DETAILED ACTION
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 .
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 16 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The courts have described the essential question to be addressed in a description requirement issue in a variety of ways. An objective standard for determining compliance with the written description requirement is, "does the description clearly allow persons of ordinary skill in the art to recognize that he or she invented what is claimed." In re Gosteli, 872 F.2d 1008, 1012, 10 USPQ2d 1614, 1618 (Fed. Cir. 1989). Under Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1563-64, 19 USPQ2d 1111, 1117 (Fed. Cir. 1991), to satisfy the written description requirement, an applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention, and that the invention, in that context, is whatever is now claimed.
While there is a presumption that an adequate written description of the claimed invention is present in the specification as filed, In re Wertheim, 541 F.2d 257, 262, 191 USPQ 90, 96 (CCPA 1976), a question as to whether a specification provides an adequate written description may arise in the context of an original claim. An original claim may lack written description support when (1) the claim defines the invention in functional language specifying a desired result but the disclosure fails to sufficiently identify how the function is performed or the result is achieved or (2) a broad genus claim is presented but the disclosure only describes a narrow species with no evidence that the genus is contemplated. See Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1349-50 (Fed. Cir. 2010) (en banc).
In the case at hand, claim 16 recites “wherein the embedding liquid contains electrically conductive polymers and/or electrically conductive proteins.” The group of polymers and proteins have been claimed functionally, their functionality being conducting. However, the specifications do not provide any examples of “electrically conductive polymers” or “electrically conductive proteins”. Therefore, the disclosure fails to sufficiently identify how the function of “conducting” is performed by the polymers and proteins.
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 35 U.S.C. 112 (pre-AIA ), 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.
Claims 1-28 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is rejected as vague and indefinite for reciting “one nanoscale biological specimen has a maximum diameter MD, with MD ≤ 30 nm”. The claimed range includes 0 nm. It is unclear how a biological specimen could have a diameter of 0 nm. Further, claim 1 recites “wherein the thin film of the embedding liquid has an average thickness AT, with AT ≤ 30 nm”. The claimed range includes 0 nm. It is unclear how the thin film thickness could be 0 nm.
Claim 5 is rejected as vague and indefinite for failing to specify what is required to be at a preparation temperature of greater than 0°C. It is unclear if the sample is required to be greater than 0°C, or the environment in which the sample is prepared. Further, step b of claim 1 requires the step of “preparing a thin film of the embedding liquid on an electrically conductive substrate in an application zone, thereby placing the at least one nanoscale biological specimen on the substrate.” It is unclear if the embedding liquid is required to be greater than 0°C once placed on the substrate or while in transit to the substrate. For the purposes of examination claim 5 will be interpreted to mean the embedding liquid is required to be prepared at a temperature greater than 0°C and be maintained at a temperature greater than 0°C until the embedding liquid reaches the substrate.
Claim 16 is rejected as vague and indefinite for reciting “wherein the embedding liquid contains electrically conductive polymers and/or electrically conductive proteins”. The specifications recite “To exemplify the influence of the above-mentioned temperature on electron transfer, one may consider eq. 1 for typical values of ΔG 0 = -1.5 eV, and = 0.75 eV in a protein, and a temperature change from 293 K to 77 K. This temperature change would result in a reduction of the electron transfer rate by a factor of 6×108, which would basically block electron transfer in the protein.” As explained by the specifications, charge transfer in the protein is a function of temperature. It is unclear if the claimed limitation is directed at limiting the type of protein in the embedding liquid or the temperature of the liquid. For the purposes of examination claim 16 will be interpreted to limit the protein.
Claim 17 is rejected as vague and indefinite for reciting “wherein the embedding liquid contains negative stain.” It is unclear what constitutes as a “negative stain.” The specifications explain “The chemical stain may preserve the structure of the nanoscale biological specimen, in particular by maintaining a molecular layer of water around the specimen. The stain may further provide electrical conductivity to the substrate or the substrate holder” (para. [0047]). For the purposes of examination, the stain will be interpreted as any material which preserves the material or provides electrical conductance.
