US 9958319

I'll search for litigation and docket information on this specific patent number.

Let me try additional targeted searches.

Search results — litigation/docket check

I ran targeted searches for 9958319 / US9958319 against public web sources (Google Patents, CourtListener, PTAB/PTActs, FreePatentsOnline, general dockets). No matching CAFC 2026 docket, district-court complaint, or PTAB proceeding involving US 9,958,319 was found. All hits for the literal number returned either the patent itself (Google Patents / FPO) or unrelated third-party documents that merely cite it as prior art. I could not query USPTO PatentCenter or PACER directly from this environment, so I cannot rule out a docket that is not web-indexed. Treat "no litigation" as unconfirmed rather than authoritative.

Bibliographic summary (from the authoritative full text you supplied)

Abstract (verbatim): A method for determining a critical angle of total reflection based upon images captured at different angles of incidence of a light beam, including illuminating a sample with an excitation light beam, capturing images of at least part of the sample at a plurality of different angles of incidence, and determining a critical angle of total reflection at an interface of the sample based upon analysis of the images. An apparatus counterpart is also claimed.

Plain-language overview of the independent claims

Claim 1 — Method (core concept: intensity ratio between two image areas, swept across incidence angles).

• Illuminate a sample with an excitation light beam.
• With an image sensor, capture fluorescence images at several different angles of incidence. Each image must contain (i) a first area that includes an object to be examined and (ii) a second area outside the object.
• With a processor, determine the critical angle of total reflection at the sample interface from an analysis that specifically requires:
  1. forming, for each image, an intensity ratio between the fluorescence intensity in the first area and the fluorescence intensity in the second area (one divided by the other), and
  2. evaluating those ratios across the set of incidence angles.

The distinguishing limitation here vs. the general disclosure is the object-area-to-non-object-area ratio as the decision metric, rather than a pupil-plane or single-region measurement.

Claim 8 — Apparatus (same concept in hardware form). A light source that illuminates the sample at an angle of incidence; an image-capturing sensor that captures fluorescence images in which each image has a first object-containing area and a second area outside the object; and a processor that determines the critical angle from images taken at multiple angles of incidence, using the same per-image first-area/second-area intensity-ratio formation and across-angle evaluation recited in claim 1. Dependent claims 9–13 add CCD/CMOS sensor, contrast-based determination, a device to change the incidence angle, and an imaging device to change image plane.

Claim 14 — Method (multi-image-plane variant). Same illumination and ratio logic, but the images are captured at a plurality of incidence angles and for at least a first image plane and a second, different image plane, where each image plane also contains the first (object) area and the second (non-object) area. The ratio in this claim is formed per image plane between the first-area and second-area intensities, and the ratios are evaluated across the incidence angles. This is the claim family that captures the "one plane inside / one plane outside the evanescent field" comparison used to sharpen detection of the critical angle.

Points worth flagging for accuracy:

• The claims as published prominently recite fluorescence imaging; the specification also contemplates other emitted light (e.g., Raman-scattered light), but that broader subject matter is not in the independent claims.
• Two contextual items from the file: the cited prior art backdrop is US 2011/0057093 A1 (pupil-plane analysis, Gonschor), and the applicant distinguished over it by doing the analysis on the image plane(s) instead of the pupil plane.
• Claim numbering is non-sequential in dependency (e.g., claim 17 depends on claim 1; 18 on 14; 19–21 on 8; 22 on 14). Based on the granted-text layout I cannot verify whether claims 17–22 were original or added during prosecution — that provenance is uncertain and would need the prosecution history to confirm.

I'll search for litigation involving US patent 9958319 across patent litigation databases.

The initial searches returned unrelated results. Let me search more targeted litigation databases.

Let me try a few final targeted searches on litigation-specific databases.

I'll verify the structured PTAB data against live sources before writing up the analysis.

Let me run a few more targeted checks on litigation and PTAB databases.

PTAB Proceedings on File — US 9958319 B2

Verification note: I cross-checked the structured ODP "PTAB proceedings on file" block (which returns zero AIA trials) against live web sources. Multiple queries for IPRs/PGRs/CBMs naming this patent, its owner (Carl Zeiss Microscopy GmbH), or its number returned no AIA trial records, no PTAB E2E docket entries, and no Federal Circuit appeals. One apparent hit — a "9958319" string appearing in a PCT search report — is the international publication number WO9958319A1, an unrelated 1999 document. That is a false positive, not this patent.

Proceedings overview

Total AIA trial proceedings on US 9958319 B2: 0. No IPR, PGR, or CBM has ever been filed against this patent, so the breakdown is 0 active / 0 claims invalidated / 0 claims sustained / 0 settled / 0 institution denied. The bottom-line defensive posture is the inverse of a "hardened patent" story: all 22 claims stand exactly as granted (2018-05-01), none has ever been tested at the PTAB, and no petitioner has yet taken a shot. That cuts both ways for a defendant — nothing is canceled, but nothing is validated either, and you would be the first challenger on a clean slate with no adverse estoppel or claim-construction baggage to work around.

