DETAILED ACTION
National Stage Application
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The listing of references in the specification (e.g., pg. 2, lines 27+) is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, “the list may not be incorporated into the specification but must be submitted in a separate paper”. Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The drawings are objected to as failing to comply with 37 CFR 1.437 and PCT Rule 11.13(l) because they do not include the following reference sign(s) 24 and 25 mentioned in the description and Figs. 10 and 11 do not include reference signs 21 and 22 mentioned in pg. 18, lines 5+ and lines 16+. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim(s) 1-17 is/are objected to because of antecedent basis informalities. Appropriate correction is required.
Claim Interpretation
The specification (e.g., see “… enhancing wavelength in the near infrared, and preferably between 700 and 1000 nm …” on pg. 6, lines 8+) serves as a glossary (MPEP § 2111.01) for the claim term “near infrared”.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(B) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of pre-AIA 35 U.S.C. 112, second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim(s) 4, 5, and 15 is/are rejected under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
Claim 4 recites the limitation “the illumination period Pi” in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 4 recites the limitation “the half-life period P1/2” in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 4 recites the limitation “the sole exciting illumination” in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 5 recites the limitation “the illumination period” in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 15 recites the limitation “the optimum enhancing wavelength λ+” in line next to the last line. There is insufficient antecedent basis for this limitation in the claim.
Claim(s) dependent on the claim(s) discussed above is/are also indefinite for the same reasons.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the [fifth paragraph of 35 U.S.C. 112 (pre-AIA )], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim(s) 9 is/are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
The limitation “the exciting and enhancing illuminations are continuous illuminations” recited in claim 9 does not appear to further limit or include the limitations “the fluorescence signal emitted by the fluorescent protein has decayed to γ times its initial value” and “γ is inferior to 1” recited in claim 1.
Claim(s) dependent on the claim(s) discussed above is/are also of improper dependent form for the same reasons. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were effectively filed absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned at the time a later invention was effectively filed in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102 of this title, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1-17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jablonski (Optically modulated fluorescent proteins, Thesis (August 2014), 193 pages).
In regard to claim 1, Jablonski discloses a method comprising:
(a) illuminating at least a region of a sample with an exciting light beam at an exciting wavelength λe being in an absorption band of a fluorescent protein, with a given exciting intensity Ie according to fluorescence microscopy technique and lower than 10 kW/cm2 (e.g., “… proteins may also have dark state action spectrums far into the red region. Studies were carried out with Padron*, EYFP, and Venus solutions and excited with a 476 nm primary laser (2 kW/cm2) …” in section 5.3); and
(b) illuminating at least partially the same region of the sample with an enhancing light beam with an enhancing intensity I+ and an enhancing wavelength λ+, wherein the enhancing intensity I+ is equal to or higher than the exciting intensity Ie, wherein the enhancing wavelength λ+ is higher than the exciting wavelength λe (e.g., “… secondary wavelength was scanned from 594 nm to 950 nm (22 kW/cm2) …” in section 5.3),
wherein the enhancing wavelength λ+ is chosen (e.g., “… first laser (primary) is chosen so that it can adequately excite the molecule of interest. The second laser (secondary) should be longer wavelength than the emission window. Table 2.3 is a collection of the primary and secondary wavelengths and systems used for each protein class … Once the excitation sources are chosen, the lasers are aligned into the back of the microscope …” in section 2.6.1),
wherein P1,γ is a characteristic period with solely the exciting light beam at the wavelength λe and the intensity Ie after which fluorescence signal emitted by the fluorescent protein has decayed to γ times its initial value (e.g., “… fluorescence lifetime decay having a single exponential component …” in section A.1 and a first characteristic period for decay to γ = e-1 (≈1/2.71828) times an initial value can be labeled as “fluorescence lifetime”),
wherein P12,γ(λ) is a characteristic period with both the enhancing light beam at the wavelength λ+ and the intensity I+ and the exciting light beam at the wavelength λe and the intensity Ie after which fluorescence signal emitted by the fluorescent protein has decayed to γ times its initial value, and wherein γ is inferior to 1 (e.g., see “… fluorescence enhancement as a function of secondary laser modulation frequency was fit to Equation 2.10 in which m is modulation depth or enhancement, νmod is the modulation frequency, and τc is the characteristic lifetime. For this project, the characteristic frequency is defined as the frequency at which the enhancement drops to 50% of its original value.
m
=
1
1
+
2
π
ν
m
o
d
τ
c
2
Equation 2.10 … individual decays were fit to the appropriate exponential decay … (Figure 4.7 A). The decay times (1/τc) are then plotted versus the primary intensity (Figure 4.7 B) …” in Figs. 4.7A-B and sections 2.6.2.2 and 4.5.2 and a second characteristic period for decay to γ = e-1 (≈1/2.71828) times an initial value can be labeled as “decay times (1/τc)”).