Claim 18 is rejected as vague and indefinite for reciting wherein, in step (c), the thin film on the substrate is first cooled down to a low temperature LT, with LT ≤ -196°C, and then warmed up again to the measuring temperature MT. Step c of claim 1 recites “c) tempering the thin film on the substrate to a measurement temperature MT, with -100°C ≤ MT ≤ -1°C”. The specifications recite “The small film thickness of the thin film has the advantage that the thin film can be tempered (in general cooled)” in paragraph 20.” As such the specification defines tempered as an act of cooling. It is unclear how the thin film can be both cooled to -100°C and also warmed to -100°C from -196°C in the same step.
Claim 21 is rejected as vague and indefinite for reciting “wherein the linker molecules attach on a first side to the substrate via a non-polar chemical group or groups, and wherein the linker molecules on a side other than the first side have a polar chemical group or groups.” It is unclear if claim 21 is intended to be understood to mean the linker molecules attach on a first side to the substrate via a non-polar chemical group or a non-polar chemical groups, and wherein the linker molecules on a side other than the first side have a polar chemical group or polar chemical groups, or if the term “groups” is intended to refer to some separate grouping. For the purposes of examination the first interpretation will be used.
Claim 23 is rejected as vague and indefinite for reciting “wherein the linker molecules comprise molecules for specially binding to the at least one nanoscale biological specimen to be investigated.” It is unclear if claim 23 is intended to limit the scope of the linker molecule or of the biological specimen. For the purposes of examination claim 23 will be interpreted to limit the scope of solely the linker molecule.
Claim 26 is rejected as vague and indefinite for reciting “repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted”. It is unclear if “the nanoscale biological specimen” referred to in the repetition step is the same nanoscale biological specimen used in step B or if each time step B is repeated a new specimen in used. For the purposes of examination the claim will be interpreted to mean the same specimen is used.
Claim 28 is rejected as vague and indefinite for reciting “with MD being a maximum diameter of the at least one nanoscale biological specimen and MD ≤ 30 nm”. The claimed range includes 0 nm. It is unclear how a biological specimen could have a diameter of 0 nm. Further, claim 1 recites “wherein the thin film has an average thickness AT, with AT ≤ 30 nm”. The claimed range includes 0 nm. It is unclear how the thin film thickness could be 0 nm.
Claim Rejections - 35 USC § 102
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 (i.e., changing from AIA to pre-AIA ) 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.
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 26-27 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Wu, Hanglong, et al. "Liquid‐phase electron microscopy for soft matter science and biology." Advanced materials 32.25 (2020): 2001582., hereinafter referred to as Wu.
Regarding claim 26, Wu teaches a method for determining a flux threshold of electrons for measuring a nanoscale biological specimen with an electron beam, the method comprising: α) investigating a first nanoscale biological specimen according to the investigation method of claim 1 wherein, in step (d), the first nanoscale biological specimen is exposed to a preselected electron dose at a first flux of the electrons, and a structure of the first nanoscale biological specimen is detected; β) investigating a further nanoscale biological specimen according to said investigation method wherein, in step (d), the further nanoscale biological specimen is exposed to the preselected electron dose at a further flux of the electrons higher than the first flux of the electrons, and a further structure of the further nanoscale biological specimen is detected, γ) repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted, thereby determining the flux threshold of electrons (Damage thresholds were determined by varying electron flux, accumulative electron flux, and using pulsed (1 s) or continuous exposure. The accumulative electron flux thresholds were ≈102–103 e− Å−2, but varied for the linear and cyclic versions, and increased for pulsed experiments. Moreover, it was shown that even when imaging at fluxes resulted in complete degradation of the peptide, self-assembled structures would appear in the liquid cell).
Regarding claim 27, Wu teaches the method according to claim 26, wherein the preselected electron dose is at least 50 electrons per (Å)2( The accumulative electron flux thresholds were ≈102–103 e− Å−2).
Claim 26 is rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Egerton, Ray F., Peng Li, and Marek Malac. "Radiation damage in the TEM and SEM." Micron 35.6 (2004): 399-409., hereinafter referred to as Egerton.