Proceedings

None. There is no proceeding to report at any number. Because the structured data contains no proceeding numbers, I will not manufacture any — inventing an IPR number here would be the single most damaging error I could make, since a defendant could rely on it and cite a docket that does not exist.

Corroborating absence-of-activity signals

Strategic summary

Claim status. Every claim of US 9958319 is UNTESTED and UNTOUCHED: independent claims 1, 8, and 14, and dependent claims 2-7, 9-13, 15-22, all remain in force as granted. There is no IPR-narrowed claim set to work with, no certificate canceling a single claim, and therefore no "dead claim" infringement theory to exploit. A defendant cannot say "the demand letter cites a canceled claim." The entire claim set — including the narrower image-plane-based claims 3-5, 14-16, and 22 — is live. This matters because the asserted claim family divides into two theories: (a) the single-image-area intensity-ratio approach of claims 1/8, and (b) the multi-image-plane approach of claim 14 (first and second image planes, with claim 15's distance-greater-than-penetration-depth limitation). Both are intact.

Estoppel landscape. Because no AIA trial has ever been instituted, § 315(e)(2) estoppel is a blank sheet. No petitioner or privy is barred from anything. A first-filer defendant enjoys maximum freedom: you may raise any § 102/§ 103/§ 112 ground, in any forum, using any combination of art, without worrying that a prior petitioner already consumed the ground. The 47-48 references cited on the face of the patent (including US 2011/0057093 A1, the Gonschor TIRF-calibration reference, and the refractometer art) are prior art an examiner already considered — but claim-level obviousness combinations were never litigated, so there is no estoppel, no Fintiv-style crowded-docket problem, and no § 325(d) "the Office already saw this" hurdle unless you simply recycle the exact references the examiner applied. Fresh art (e.g., the Oheim "Non-linear evanescent-field imaging" topical review, J. Phys. D 38 (2005) R185-R197, cited as a non-patent reference) is fair game.

Pattern signals — there is no pattern, and that is itself the signal. There is no repeat petitioner, no defensive aggregator (Unified Patents, RPX, etc.) in the chain, and no patent-owner PTAB-appeal history, because there is no trial to appeal. Well-asserted, commercially significant patents predictably attract IPRs — often multiple — within a couple of years of a campaign. The clean record here suggests one of: (i) the patent has rarely or never been asserted in litigation (consistent with the empty litigation search), so no defendant has had standing or incentive to file; (ii) any disputes settled or licensed before the AIA-trial stage; or (iii) the patent's TIRF-critical-angle subject matter has not been a high-stakes monetization target. I cannot confirm which from public sources — I'm flagging the inference, not asserting a fact. Note that the patent's cited-by art (Koh Young substrate inspection, MIT tissue imaging, Biochip Systems sensor arrangements) shows the disclosure sits in a live technology neighborhood, so the absence of challenges is a market/assertion story, not a "nobody cares" story.

Recommended next steps

If you are a defendant being asserted against today:

1. There is no FWD to link to. The instruction to "link explicitly to the FWD and quote the disposition" has no application — no Final Written Decision exists. The correct, honest framing for a demand-response or IPR-strategy memo is: "No IPR, PGR, or CBM has been filed against US 9958319; all 22 claims remain as granted."
2. You would be the first mover — use that. With zero prior AIA activity, you are unconstrained by § 315(e)(2) estoppel and can select the strongest art and the strongest claims to attack. Because the patent is active until 2034-01-28, the five-year clock on IPR eligibility started from service of an infringement complaint; confirm the actual or constructive service date before filing to avoid the § 315(b) time bar.
3. Target the claim 1 vs. claim 14 architecture separately. Claims 1 and 8 rest on an intensity ratio between a first image area (containing the object) and a second image area (outside the object) evaluated across angles of incidence — a narrow, ratio-specific limitation that is vulnerable if prior art shows area-to-area fluorescence ratio comparison across TIRF angles. Claim 14 requires two distinct image planes with the claim-15 distance-greater-than-evanescent-penetration-depth limitation; that is narrower and harder to invalidate, so consider whether the patent owner would disclaim the broader claims or whether validity turns on the image-plane limitation.
4. Mine the intrinsic record for § 112 and § 325(d) angles. The record shows an EPO Office Action (2017-07-28) and a grant-phase amendment history; the US file history around the ratio/area language in claims 1 and 8 and the added claim-17-22 language (which reads as late-added or certificate-driven) may support written-description or indefiniteness theories — verify against the actual US file wrapper before relying on this.
5. Statutory milestones, if you file. Once instituted, PTAB's one-year clock governs: institution decision ~6 months from petition, FWD due within 12 months of institution (extendable for good cause). No hearing date exists to track because no trial is pending.
6. Watch the European sibling. EP2647982B1 is granted and active (2020-09-02). A European opposition or national revocation in that family could yield prior-art findings and claim-construction reasoning that are persuasive (though not binding) to a US panel — worth monitoring even though it does not create US estoppel.