The method of Jablonski lacks an explicit description of details of the “… chosen …” wavelength such as a ratio Tγ(λ)=P12,γ(λ)/P1,γ is higher than 1+(Tmax−1)/2 where Tmax is the maximum value of the ratio Tγ(λ). However, “… chosen …” wavelength details are known to one of ordinary skill in the art (e.g., see “… this work studies the optical states involved in modulation for fluorescent proteins for the determination of a possible mechanism and optimization pathway … number of frames can vary based on modulation signal, modulation frequency, and/or photostability … determining optimal enhancement and modulation frequency … other wavelengths may be more optimally absorbed by the optical dark state … another parameter that dictates its utility is the rate at which the protein can be modulated … secondary laser excitation alters relative emissive and dark state populations to establish a new steady-state fluorescence intensity in a time that directly depends on the new rates in and out of the dark state manifold …” in sections 1.1, 2.8.3.1, 3.4, 4.3, and 4.4 of Jablonski). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional chosen wavelength (e.g., comprising details such as “optimization” “based on modulation signal, modulation frequency, and/or photostability” and additional variables such as some “wavelengths may be more optimally absorbed by the optical dark state”) for the unspecified chosen wavelength of Jablonski and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional chosen wavelength (e.g., comprising details such as a ratio Tγ(λ)=P12,γ(λ)/P1,γ is higher than 1+(Tmax−1)/2 where Tmax is the maximum value of the ratio Tγ(λ)) as the unspecified chosen wavelength of Jablonski.
In regard to claim 2 which is dependent on claim 1, the method of Jablonski lacks an explicit description of details of the “… chosen …” wavelength such as the enhancing wavelength is an optimum enhancing wavelength chosen so as to maximize the ratio Tγ(λ). However, “… chosen …” wavelength details are known to one of ordinary skill in the art (e.g., see “… this work studies the optical states involved in modulation for fluorescent proteins for the determination of a possible mechanism and optimization pathway … number of frames can vary based on modulation signal, modulation frequency, and/or photostability … determining optimal enhancement and modulation frequency … other wavelengths may be more optimally absorbed by the optical dark state … another parameter that dictates its utility is the rate at which the protein can be modulated … secondary laser excitation alters relative emissive and dark state populations to establish a new steady-state fluorescence intensity in a time that directly depends on the new rates in and out of the dark state manifold …” in sections 1.1, 2.8.3.1, 3.4, 4.3, and 4.4 of Jablonski). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional chosen wavelength (e.g., comprising details such as “optimization” “based on modulation signal, modulation frequency, and/or photostability” and additional variables such as some “wavelengths may be more optimally absorbed by the optical dark state”) for the unspecified chosen wavelength of Jablonski and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional chosen wavelength (e.g., comprising details such as the enhancing wavelength is an optimum enhancing wavelength chosen so as to maximize the ratio Tγ(λ)) as the unspecified chosen wavelength of Jablonski.
In regard to claim 3 which is dependent on claim 1, Jablonski also discloses that the enhancing illumination is conducted at an optimum enhancing intensity (e.g., see “… appropriate excitation intensities for the primary and secondary laser were found …” in Fig. 3.3B and section 3.4.1), wherein P12,γ’(I) is a characteristic period when illuminated by both the enhancing illumination with the enhancing wavelength λ+ and intensity I and the exciting illumination with the exciting wavelength λe and the exciting intensity Ie after which the fluorescence signal has decayed to γ times its initial value (e.g., see “… fluorescence enhancement as a function of secondary laser modulation frequency was fit to Equation 2.10 in which m is modulation depth or enhancement, νmod is the modulation frequency, and τc is the characteristic lifetime. For this project, the characteristic frequency is defined as the frequency at which the enhancement drops to 50% of its original value.