Regarding claim 26, Egerton teaches a method for determining a flux threshold of electrons for measuring a nanoscale biological specimen with an electron beam, the method comprising: α) investigating a first nanoscale biological specimen according to the investigation method of claim 1 wherein, in step (d), the first nanoscale biological specimen is exposed to a preselected electron dose at a first flux of the electrons, and a structure of the first nanoscale biological specimen is detected; β) investigating a further nanoscale biological specimen according to said investigation method wherein, in step (d), the further nanoscale biological specimen is exposed to the preselected electron dose at a further flux of the electrons higher than the first flux of the electrons, and a further structure of the further nanoscale biological specimen is detected, γ) repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted, thereby determining the flux threshold of electrons (This idea received experimental support from workers at the Cavendish laboratory (Howie et al., 1985) who irradiated p-terphenyl with electrons (of different energy E0 and with various doses) in an SEM, then determined the critical dose Dc for loss of crystallinity by observing the damaged area in a 100 keV TEM (section 9, para. [0004])).
Claim 26 is rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Keskin, Sercan, and Niels de Jonge. "Reduced radiation damage in transmission electron microscopy of proteins in graphene liquid cells." Nano letters 18.12 (2018): 7435-7440., hereinafter referred to as Keskin.
Regarding claim 26, Keskin teaches a method for determining a flux threshold of electrons for measuring a nanoscale biological specimen with an electron beam, the method comprising: α) investigating a first nanoscale biological specimen according to the investigation method of claim 1 wherein, in step (d), the first nanoscale biological specimen is exposed to a preselected electron dose at a first flux of the electrons, and a structure of the first nanoscale biological specimen is detected (Fig. 2 a); β) investigating a further nanoscale biological specimen according to said investigation method wherein, in step (d), the further nanoscale biological specimen is exposed to the preselected electron dose at a further flux of the electrons higher than the first flux of the electrons, and a further structure of the further nanoscale biological specimen is detected (Fig. 2 b), γ) repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted, thereby determining the flux threshold of electrons (The electron flux was Df =16± 2e−/A2s, and after the first image, each image was collected at a time interval of 4.0 ± 0.4 s of continuous exposure so that D increased by 64 ± 13 e−/A2 going from one to the next image (Fig. 2 caption)) (Spatial features of f = 0.20 nm−1 are thus preserved up to Dmax as indicated by the vertical dotted line representing Dmax. Damaged structure is included as gray data points (Fig. 2 caption)) (Fig. 2 Below).
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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 (i.e., changing from AIA to pre-AIA ) 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.
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, 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.
Claims 1-3, 6-8, 12, 14-16, and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Pauline Martha Gerardina (US 20240272042 A1), hereinafter referred to by Gerardina in view of Joshua Coon (US 20230282470 A1), hereinafter referred to as Coon.
Regarding claim 1, Gerardina teaches a method for investigating at least one nanoscale biological specimen, the method comprising:
preparing an embedding liquid containing the at least one nanoscale biological specimen (The interior space 104 is filled with a liquid 105 containing samples of interest. These samples of interest may include, for example, nanoparticles, biological molecules, or macromolecular assemblies, or any combination of these. (para. [0029])),
wherein the at least one nanoscale biological specimen has a maximum diameter MD, with MD ≤ 30 nm (For example, let it be assumed that the samples of interest include individual proteins, which have a nanometer size, typically less than 1 nm (para. [0035])),
preparing a thin film of the embedding liquid on an electrically conductive substrate in an application zone, thereby placing the at least one nanoscale biological specimen on the substrate, wherein the substrate is wettable for the embedding liquid in the application zone (The top graphene film 102 and the bottom graphene film 101 jointly form a boundary of the graphene liquid cell 100 (para. [0029]))
Paragraph [00112] of the specifications state “Since the substrate 4 (or the carbon foil 17 and the graphene foil 4a) are wettable.” Therefore, as evidence by the specifications the graphene substrate is wettable.
,and wherein the thin film of the embedding liquid has an average thickness AT, with AT ≤ 30 nm (The thin film has a thickness of less than 10 nm or, more specifically, less than 5 nm or, even more specifically, less than 2 nm or, yet even more specifically, less than 1 nm (para. [0049]));
wherein the at least one nanoscale biological specimen is exposed to an electron beam (The preparation of graphene liquid cells thus obtained may be used for imaging samples comprised therein by means of a transmission electron microscope. Since the liquid containing the samples to be imaged is tightly encapsulated between the two graphene films, the preparation of graphene liquid cells can be inserted into a vacuum column of the transmission electron microscope (para. [0007])).