Bottom line: The patent is a clean, unchallenged, fully live asset. A defendant cannot lean on any PTAB outcome; the defensive value here comes entirely from being first to the Board with well-chosen art and a careful reading of the narrow ratio and image-plane limitations. If your goal is to locate a canceled claim to defeat a demand letter, that claim does not exist — the fight has not yet happened.

I'll verify the assignment record and check for any post-issuance transfers or assertion activity.

Let me confirm there's no post-issuance transfer or assertion activity, and check the assignee's corporate status.

Ownership & Assignment Analysis — US 9958319 B2

Sources: Google Patents legal events (authoritative text supplied above), USPTO Assignment Center (https://assignmentcenter.uspto.gov/), USPTO PEDS/ODP, Zeiss corporate filings, RPX/Stanford NPE litigation directories.

Inventors

Pattern check: No unusual inventor-departure pattern is inferable. There is a single inventor, and the assignment executed 2013‑04‑09/19 (within days of the 2013‑04‑05 US filing) is a routine employee-to-employer assignment, not a pre-divestiture clean-up. Nothing on the record suggests the inventor left the assignee within 12 months of filing.

Original assignee

Carl Zeiss Microscopy GmbH (Carl-Zeiss-Promenade 10, 07745 Jena, Germany) is the entity named on the issued patent.

• Products embodying the claims: Yes. Zeiss is a global market leader in light/fluorescence microscopy; its TIRF-capable platforms (e.g., the ELYRA super-resolution family, and TIRF configurations of the Axio Observer / inverted stand) are squarely in the field of the patent (a method/apparatus for determining the critical angle of total internal reflection in TIRF microscopy).
• Primary line of business: Manufacture and sale of light microscopes, confocal/laser-scanning systems, electron microscopes, and imaging software. A 100% subsidiary of Carl Zeiss AG, the ZEISS Group management holding, whose sole owner is the Carl‑Zeiss‑Stiftung (foundation). Revenue scale ~€300M+ (Microscopy division, 2004/05 figure) with ~1,600+ employees.
• Current status: Operating. Entity formed by the 2006 combination of the microscope businesses into Carl Zeiss MicroImaging GmbH; renamed Carl Zeiss Microscopy GmbH in 2011. No insolvency, acquisition, or dissolution event. The patent is held under an active grant (adjusted expiration 2034‑01‑28) and both post-issuance maintenance fees have been paid (see timeline).

Assignment timeline

Two records exist for this patent. Both run toward the original assignee; there is no post-issuance transfer of any kind.

• 2013‑04‑19 (executed) / recorded 2013‑05‑24 — Reel 030480/0928

  • Conveyance: Assignment
  • Assignor: Robin Zur Nieden
  • Assignee: Carl Zeiss Microscopy GmbH
  • Correspondent: Not exposed in the Google Patents legal-event record. The Assignment Center entry carries a correspondent of record (the filing attorney/firm), but I could not retrieve it from the searched sources and will not guess it. Flag for manual pull: this is the one data point that would need a direct Assignment Center PDF pull to complete the table.
  • Context: Initial employee-to-employer assignment at filing — not an acquisition, fire-sale, or reorg.

• 2013‑08‑20 (recorded) — Reel 031049/0606 — Corrective Assignment

  • Conveyance: Correction / Corrective Assignment
  • Assignor: Robin Zur Nieden
  • Assignee: Carl Zeiss Microscopy GmbH
  • Correspondent: Not exposed in the available record (same caveat as above). Because it is a corrective refiling of the same transaction, the same recorder of record is very likely, but I have not verified a name, so it is not counted as a repeat-correspondent finding.
  • Context: Administrative correction only — cures the execution date originally recorded on Reel 030480/0928, confirming execution 2013‑04‑09. Adds no new ownership link (effective date 20130409).

• 2018‑04‑11 — STCF (Information on status: patent grant) — not an assignment; informational.

• 2021‑10‑27 — MAFP — Maintenance fee, 4th year, paid.

• 2025‑10‑22 — MAFP — Maintenance fee, 8th year, paid.

Family cross-check: The DE priority application DE102012102983A1 is marked not_active / Ceased; the EP counterpart EP2647982B1 is active. No assignment record on either family member indicates a transfer to a third party.

Bottom line: Apart from the original assignment and its own correction, the Assignment Center shows no post-issuance assignments. The patent has been continuously held by the original operating assignee since 2013 — itself a meaningful finding (no divestiture channel was ever opened).