m
=
1
1
+
2
π
ν
m
o
d
τ
c
2
Equation 2.10 … individual decays were fit to the appropriate exponential decay … (Figure 4.7 A). The decay times (1/τc) are then plotted versus the primary intensity (Figure 4.7 B) …” in Figs. 4.7A-B and sections 2.6.2.2 and 4.5.2 and a third characteristic period for decay to γ = e-1 (≈1/2.71828) times an initial value can be labeled as “decay times (1/τc)”). The method of Jablonski lacks an explicit description of details of the “… appropriate excitation intensities …” such as smallest intensity, α≤2, and Tγ’(2I)≤α·Tγ’(I) that can be rewritten as P12,γ’(2I) ≤ α·P12,γ’(I). However, “… appropriate excitation intensities …” wavelength details are known to one of ordinary skill in the art (e.g., see “… this work studies the optical states involved in modulation for fluorescent proteins for the determination of a possible mechanism and optimization pathway … number of frames can vary based on modulation signal, modulation frequency, and/or photostability … determining optimal enhancement and modulation frequency … As the secondary laser intensity is increased, there is an increase in enhancement reaching somewhat of a plateau at 30 kW/cm2 … other wavelengths may be more optimally absorbed by the optical dark state … another parameter that dictates its utility is the rate at which the protein can be modulated … secondary laser excitation alters relative emissive and dark state populations to establish a new steady-state fluorescence intensity in a time that directly depends on the new rates in and out of the dark state manifold …” in sections 1.1, 2.8.3.1, 3.4, 3.4.1 4.3, and 4.4 of Jablonski). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional appropriate intensities (e.g., comprising details such as “optimization” “based on modulation signal, modulation frequency, and/or photostability” and additional variables such as “the secondary laser intensity is increased, there is an increase in enhancement reaching somewhat of a plateau at 30 kW/cm2”) for the unspecified appropriate intensities of Jablonski and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional appropriate intensities (e.g., comprising details such as smallest value of the enhancing intensity I+ respecting Tγ’(2I)≤α·Tγ’(I) where α is inferior or equal to 2 and Tγ’(I)=P12,γ’(I)/P1,γ) as the unspecified appropriate intensities of Jablonski.
In regard to claim 4 which is dependent on claim 1 in so far as understood, Jablonski also discloses that an illumination period Pi of only the exciting light beam is longer than a half-life period P1/2 of the fluorescent protein (e.g., see “… BIFL Data Analyzer is a software program that allows for the import of time correlated single photon counting (TCSPC) data and has the subsequent analysis models for fluorescence decays, anisotrophy, and autocorrelation … Figure 4.5 Single laser FCS curves of AcGFP. (A) FCS fits for varying primary intensities in the absence of the 561 nm secondary …” in Fig. 4.5 and sections 2.4 and 4.5.1).
In regard to claim 5 which is dependent on claim 1 in so far as understood, Jablonski also discloses that an illumination period is longer than 1 s (e.g., “… variety of exposure rates and total frames were investigated and in order to collect enough data for demodulation and not have significant photobleaching ~100 frames at an 80 millisecond exposure rate (total time was ~8 seconds) …” in section 3.7).
In regard to claim 6 which is dependent on claim 1, Jablonski also discloses that the exciting intensity Ie is inferior or equal to 1000 W·cm-2 (e.g., “… proteins may also have dark state action spectrums far into the red region. Studies were carried out with Padron*, EYFP, and Venus solutions and excited with a 476 nm primary laser (2 kW/cm2) while the secondary wavelength was scanned from 594 nm to 950 nm (22 kW/cm2) …” in section 5.3).
In regard to claim 7 which is dependent on claim 1, Jablonski also discloses that the enhancing intensity I+ fulfills the relation: 3Ie ≤ I+ ≤ 100Ie (e.g., 3x2 kW/cm2 ≤ 22 kW/cm2 ≤ 100x2 kW/cm2 from “… proteins may also have dark state action spectrums far into the red region. Studies were carried out with Padron*, EYFP, and Venus solutions and excited with a 476 nm primary laser (2 kW/cm2) while the secondary wavelength was scanned from 594 nm to 950 nm (22 kW/cm2) …” in section 5.3).
In regard to claim 8 which is dependent on claim 1, Jablonski also discloses that the enhancing wavelength λ+ is chosen in the near infrared (e.g., see data plotted in Fig. 5.4 and “… proteins may also have dark state action spectrums far into the red region. Studies were carried out with Padron*, EYFP, and Venus solutions and excited with a 476 nm primary laser (2 kW/cm2) while the secondary wavelength was scanned from 594 nm to 950 nm (22 kW/cm2) …” in section 5.3).