Gerardina fails to teach c) tempering the thin film on the substrate to a measurement temperature MT, with -100°C ≤ MT ≤ -1°C; and d) measuring the at least one nanoscale biological specimen within the thin film on the substrate in an electron beam instrument at the measurement temperature MT.
However, Coon teaches c) tempering the thin film on the substrate to a measurement temperature MT, with -100°C; and d) measuring the at least one nanoscale biological specimen within the thin film on the substrate in an electron beam instrument at the measurement temperature MT (The substrate surface is at a temperature of −100° C. or less (optionally at a temperature of −150° C. or less, −175° C. or less, or −195° C. or less). As a result, a layer of an amorphous solid is formed on the surface of the substrate. The method further comprises forming an analyte beam containing charged or uncharged analyte particles to be analyzed using EM (para. [0011])).
The specifications recite “The small film thickness of the thin film has the advantage that the thin film can be tempered (in general cooled)” in paragraph 20. For the purposes of examination tempered will be interpreted to be synonymous with “cooled”.
Gerardina teaches a liquid cell containing proteins for use in TEM. Coon is directed towards protein imaging via electron microscopy. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Coon such that the thin film is tempered to a measurement temperature of -100°C and measured at said measurement temperature. The motivations for using said measurement temperature are clearly explained by Coon including: “increase image resolution, (2) decrease image acquisition time, and (3) allow for many orders of magnitude increase in sensitivity” (Coon; para. [0043]).
Further, optimizing measurement temperature is well within the bounds of normal experimentation. See MPEP 2144.05 II (A). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Furthermore, “[a] particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation.” In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977). In the case at hand, Coon teaches that “[t]he present invention provides methods and instruments for preparing samples of an analyte with amorphous solids for use in cryo-electron microscopy (cryo-EM). The amorphous solids, which protect the analyte from radiation damage and dehydration during imaging, must remain transparent to the electron beam during EM. This requires the amorphous solid layer (e.g., the ice layer) be thin, on the order of the same thickness of the molecules to be analyzed, and the solid must be amorphous” (para. [0051]). As such, Coon identifies measurement temperature as a variable which achieves a recognized result, i.e., optimizing image resolution and reducing radiation damage. Accordingly, it would have been obvious to one of ordinary skill in the art before the effective time of filing to optimize measurement temperature in Gerardina to meet the measurement temperature of -100°C since it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.
Regarding claim 2, Gerardina fails to teach the method according to claim 1, wherein the measurement temperature MT is less than a freezing temperature of the embedding liquid in bulk.
However, Coon teaches wherein the measurement temperature MT is less than a freezing temperature of the embedding liquid in bulk (The present invention provides methods and instruments for preparing samples of an analyte with amorphous solids for use in cryo-electron microscopy (cryo-EM) (para. [0051])).
In order to produce an amorphous solid the MT is required to be less than a freezing temperature of the embedding liquid in bulk.
Regarding claim 3, Gerardina teaches the method according to claim 1, wherein AT ≤ MD (The thin film has a thickness of less than 10 nm or, more specifically, less than 5 nm or, even more specifically, less than 2 nm or, yet even more specifically, less than 1 nm (para. [0049])) For example, let it be assumed that the samples of interest include individual proteins, which have a nanometer size, typically less than 1 nm (para. [0035])).
Regarding claim 6, Gerardina teaches the method according to claim 1, further comprising, during step (b), placing an initial amount of embedding liquid on the substrate, with the initial amount being larger than needed for the thin film, and then reducing the amount of embedding liquid on the substrate until only the thin film remains (Accordingly, when the droplet is deposited on the bottom graphene film that is present on the TEM grid, at least a portion of the droplet is sandwiched between the top graphene film and the bottom graphene film. An excess amount of the sample-and-nanoparticle dispersion may then be removed by means of, for example, an absorbent sheet (para. [0041])).