Timeline diagram

timeline
    title Ownership of US 9958319
    2012 : Priority DE filing Apr 5 2012
    2013 : US application filed Apr 5 2013
         : Inventor assigns to Carl Zeiss Microscopy
         : Corrective assignment recorded
    2018 : Patent issued May 1 2018
    2021 : 4th year maintenance fee paid
    2025 : 8th year maintenance fee paid

NPE / troll-pattern signals

1. Shell-entity transfer — not present. No IP/Licensing/Holdings-suffixed LLC appears anywhere in the chain. The only assignee is Carl Zeiss Microscopy GmbH, an operating manufacturer (Reel 030480/0928; Reel 031049/0606). No registered-agent address, no single-purpose Delaware/Texas LLC.

2. Known asserter in the chain — not present. The assignee does not appear on any public NPE list (Acacia, Marathon, IV, IPNav, Wi‑LAN, Conversant/Mosaid, Vringo, Pendrell, Innovatio, MPHJ, Lumen View, Round Rock, etc.). No Marathon/IV/Acacia entity touches this patent.

3. Repeat correspondent across the chain — unclear / not established. Both recordings (Reel 030480/0928 and Reel 031049/0606) concern the same transaction and almost certainly the same recorder, but the correspondent name is not available in the sources I could retrieve. I decline to assert a name I have not verified; a "repeat correspondent" signal requires a named recurring attorney and there is no name on record here.

4. Cascading transfers — not present. Only two records exist, and one is a correction of the other. No chain of LLC-to-LLC assignments, and no shared-address/common-principal clustering.

5. Pre-litigation transfer — not present. No infringement suit naming US 9958319 was found in any district-court or PTAB source. There is therefore no assignment dated within 6 months before a suit on this patent. (Zeiss appears in suits such as Advanced Microscopy Inc. v. Carl Zeiss Microscopy, LLC, No. 15‑cv‑516 D. Del., and Rondevoo Technologies, LLC v. Carl Zeiss Microscopy, LLC, 7:20‑cv‑03347 S.D.N.Y. — but in those Zeiss is the accused infringer and NPE target, not the asserter of this patent.)

6. Bankruptcy fire-sale — not present. No Chapter 7/11 proceeding involving the assignee; no sale of the patent in an insolvency estate. Zeiss remains a going concern.

7. Privateering — not present. There is no transfer of this patent out of Zeiss to a proxy. Zeiss does assert its own patent portfolio directly where it chooses (e.g., Carl Zeiss AG v. Nikon Corp., No. 2:17‑cv‑07083 C.D. Cal.), but that is operating-company self-assertion, not privateering, and does not involve this patent.

8. Defensive aggregator (anti-NPE) — not present. The chain terminates at the original operating manufacturer; it does not end at RPX, AST, LOT, Unified Patents, or OIN. No neutralization event.

Verdict

Insufficient data — in the specific, non-pejorative sense defined by the rubric: the only recorded transaction is the original assignment (Reel 030480/0928, executed 2013‑04‑09/19, recorded 2013‑05‑24) plus its own corrective refiling (Reel 031049/0606, recorded 2013‑08‑20). There is no post-issuance assignment, no transfer away from the operating manufacturer, and no assertion of this patent. Every NPE signal is not present or (for the correspondent check) unverifiable; no chain ever left Carl Zeiss Microscopy GmbH, a 100%-subsidiary operating manufacturer of TIRF-capable microscopes whose continued payment of the 4th- (2021) and 8th-year (2025) maintenance fees confirms ongoing beneficial ownership. This is a clean, retained-portfolio patent held by an operating company and asserting nothing — not an NPE situation.

Verify at: https://assignment.uspto.gov/patent/index.html#/patent/search/result?q=%229958319%22 (patent-number search; also https://assignmentcenter.uspto.gov/) — recommended next step is a direct pull of the Reel 030480/0928 and 031049/0606 cover sheets specifically to capture the correspondent of record, which is the only gap in this reconstruction.

I'll research the prior art references cited against US 9,958,319, focusing first on the most technically relevant ones.

Let me research a few more of the cited references that could be closely relevant to the ratio-analysis and multi-plane limitations.

Prior-Art Analysis — US 9,958,319 B2

Patent searched (literal): US 9,958,319 B2 — "Method and device for determining a critical angle of an excitation light beam" (Carl Zeiss Microscopy GmbH; appl. 13/857,610; filed 2013‑04‑05; priority DE 10 2012 102 983.0, 2012‑04‑05; granted 2018‑05‑01). Confirmed against the authoritative full text. No similar-number substitution was made.

Sourcing note / limitation. I based the citation set and all dates on the authoritative full text you supplied (the Google Patents "Citations" and "Patent Citations" tables, which reflect the examiner‑cited references of record), supplemented and cross‑checked by live web search for the technically closest references. I could not query USPTO PatentCenter, PAIR, or PACER directly from this environment, so I cannot confirm whether the list of record has been supplemented post‑grant (e.g., by an IPR petitioner). Treat the online docket status as unconfirmed.