In regard to claim 9 which is dependent on claim 1 in so far as understood, the cited prior art is applied as in claim 1 above.
In regard to claim 10 which is dependent on claim 1, Jablonski also discloses simultaneous illumination of the exciting and enhancing light beams (e.g., “… first laser (primary) is chosen so that it can adequately excite the molecule of interest. The second laser (secondary) should be longer wavelength than the emission window. Table 2.3 is a collection of the primary and secondary wavelengths and systems used for each protein class … Once the excitation sources are chosen, the lasers are aligned into the back of the microscope …” in section 2.6.1).
In regard to claim 11 which is dependent on claim 1, Jablonski also discloses that the illuminations are time-modulated illuminations (e.g., “… primary and secondary laser are modulated …” in section 2.7).
In regard to claim 12 which is dependent on claim 1, Jablonski also discloses that the sample comprises living species and is maintained at a non-lethal temperature (e.g., “… Live cells must be held at 37°C. Thus, a heated stage is placed on the piezo- stage and secured to the moving part of the stage. The controller is set to 47°C because the transfer of heat from the stage to the liquid covering the cells is through the lip of plastic on the imaging dish (Figure 2.7). This results in a heterogeneous heating of the dish with a higher ~45°C temperature of solution/dish at the edge and ~37°C temperature in the 14 mm imaging area …” in section 2.8.2.1).
In regard to claim 13 which is dependent on claim 1, Jablonski also discloses that the enhancing wavelength λ+ is chosen (e.g., “… first laser (primary) is chosen so that it can adequately excite the molecule of interest. The second laser (secondary) should be longer wavelength than the emission window. Table 2.3 is a collection of the primary and secondary wavelengths and systems used for each protein class … Once the excitation sources are chosen, the lasers are aligned into the back of the microscope …” in section 2.6.1), wherein Q1 is quantity of fluorescence light emitted by the fluorescent protein during an illumination period Pi when solely illuminated by the exciting light beam at the exciting wavelength λe with the exciting intensity Ie (e.g., “… no emission is detected from secondary illumination alone …” in section 2.6.2.1), Q2(λ) is quantity of fluorescence light emitted by the fluorescent protein during the illumination period Pi when solely illuminated by the enhancing light beam with a wavelength λ at the enhancing intensity I+ (e.g., “… fluorescence intensity recorded in photon counts per second and the subscript designation is whether the illumination is with the primary only (1 laser) or both lasers (2 laser). No enhancement is equal to 1. This method of calculating enhancement is good for real-time evaluation of intensity change.
E
n
h
a
n
c
e
m
e
n
t
=
I
F
2
l
a
s
e
r
-
I
F
1
l
a
s
e
r
I
F
1
l
a
s
e
r
Equation 2.8 …” in section 4.2), Q1+2(λ) is quantity of fluorescence light emitted by the fluorescent protein during the illumination period Pi when illuminated by both the enhancing light beam with the wavelength λ and the enhancing intensity I+ and the exciting light beam with the exciting wavelength λe and the exciting intensity Ie (e.g., “… fluorescence intensity recorded in photon counts per second and the subscript designation is whether the illumination is with the primary only (1 laser) or both lasers (2 laser). No enhancement is equal to 1. This method of calculating enhancement is good for real-time evaluation of intensity change.
E
n
h
a
n
c
e
m
e
n
t
=
I
F
2
l
a
s
e
r
-
I
F
1
l
a
s
e
r
I
F
1
l
a
s
e
r
Equation 2.8 …” in section 4.2).