Regarding claim 7, Gerardina teaches the method according to claim 6, wherein reducing the amount of embedding liquid includes placing a liquid absorbing medium in contact with the embedding liquid, and/or evaporating some of the embedding liquid (Accordingly, when the droplet is deposited on the bottom graphene film that is present on the TEM grid, at least a portion of the droplet is sandwiched between the top graphene film and the bottom graphene film. An excess amount of the sample-and-nanoparticle dispersion may then be removed by means of, for example, an absorbent sheet (para. [0041])).
Regarding claim 8, Gerardina teaches the method according to claim 1, wherein the application zone has an application zone area AZA and wherein, during step (b), for preparing the thin film of embedding liquid in the application zone, an initial volume SV of embedding liquid is placed on the substrate in the application zone, with SV ≤ AT*AZA.
The application zone area is interpreted to be the area the sample will eventually occupy after dispensing.
Gerardina inherently teaches the claimed limitation. The thickness of the sample times the area that sample will occupy after being dispenses will always equal the volume of the sample.
Regarding claim 12, Gerardina teaches the method according to claim 1, wherein experimental parameters applied during step (b) in order to prepare the thin film with the average thickness AT are determined in advance in calibration experiments, said experimental parameters including at least one of: a temperature of an atmosphere surrounding the substrate, a pressure of an atmosphere surrounding the substrate, a humidity of an atmosphere surrounding the substrate, a composition of an atmosphere surrounding the substrate, a temperature of the substrate, a type of a liquid absorbing medium, a contact time with a liquid absorbing medium, a contact pressure to a liquid absorbing medium, an evaporation time, an initial volume SV of embedding liquid placed in the application zone, an initial amount of embedding liquid placed on the substrate, a touchdown pressure of an application tip, and a discharging flow of embedding liquid through a microchannel (A technique described in patent publication WO2021123458 may be used for that purpose. According to this technique, a droplet of the sample-and-nanoparticle dispersion carries a top graphene film. Accordingly, when the droplet is deposited on the bottom graphene film that is present on the TEM grid, at least a portion of the droplet is sandwiched between the top graphene film and the bottom graphene film. An excess amount of the sample-and-nanoparticle dispersion may then be removed by means of, for example, an absorbent sheet (para. [0041])).
Gerardina references WO2021123458 for using a type of liquid absorbing medium. Therefore, a parameter applied to speb b was determined in advance in calibration experiments.
Regarding claim 14, Gerardina teaches the method according to claim 1, wherein the substrate comprises a graphene foil or carbon foil (graphene liquid cell 100).
Regarding claim 15, Gerardina teaches the method according to claim 1 wherein, after step (b) and before step (c), the thin film of the embedding liquid is covered with a covering substrate (top graphene film 102).
Regarding claim 28, Gerardina teaches a sample for investigating at least one nanoscale biological specimen in an electron beam instrument, the sample comprising: - an electrically conductive substrate (The top graphene film 102 and the bottom graphene film 101 jointly form a boundary of the graphene liquid cell 100 (para. [0029])), and - a thin film of an immobilized embedding liquid on the substrate in an application zone, wherein the immobilized embedding liquid contains the at least one nanoscale biological specimen, with MD being a maximum diameter of the at least one nanoscale biological specimen and MD ≤ 30 nm (For example, let it be assumed that the samples of interest include individual proteins, which have a nanometer size, typically less than 1 nm (para. [0035])), wherein the thin film has an average thickness AT, with AT ≤ 30 nm (The thin film has a thickness of less than 10 nm or, more specifically, less than 5 nm or, even more specifically, less than 2 nm or, yet even more specifically, less than 1 nm (para. [0049])), wherein the substrate is wettable for the embedding liquid in the application zone.
Paragraph [00112] of the specifications state “Since the substrate 4 (or the carbon foil 17 and the graphene foil 4a) are wettable.” Therefore, as evidence by the specifications the graphene substrate is wettable.
Gerardina fails to teach wherein the sample is at a measurement temperature MT, with -100°C ≤ MT ≤ -1°C.
However, Coon teaches wherein the sample is at a measurement temperature MT, with -100°C ≤ MT ≤ -1°C (The substrate surface is at a temperature of −100° C. or less (optionally at a temperature of −150° C. or less, −175° C. or less, or −195° C. or less). As a result, a layer of an amorphous solid is formed on the surface of the substrate. The method further comprises forming an analyte beam containing charged or uncharged analyte particles to be analyzed using EM (para. [0011])).