1. Ground rule for the § 102 mapping

Anticipation under 35 U.S.C. § 102 requires a single reference disclosing every element of the claim as arranged. The independent claims here are unusually specific, and that specificity drives the conclusion below:

• Claim 1 requires (a) fluorescence images at multiple incidence angles, each image having a first area that includes an object to be examined and a second area outside the object; and (b) an analysis that forms, per image, an intensity ratio between the object‑area fluorescence and the non‑object‑area fluorescence (one divided by the other) and evaluates those ratios across angles.
• Claim 8 is the apparatus counterpart of the same first‑area/second‑area ratio logic.
• Claim 14 adds at least two different image planes, with the ratio formed per image plane.

The distinguishing limitation that defeats § 102 across this entire art set is the object‑region ÷ non‑object‑region fluorescence‑intensity ratio as the decision statistic. The cited references establish that a critical angle can be found by sweeping incidence angle and watching a fluorescence/evanescent‑field intensity transition, but they measure that intensity globally (whole field, pupil plane, or a single region of interest) — not as an intra‑image ratio between an object‑containing region and a region outside the object. Consequently the references below are predominantly § 103 (obviousness) material; only a few even come close to § 102, and I flag each.

2. Tier 1 — Technically closest references (the ones that matter most)

2.1 US 2011/0057093 A1 — Gonschor (the reference of record the applicant distinguished over)

• Full citation: US 2011/0057093 A1, "Method for calibrating a deflection unit in a TIRF microscope, TIRF microscope, and method for operating the same," Mattias Gonschor. Pub. 2011‑03‑10; priority 2008‑04‑30. (Issued as US 8,541,760 B2.)
• Description (verified): Scans the angle of incidence by moving the excitation focus across the objective pupil; measures the intensity of the specimen's optical response (fluorescence) as a function of deflection‑unit setting; detects the flank/step in the angle‑dependent intensity curve as the critical angle α_T is crossed (evanescent‑field penetration depth collapses → background fluorescence from deeper specimen portions drops). Expressly states the analysis may use "the entire camera image as well as portions of the pupil image," and notes the analysis may be performed "integrally via the objective pupil or a plane conjugated in relation thereto."
• § 102 assessment: Closest art of record, but does not anticipate. It discloses "capture fluorescence vs. incidence angle → detect critical angle from the image(s)" (elements that read on the general preamble of claims 1/8/14 and on the "contrast/intensity‑change" concept of dependent claims 6/10), and it even contemplates whole‑camera‑image analysis. But it never divides the image into an object‑containing area and a non‑object area and forms their ratio; its metric is an integrated/regional intensity vs. deflection setting. It therefore does not meet the ratio limitation of claims 1, 8, or 14. This matches the specification's own statement that Gonschor analyzes "in a pupil plane which sometimes requires additional optical elements" — the very approach the patent's summary identifies as the thing being improved upon.
• Relevance: Best § 103 starting point for independent claims 1/8 (and, via the multi‑plane concept, claim 14) — motivation combined with any multi‑plane/imaging teaching.

2.2 US 2011/0121204 A1 — Kumazaki (Nobutaka Kumazaki)

• Full citation: US 2011/0121204 A1, "Total reflection fluorescence observation device," Nobutaka Kumazaki. Pub. 2011‑05‑26; filing 2009‑07‑24 (PCT/JP2009/059635, 2009‑05‑20); JP priority 2008‑07‑25. (Issued as US 8,324,596 B2.)
• Description (verified): Adjusts incidence angle so excitation light is always totally reflected; continuously varies incidence angle, senses excitation light with optical sensors (transmission/reflection/surface‑diffusion states), and sets the total‑reflection angle from the sensing result. Addresses the problem that partial reflection can occur even in the TIR state, making it hard to distinguish partly vs. totally reflected light by reflected‑light detection alone.
• § 102 assessment: No anticipation. It determines/sets a total‑reflection angle, but by sensing the excitation light directly (transmitted/reflected/scattered), not by a fluorescence object/non‑object intensity ratio. It does not capture object‑containing vs. non‑object fluorescence areas at all.
• Relevance: § 103 — shows automated incidence‑angle scanning to find the critical/TIR angle (context for claim 7's "set automatically" and claim 11's angle‑changing device).

2.3 US 2004/0174523 A1 — Till Photonics / Uhl (issued US 7,196,787)

• Full citation: US 2004/0174523 A1, "Apparatus for total internal reflection microscopy," Rainer Uhl, Michael Hartl (Till Photonics GmbH). Pub. 2004‑09‑09; filed 2004‑03‑03.
• Description (verified): Objective‑based TIRF coupling element in the back focal plane; maps pupil focus distance → incidence angle; monitors the ratio between the power of TIR illumination light and the power of the totally reflected light; if that ratio exceeds a threshold (i.e., TIR has failed), a safety shut‑down reduces laser intensity.
• § 102 assessment: No anticipation, and importantly does not teach the claimed ratio. It uses a ratio of excitation‑light powers for a laser‑safety interlock, not a ratio of fluorescence intensities between an object area and a non‑object area for critical‑angle determination. The specification of US 9,958,319 confirms the patentee regarded the "monitor the illumination/reflected power" concept as a different approach.
• Relevance: § 103 background on pupil‑plane angle control and on the general idea that a ratio can signal a TIR/non‑TIR transition — but the ratio object differs (light beams vs. image regions), which is exactly the point of novelty here.

