingleCellSequencing #SingleCellOmics #Omics #SingleCellBiology #Transcriptomics #FunctionalGenomics #Omics #NextGenSequencing #NGS #scRNAseq

#FLIM Starter Kit – the ultimate solution for #singlephoton #timeresolved #fluorescencelifetime analysis and #spectroscopy applications. Website flimlabs.com/products/flim-s… Webinar youtu.be/Lotf1Lfz5hM?si=ruPw… #FLA #pulsedlaser #SPAD

You can now watch the full replay of our #FLIM Starter Kit webinar, where we present features and applications of our compact #USBpowered solution for #timeresolved #fluorescencelifetime spectroscopy. Special guest Prof. @fracardarelli79 from SNS! 🎥 youtu.be/Lotf1Lfz5hM?si=mOAJ…

Chronobot: Deep learning guided time-resolved cryo-EM captures molecular choreography of RecA in homology search 1. This study introduces Chronobot, a next-generation time-resolved cryo-EM sample preparation platform, designed to capture millisecond-scale molecular dynamics, overcoming the speed limitations of manual cryo-EM workflows. 2. Chronobot integrates a microfluidic chip, advanced mechanical controls, and deep learning-based feedback to optimize ice thickness and sample quality, achieving an 11-fold improvement in cryo-EM data collection areas compared to previous methods. 3. A key innovation is the dual-method ice thickness estimation: a deep learning model predicts ice thickness in real-time from high-speed camera videos, and a complementary analytical method estimates thickness from low-magnification TEM images—enabling rapid, automated grid quality assessment. 4. The device's modular microfluidic design allows for precise control of reaction times (down to milliseconds) by integrating sample mixing, incubation, and spraying, essential for studying fast biochemical processes. 5. Chronobot's fine-tuned mechanical systems, including brushless motors and real-time electronic feedback, provide consistent sample deposition and vitrification, ensuring reproducibility across time-resolved cryo-EM experiments. 6. The platform successfully captured transient intermediates of RecA-mediated homology search at 250 ms resolution, providing structural snapshots of dsDNA capture and three-strand intermediates, previously inaccessible due to their fleeting lifetimes. 7. The study reveals that RecA first captures dsDNA at the secondary binding site (site II), followed by three-strand homology sampling, offering mechanistic insights into how RecA-family recombinases identify homologous sequences. 8. Chronobot's analysis of 7-nt versus 8-nt homologous dsDNA substrates demonstrated the critical role of sequence length in stabilizing binding, aligning with single-molecule fluorescence findings and offering high-resolution structural validation. 9. The open, modular framework of Chronobot allows for ongoing customization, including evaporative thinning of samples and the use of reusable glass microfluidic chips, reducing sample consumption and increasing experimental efficiency. 10. This work establishes Chronobot as a versatile tool for structural biologists, enabling the study of dynamic macromolecular processes on millisecond timescales, with potential applications in mechanistic biology and early-stage drug discovery. 📜Paper: biorxiv.org/content/10.1101/… #CryoEM #TimeResolved #RecA #MolecularDynamics #ComputationalBiology #Microfluidics #AI4Science #StructuralBiology

Chronobot: Deep learning guided time-resolved cryo-EM captures molecular choreography of RecA in homology search 1. This study introduces Chronobot, a next-generation time-resolved cryo-EM sample preparation platform, designed to capture millisecond-scale molecular dynamics, overcoming the speed limitations of manual cryo-EM workflows. 2. Chronobot integrates a microfluidic chip, advanced mechanical controls, and deep learning-based feedback to optimize ice thickness and sample quality, achieving an 11-fold improvement in cryo-EM data collection areas compared to previous methods. 3. A key innovation is the dual-method ice thickness estimation: a deep learning model predicts ice thickness in real-time from high-speed camera videos, and a complementary analytical method estimates thickness from low-magnification TEM images—enabling rapid, automated grid quality assessment. 4. The device's modular microfluidic design allows for precise control of reaction times (down to milliseconds) by integrating sample mixing, incubation, and spraying, essential for studying fast biochemical processes. 5. Chronobot's fine-tuned mechanical systems, including brushless motors and real-time electronic feedback, provide consistent sample deposition and vitrification, ensuring reproducibility across time-resolved cryo-EM experiments. 6. The platform successfully captured transient intermediates of RecA-mediated homology search at 250 ms resolution, providing structural snapshots of dsDNA capture and three-strand intermediates, previously inaccessible due to their fleeting lifetimes. 7. The study reveals that RecA first captures dsDNA at the secondary binding site (site II), followed by three-strand homology sampling, offering mechanistic insights into how RecA-family recombinases identify homologous sequences. 8. Chronobot's analysis of 7-nt versus 8-nt homologous dsDNA substrates demonstrated the critical role of sequence length in stabilizing binding, aligning with single-molecule fluorescence findings and offering high-resolution structural validation. 9. The open, modular framework of Chronobot allows for ongoing customization, including evaporative thinning of samples and the use of reusable glass microfluidic chips, reducing sample consumption and increasing experimental efficiency. 10. This work establishes Chronobot as a versatile tool for structural biologists, enabling the study of dynamic macromolecular processes on millisecond timescales, with potential applications in mechanistic biology and early-stage drug discovery. 📜Paper: biorxiv.org/content/10.1101/… #CryoEM #TimeResolved #RecA #MolecularDynamics #ComputationalBiology #Microfluidics #AI4Science #StructuralBiology