The method of Jablonski lacks an explicit description of details of the “… chosen …” wavelength such as a ratio R(λ)=[Q1+2(λ)−Q2(λ)]/Q1 is higher than 1+( Rmax−1)/2 wherein Rmax is the maximum value of R(λ). However, “… chosen …” wavelength details are known to one of ordinary skill in the art (e.g., see “… this work studies the optical states involved in modulation for fluorescent proteins for the determination of a possible mechanism and optimization pathway … number of frames can vary based on modulation signal, modulation frequency, and/or photostability … determining optimal enhancement and modulation frequency … other wavelengths may be more optimally absorbed by the optical dark state … another parameter that dictates its utility is the rate at which the protein can be modulated … secondary laser excitation alters relative emissive and dark state populations to establish a new steady-state fluorescence intensity in a time that directly depends on the new rates in and out of the dark state manifold …” in sections 1.1, 2.8.3.1, 3.4, 4.3, and 4.4 of Jablonski). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional chosen wavelength (e.g., comprising details such as “optimization” “based on modulation signal, modulation frequency, and/or photostability” and additional variables such as some “wavelengths may be more optimally absorbed by the optical dark state”) for the unspecified chosen wavelength of Jablonski and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional chosen wavelength (e.g., comprising details such as a ratio R(λ)=[Q1+2(λ)−Q2(λ)]/Q1 is higher than 1+( Rmax−1)/2 wherein Rmax is the maximum value of R(λ)) as the unspecified chosen wavelength of Jablonski.
In regard to claim 14, the cited prior art is applied as in claim 1 above. Jablonski discloses a method for implementing the method on a fluorescence microscopy system implementing the fluorescence microscopy technique, the system comprising an illumination system able to deliver at least two light beams in a same illumination region which each wavelength λe and λ+ may be chosen among n different illumination wavelengths λk and each intensity Ie and I+ is lower or equal to the maximum intensity Imax(λk) reachable by the illumination system at wavelength λk (e.g., “… first laser (primary) is chosen so that it can adequately excite the molecule of interest. The second laser (secondary) should be longer wavelength than the emission window. Table 2.3 is a collection of the primary and secondary wavelengths and systems used for each protein class … Table 2.3 Excitation Sources for Optical Modulation … 700-950 … Once the excitation sources are chosen, the lasers are aligned into the back of the microscope (Figure 2.2). Normally, the secondary laser intensity will need to be dynamically changed at a specified frequency. To do this, a modulator is placed in the path of the secondary laser as it is aligned to the microscope. The modulator can be a shutter, chopper wheel, or electro-optical modulator (EOM) as shown in Figure 2.2 …” in section 2.6.1), the method comprising:
(a) illuminating a region of a sample containing several molecules of the fluorescent protein with an exciting light beam at an exciting wavelength λe being both in an absorption band of the fluorescent protein and among the n different illumination wavelengths, with a given intensity Ie according to the fluorescence microscopy technique (e.g., “… first laser (primary) is chosen so that it can adequately excite the molecule of interest. The second laser (secondary) should be longer wavelength than the emission window. Table 2.3 is a collection of the primary and secondary wavelengths and systems used for each protein class … Table 2.3 Excitation Sources for Optical Modulation … 700-950 … Once the excitation sources are chosen, the lasers are aligned into the back of the microscope(Figure 2.2). Normally, the secondary laser intensity will need to be dynamically changed at a specified frequency. To do this, a modulator is placed in the path of the secondary laser as it is aligned to the microscope. The modulator can be a shutter, chopper wheel, or electro-optical modulator (EOM) as shown in Figure 2.2 …” in section 2.6.1) and measuring the quantity of fluorescence light emitted by the fluorescent protein during an illumination period Pi (e.g., “… performed with primary-only and with dual-laser excitation. Each time trace for the fluorescent protein is collected until photobleaching occurs …” in section 2.5),
(b) determining an enhancing wavelength λ+ for an enhancing illumination as the enhancing wavelength λ+ which is higher than the exciting wavelength λe and among the n illumination wavelengths (e.g., “… first laser (primary) is chosen so that it can adequately excite the molecule of interest. The second laser (secondary) should be longer wavelength than the emission window. Table 2.3 is a collection of the primary and secondary wavelengths and systems used for each protein class … Table 2.3 Excitation Sources for Optical Modulation … 700-950 … Once the excitation sources are chosen, the lasers are aligned into the back of the microscope(Figure 2.2). Normally, the secondary laser intensity will need to be dynamically changed at a specified frequency. To do this, a modulator is placed in the path of the secondary laser as it is aligned to the microscope. The modulator can be a shutter, chopper wheel, or electro-optical modulator (EOM) as shown in Figure 2.2 …” in section 2.6.1),
wherein P1,γ is a characteristic period with the sole exciting light beam at wavelength λe and intensity Ie after which the fluorescence signal has decayed to γ times its initial value (e.g., “… fluorescence lifetime decay having a single exponential component …” in section A.1 and a first characteristic period for decay to γ = e-1 (≈1/2.71828) times an initial value can be labeled as “fluorescence lifetime”),
wherein P12,γ(λ) is characteristic period with both the enhancing illumination at wavelength λ+ and intensity I+ and the exciting illumination at wavelength λe and intensity Ie after which the fluorescence signal has decayed to γ times its initial value; and γ is inferior to 1 (e.g., see “… fluorescence enhancement as a function of secondary laser modulation frequency was fit to Equation 2.10 in which m is modulation depth or enhancement, νmod is the modulation frequency, and τc is the characteristic lifetime. For this project, the characteristic frequency is defined as the frequency at which the enhancement drops to 50% of its original value.