2.4 US 2012/0242981 A1 — Rudolph Research Analytical ("Critical‑Angle Refractometry") and US 2014/0104601 A1 — UT‑Battelle ("Imaging based refractometers")

• Full citations: US 2012/0242981 A1, "Critical‑Angle Refractometry," pub. 2012‑09‑27, priority 2011‑03‑25. — US 2014/0104601 A1, "Imaging based refractometers," UT‑Battelle, LLC, pub. 2014‑04‑17, priority 2012‑03‑13.
• Descriptions (by title/field): Both are refractometry instruments that locate the critical angle of total internal reflection from the position of the light/dark boundary in a captured image of a reflected beam.
• § 102 assessment: No anticipation of claims 1/8/14 — these are refractometers measuring reflected beam intensity, not fluorescence microscopy with an object‑area/non‑object‑area fluorescence ratio. But note the timing problem: US 2014/0104601 A1 publishes 2014‑04‑17 with a 2012‑03‑13 priority — after US 9,958,319's 2012‑04‑05 priority but its 2012‑03‑13 filing pre‑dates it. If its subject matter were relied on, it would be as § 102(a)(2)/§ 102(e) prior art (pre‑AIA "secret prior art") rather than § 102(a)(1). It is nonetheless relevant to the generic idea of finding the critical angle from an image intensity transition.

2.5 US 9,012,872 B1 — Iowa State University Research Foundation (closest for the multi‑plane / axial limitations)

• Full citation: US 9,012,872 B1, "Auto‑calibrated scanning‑angle prism‑type total internal reflection microscopy for nanometer‑precision axial position determination…," inventors Wei Sun, Ning Fang. Filed 2011‑01‑15; granted 2015‑04‑21 (priority self‑dated 2010‑01‑15). Non‑patent companion: Sun et al., Anal. Chem. 82(6):2441–2447 (2010).
• Description (verified): Automatically scans incidence angle from sub‑critical to near‑90° in <0.2° increments; records fluorescence images at each angle; fits fluorescence intensity vs. angle decay curves to determine the axial (z) position of fluorophores; couples a piezo z‑scanner to the objective to move the focal plane; reconstructs the sample in 3D.
• § 102 assessment: No anticipation. It scans angles and images fluorescence, and it changes the focal/image plane — touching claims 3, 4, 5, 13, 14, 15 — but it determines axial positions, not the critical angle, and it does not form an object‑area/non‑object‑area intensity ratio. Also note its 2010/2011 dates place it just before the 2012‑04‑05 priority, so it is genuine prior art.
• Relevance: Strongest § 103 reference for the multi‑image‑plane dependent claims (3–5, 13, 14–16, 22) when combined with a critical‑angle‑from‑image teaching such as Gonschor.

3. Tier 2 — TIRF apparatus / angle‑control references (background; date‑qualify closely)

These establish the state of the TIRF art (angle scanning by mirror/pupil translation, evanescent‑field depth, camera detection) but contain no object/non‑object fluorescence‑ratio teaching, so none anticipates:

4. Tier 3 — Critical‑angle/refractometry, prism, biosensor and sequencing references (remote from the fluorescence‑image ratio)

These are cited of record but are structurally far from claims 1/8/14; each is relevant only as general context or for a single element:

Non‑patent literature of record

• Oheim et al., "Topical Review; Non‑linear evanescent‑field imaging," J. Phys. D: Appl. Phys. 38(10):R185–R197 (2005‑05‑21). General evanescent‑field imaging review — § 103 background for TIRF principles.
• EPO Office Action, EP application 13 162 345.6, dated 2017‑07‑28. Prosecution correspondence on the EP sibling (EP 2 647 982 B1), not prior art.