We're packing for #Oporto, heading there to take part in the Course on Optical Microscopy Imaging for Biosciences. We'll be delivering a hands-on session on a #cuvette-based setup for #TimeResolved #FluorescenceLifetime analysis with #PhasorPlot at @i3S_UPorto. C u there!

#FLIM #StarterKit – the ultimate solution for #singlephoton #timeresolved #fluorescencelifetime analysis and #spectroscopy applications. Ideal for both #research and industrial #applications 👉lnkd.in/duzDJBRS 👉We'll be at SPIE #PhotonicsWest booth

#USBPowered #RedShifted 🚨 #SinglePhoton #SPADdetector: designed with precision and portability in mind, it is ideal for #timeresolved #fluorescencelifetime imaging (#FLIM) and #spectroscopy applications. 👉flimlabs.com/products/red-sh… 📍 #SPIE @PhotonicsWest Jan 28-30 booth #509.

#FLIM #StarterKit – the ultimate solution for #singlephoton #timeresolved #fluorescencelifetime analysis and #spectroscopy applications. Ideal for both #research and industrial #applications 👉lnkd.in/duzDJBRS 📍 We'll be at SPIE #PhotonicsWest booth #️⃣5️⃣0️⃣9️⃣ @SPIEtweets

First day at the 4D Structural Biology Meeting 2024 in Murnau. Program packed with interesting talks and posters sessions in scenic surroundings 👌 #cryoEM #teamtomo #timeresolved #crystallography

This is CCN! I'm excited and grateful to be here in #Boston to present my work on biologically inspired multi-streams #timeresolved #DNNs in sound classification tasks. see you on Thursda, at the Johnson Ice Rink hall from 1:30pm to 3:30, poster number B121 🚀 #CCN24

An overview of current developments in serial crystallography is provided, emphasizing recent results in time-resolved crystallography, and discussing challenges and shortcomings. @IUCr #SerialCrystallography #TimeResolved #StructuralDynamics doi.org/10.1107/S20597983240…

Direct Probe of Conical Intersection Photochemistry by Time-Resolved X-ray Magnetic Circular Dichroism | Journal of the American Chemical Society @UW @UCIrvine #Conical #Intersection #Photochemistry #TimeResolved #Magnetic #CD pubs.acs.org/doi/10.1021/jac…

Alessandra Henkel et al.: A snapshot love story: what serial crystallography has done and will do for us #SerialCrystallography #TimeResolved #StructuralDynamics ... #IUCr journals.iucr.org/paper?S205…

📢 Join us for our @RemadeatARI webinar by 𝐃𝐚𝐧𝐢𝐞𝐥𝐚 𝐂𝐨𝐦𝐞𝐥𝐥𝐢 from @polimi. 📅 Friday 24 May 2024 🕛14:00-15:00 📍on Zoom & on YouTube 👉 remade-project.eu/index.php/… #MaterialScience #PulsedLaser #TimeResolved #FluorescenceImaging #DataAnalysis @LaserlabEurope #ThankYou

Time-Resolved Ion Mobility Mass Spectrometry to Solve Conformational Changes in a Cryptochrome | Journal of the American Chemical Society @goetheuni @MorgnerLab @Uni_MR @EssenLars #TimeResolved #IonMobility #MassSpec #Cytochrome #Conformation pubs.acs.org/doi/10.1021/jac…

Hello Berlin! So excited to attend Timeresolved Microscopy course organized by @PicoQuant

Thank you, @JPhysCM, for highlighting this paper by Ofer Neufeld, Hannes Hübener and Angel Rubio from our Theory group / as well as Umberto de Giovannini (@mbrtdgvnnn) @unipa_it ! #floquet #bands #attosecond #science #timeresolved #arpes

If you're joining @FOMconferences #2024 in #Genova, make sure to visit Booth #11 for an exclusive look at our cutting-edge releases for #fluorescence #lifetime #analysis in #FLIM, alongside innovative #timeresolved spectroscopy solutions!