m
=
1
1
+
2
π
ν
m
o
d
τ
c
2
Equation 2.10 … individual decays were fit to the appropriate exponential decay … (Figure 4.7 A). The decay times (1/τc) are then plotted versus the primary intensity (Figure 4.7 B) …” in Figs. 4.7A-B and sections 2.6.2.2 and 4.5.2 and a second characteristic period for decay to γ = e-1 (≈1/2.71828) times an initial value can be labeled as “decay times (1/τc)”),
wherein setting the system so that when using the fluorescent protein, the illumination system delivers at least: an exciting beam having the exciting wavelength λe and the exciting intensity Ie, an enhancing beam having the enhancing wavelength λ+ and the maximum enhancing intensity Imax(λ+) reachable for the enhancing wavelength λ+ (e.g., “… first laser (primary) is chosen so that it can adequately excite the molecule of interest. The second laser (secondary) should be longer wavelength than the emission window. Table 2.3 is a collection of the primary and secondary wavelengths and systems used for each protein class … Table 2.3 Excitation Sources for Optical Modulation … 700-950 … Once the excitation sources are chosen, the lasers are aligned into the back of the microscope (Figure 2.2). Normally, the secondary laser intensity will need to be dynamically changed at a specified frequency. To do this, a modulator is placed in the path of the secondary laser as it is aligned to the microscope. The modulator can be a shutter, chopper wheel, or electro-optical modulator (EOM) as shown in Figure 2.2 …” in section 2.6.1).
The method of Jablonski lacks an explicit description of details of the “… chosen …” wavelength such as maximizes a ratio Tγ(λ)=P12,γ(λ)/P1,γ. However, “… chosen …” wavelength details are known to one of ordinary skill in the art (e.g., see “… this work studies the optical states involved in modulation for fluorescent proteins for the determination of a possible mechanism and optimization pathway … number of frames can vary based on modulation signal, modulation frequency, and/or photostability … determining optimal enhancement and modulation frequency … other wavelengths may be more optimally absorbed by the optical dark state … another parameter that dictates its utility is the rate at which the protein can be modulated … secondary laser excitation alters relative emissive and dark state populations to establish a new steady-state fluorescence intensity in a time that directly depends on the new rates in and out of the dark state manifold …” in sections 1.1, 2.8.3.1, 3.4, 4.3, and 4.4 of Jablonski). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional chosen wavelength (e.g., comprising details such as “optimization” “based on modulation signal, modulation frequency, and/or photostability” and additional variables such as some “wavelengths may be more optimally absorbed by the optical dark state”) for the unspecified chosen wavelength of Jablonski and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional chosen wavelength (e.g., comprising details such as maximizes a ratio Tγ(λ)=P12,γ(λ)/P1,γ) as the unspecified chosen wavelength of Jablonski.
In regard to claim 15 which is dependent on claim 14, Jablonski also discloses choosing an optimum enhancing intensity for the enhancing intensity I+ (e.g., see “… appropriate excitation intensities for the primary and secondary laser were found …” in Fig. 3.3B and section 3.4.1), wherein P12,γ’(I) is a characteristic period when illuminated by both the enhancing illumination with the enhancing wavelength λ+ and intensity I and the exciting illumination with the exciting wavelength λe and the exciting intensity Ie after which the fluorescence signal has decayed to γ times its initial value (e.g., see “… fluorescence enhancement as a function of secondary laser modulation frequency was fit to Equation 2.10 in which m is modulation depth or enhancement, νmod is the modulation frequency, and τc is the characteristic lifetime. For this project, the characteristic frequency is defined as the frequency at which the enhancement drops to 50% of its original value.
m
=
1
1
+
2
π
ν
m
o
d
τ
c