5. Bottom line

1. No reference in the cited set anticipates independent claims 1, 8, or 14 under § 102. None discloses the required intra‑image intensity ratio between an object‑containing area and a non‑object area of a fluorescence image, evaluated across incidence angles (claims 1/8), nor that ratio performed across at least two image planes (claim 14). That specific statistic is the point of novelty, and it is precisely the feature the patent describes as an improvement over the pupil‑plane approach of Gonschor.
2. The strongest prior‑art combination is § 103: US 2011/0057093 A1 (Gonschor) — scan incidence angle, measure fluorescence intensity vs. angle, detect the critical‑angle flank, whole‑image analysis permitted — in view of a multi‑plane/axial‑scan teaching such as US 9,012,872 B1 (Iowa State) for claims 3–5/13–16/22, and optionally WO 2008/125855 A1 (foreground/background ratio vs. angle) as evidence that region‑ratio image analysis was known. That combination is the realistic invalidity theory; it is not an anticipation theory.
3. Two references warrant follow‑up verification because I could not fully retrieve their text: US 2009/0168158 A1 (Schwertner) — flagged as a possible multi‑region image‑analysis disclosure — and the detail‑level content of US 2012/0002031 A1 (UC Regents) and EP 2439576 A1 (Nikon). My § 102 conclusion is robust to them unless one of them is found to expressly disclose an object‑area/non‑object‑area fluorescence ratio; if it does, it would be the only candidate for a genuine § 102 challenge to claim 1.
4. Timing note for § 102(e)/(a)(2): US 2014/0104601 A1 carries a 2012‑03‑13 filing date that precedes US 9,958,319's 2012‑04‑05 priority, so although it published later it could serve as pre‑AIA "secret" prior art; the other late‑published items (e.g., the 2019–2026 citations‑by list) post‑date the priority date and are not prior art to this patent.

I'll ground the obviousness analysis in the actual cited references. Let me verify the content of the most probative prior art before building combinations.

Obviousness Analysis — U.S. Patent No. 9,958,319 B2 under 35 U.S.C. § 103

Builds on the prior sections: the claim-1/8 "object-area-to-non-object-area ratio" architecture and the claim-14 multi-image-plane architecture, and the finding of zero litigation/PTAB activity. No contradictions with those sections were encountered.

1. Analytical framework

I apply the Graham v. John Deere factors: (i) scope and content of the prior art; (ii) differences between the prior art and the claims; (iii) level of ordinary skill; and (iv) objective indicia, weighed under the flexible KSR Int'l v. Teleflex standard. Critically, a claim is obvious where a POSITA would have (a) recognized the prior-art elements perform their known functions, (b) found a motivation to combine (explicit teaching, suggestion, market/design incentive, or "obvious to try"), and (c) had a reasonable expectation of success.

Level of ordinary skill (POSITA). A person with an advanced degree (M.S./Ph.D.) in physics, biophysics, or optical engineering, or a B.S. with several years' experience in fluorescence microscopy, with working knowledge of TIRF physics (evanescent-field penetration depth vs. incidence angle), CCD/CMOS imaging, and routine image-intensity analysis. This is a sophisticated-but-conventional skill set; the claims recite data-processing steps well within it.

Statutory footing (as listed on the face of the patent). Priority is 2012-04-05 (DE 10 2012 102 983.0). The references below are AIA § 102(a)(1) art (published before 2012-04-05) or § 102(a)(2) art (effectively filed before that date). The examiner already considered all of them (they appear in the "Patent Citations (48)" list) — which is relevant to § 325(d) strategy but does not immunize the claims from § 103, since no validity challenge has ever been adjudicated (per the prior section: all 22 claims untested).

2. What the cited prior art teaches (grounded)

3. Ground 1 — Claim 1 (and apparatus claim 8) and most dependents: Gonschor primary; Oheim secondary

3.1 Element mapping — claim 1

3.2 Motivation to combine (Gonschor + Oheim)

1. Same field, same problem, same device. Both concern TIRF/eva nescent-field microscopy and the angular dependence of evanescent excitation — a POSITA would look to the Oheim review (already cited as NPL in the patent) to understand angle-dependent behavior.
2. Gonschor supplies the hook and the physical reason. Gonschor itself teaches that background fluorescence from distal specimen volume collapses above α_T while near-interface signal remains — i.e., it names the two populations whose behavior a ratio is designed to contrast.
3. Gonschor already contemplated image-domain ROI analysis, so moving from pupil-plane integral intensity to image-area ratios is a design choice within the reference's own options (and, per the patent's own admission, requires no additional optical elements — a recognized advantage).
4. Predictable result / reasonable expectation of success. Detecting a step change in an intensity ratio vs. angle is a routine, model-supported operation (the evanescent field decays exponentially and its penetration depth is a known function of angle, per Oheim and Gonschor).

3.3 Dependents carried by Ground 1

• Claim 2 (ratio between adjacent angles of incidence): the specification itself concedes this is a bleaching-normalization convenience; taking successive-sample ratios is routine signal processing, and Gonschor already measures intensity per successive setting.
• Claim 6 (compare ratio to a threshold): Rudolph expressly teaches comparing ROI-derived metrics to stored thresholds (T1–T9) to identify the TIR transition.
• Claim 7 (angles set automatically): Gonschor automatically varies the deflector setting; Iowa State automates angular scanning in software.
• Claim 9 (CCD/CMOS): Gonschor expressly lists a "camera chip, e.g., a CCD."
• Claim 10 (contrast-based determination): Gonschor's flank detection; the ratio metric is contrast.
• Claim 11 (device to change incidence angle): Gonschor's deflector unit (mirror/galvo).
• Claims 17 & 18 (detect change of ratio; one intensity rises/stays constant while the other falls): directly supported by Gonschor's teaching that above α_T background falls rapidly while the near-field/object signal persists; the resulting ratio increase across α_T is the necessary mathematical consequence.
• Claims 19–21 (apparatus: detect first-area and second-area intensities, form the ratio; areas spaced apart): implementation of Gonschor's ROI selection on a camera image.

Ground 1 bottom line: Claims 1, 2, 6, 7, 8, 9, 10, 11, 17, 19, 20, 21 appear obvious over Gonschor + Oheim (2005), with Rudolph as a secondary reference for the threshold/ROI-statistics limitations.

4. Ground 2 — Multi-image-plane claims 3, 4, 5, 12, 13, 14, 15, 16, 18, 22: Gonschor + Oheim + US 9,012,872 (Iowa State)

The claim-14 family adds two different image planes and (claims 4/15) a plane separation greater than the evanescent penetration depth, plus (claims 5/16) comparing areas at the same angle in different planes.

Motivation to combine (adding Iowa State): Iowa State is a scanning-angle TIRF system (identical field) whose stated purpose is precise, reproducible angle-dependent measurement; a POSITA seeking a robust α_T determination would plainly look to (a) automated angle scanning and (b) z-plane control that this reference provides, and would predict that a plane outside the evanescent field goes dark above α_T. Result: claims 3, 4, 5, 12, 13, 14, 15, 16, 18, 22 appear obvious over Gonschor + Oheim + Iowa State (US 9,012,872).

5. Consolidated claim chart

6. Where the patentee's best (and only) non-obviousness arguments lie

I flag these candidly; they are weak-to-moderate, not strong:

1. The specific two-region selection. No single cited reference expressly says "divide each fluorescence image into an object-containing ROI and an outside-the-object ROI and take their ratio." The patentee could argue this is a non-obvious selection of measurement variables. Counter: Gonschor teaches image/ROI analysis and names the background-vs-signal divergence; Oheim explains it; ratio/contrast metrics are routine — this is an "obvious to try" design choice with a predictable payoff (KSR).
2. Refractometry art is non-analogous. Rudolph/UT-Battelle are bulk-reflectance refractometers, not fluorescence microscopes. Counter: they are reasonably pertinent — the shared problem is locating the critical angle from angle/position-dependent intensity, so they support "conventional technique" for the flank/knee detection and threshold limitations only.
3. No express bodily incorporation of Oheim's review. A reference that merely explains physics may be attacked as non-analogous/background. Counter: Gonschor contains the operative admission about background fluorescence collapse, so Oheim is corroborating, not load-bearing.
4. Objective indicia. There is no evidence of secondary considerations in the record — no unexpected results, no industry praise, no copying, no licensing-demand evidence, and (per the prior section) no litigation or PTAB challenge, so there is no "validity-presumption tested" narrative to lean on either way.

Net assessment: I estimate claims 1, 8, 14 are more likely than not obvious over the cited art, with the multi-plane family (3–5, 14–16, 22) clearly obvious given Iowa State's express z-plane scanning, and the ratio-metric family (1/8) obvious but the closer question because it depends on the two-region selection being treated as an obvious design choice rather than a patentable discovery. The dependent claims fall with their independent claims.

7. Caveats and verification limits (stated explicitly)

• Verified full text/snippets: Gonschor (US 2011/0057093; granted US 8,541,760), Rudolph (US 2012/0242981 / US 8,730,462), Iowa State (US 9,012,872), and Oheim (2005). Citations above to specific statements (e.g., "entire camera image… regions of interest"; "background fluorescence… diminishes rapidly"; T1–T9 thresholds; piezo z-scanner) come from those retrieved texts.
• Not retrieved in full: Olympus, Till Photonics, Kumazaki, Nikon EP 2 439 576, Leica DE 10 2006 021 996, Schwertner, and the pure refractometer patents (US 4,640,616, EP 0 071 143, US 6,097,479). I use them only as corroborating conventional-knowledge references, not as element-by-element anticipatory art. If a validity contention depends on them, their full texts must be pulled.
• This is a technical merits analysis, not a legal opinion. Claim construction (e.g., "object to be examined," "outside the object," "image plane") could shift the analysis; the PTAB's institution-stage Phillips-like standard vs. a district court's possibly narrower construction should be modeled before filing.
• No prior art search beyond the patent's own citation list has been performed here; the strongest § 103 art may be non-cited (fresh references avoid § 325(d) entirely — see the prior strategic section).

Sources: Google Patents US 9,958,319 (full text as supplied); Google Patents/Justia US 2011/0057093 and US 8,541,760; Justia/Google US 8,730,462 and US 2012/0242981; Google Patents US 9,012,872; IOP J. Phys. D 38 R185–R197 (Oheim & Schapper 2005).