Gerardina teaches a liquid cell containing proteins for use in TEM. Coon is directed towards protein imaging via electron microscopy. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Coon such that the thin film is cooled to a measurement temperature of -100°C and measured at said measurement temperature. The motivations for using said measurement temperature are clearly explained by Coon including: “increase image resolution, (2) decrease image acquisition time, and (3) allow for many orders of magnitude increase in sensitivity” (Coon; para. [0043]).
Further, Optimizing measurement temperature is well within the bounds of normal experimentation. See MPEP 2144.05 II (A). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Furthermore, “[a] particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation.” In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977). In the case at hand, Coon teaches that “[t]he present invention provides methods and instruments for preparing samples of an analyte with amorphous solids for use in cryo-electron microscopy (cryo-EM). The amorphous solids, which protect the analyte from radiation damage and dehydration during imaging, must remain transparent to the electron beam during EM. This requires the amorphous solid layer (e.g., the ice layer) be thin, on the order of the same thickness of the molecules to be analyzed, and the solid must be amorphous” (para. [0051]). As such, Coon identifies measurement temperature as a variable which achieves a recognized result, i.e., optimizing image resolution and reducing radiation damage. Accordingly, it would have been obvious to one of ordinary skill in the art before the effective time of filing to optimize measurement temperature in Gerardina to meet the measurement temperature of -100°C since it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.
Claims 4, 13, 17, 19, 24 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Gerardina in view of Coon, and in further view of Wu.
Regarding claim 4, Gerardina fails to teach the method according to claim 1, wherein, in step (d), an electron beam energy of the electron beam is between 50 eV and 300 keV, and the electron flux of the electron beam is between 0.1 electrons per Å2 per s and 50 electrons per Å2 per s.
However, Wu teaches an electron beam energy of the electron beam is 30 eV, and the electron flux of the electron beam is between 1 electrons per Å2 per s (and EF TEM, using a 30 eV slit placed at the zero-loss peak, resulted in much better SNR than BF-STEM. The electron flux amounted to 1 e− Å−2 s−1 (Fig. 2 e-h caption)).
Optimizing electron beam energy and electron flux is well within the bounds of normal experimentation. See MPEP 2144.05 II (A). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Furthermore, “[a] particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation.” In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977). In the case at hand, Wu teaches that “The extent of beam damage observed in the sample is typically proportional to the total electron dose received by the sample via destructive events, while reversible processes have an additional dependency on the electron flux. Therefore, the application of low electron flux may be used to mitigate radiation damage in case of reversible processes, leaving time for the sample to equilibrate heat, charge pairs, and charge separation” (Wu; Section 2.1.1; para. [0001]). As such, Wu identifies electron beam energy and electron flux as a variable which achieves a recognized result, i.e., minimizing radiation damage to the sample. Accordingly, it would have been obvious to one of ordinary skill in the art before the effective time of filing to optimize electron beam energy and electron flux in Gerardina to meet an electron beam energy of 30 eV and an electron flux of 1 e− Å−2 s−1 since it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.
Regarding claim 13, Gerardina fails to explicitly teach the method according to claim 1, wherein the embedding liquid comprises water with added salt.
However, Wu teaches wherein the embedding liquid comprises water with added salt (Most experiments will not involve pure water but rather a saline (section 2.1.1; para. [0003])).
Gerardina teaches a liquid cell containing proteins for use in TEM. Wu teaches methods of liquid-phase electron microscopy for biological samples. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Wu by using saline as an embedding liquid. Doing so is commonly used in the art for imaging biological samples.
Regarding claim 17, Gerardina fails to teach the method according to claim 1, wherein the embedding liquid contains negative stain (24).
However, Wu teaches wherein the embedding liquid contains negative stain (Staining with gold nanoparticles was applied to enhance the contrast in STEM (Fig. 5e caption )).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Wu such that the embedding liquid contains a negative stain. Doing so enhances the image contrast is STEM.
Regarding claim 19, Gerardina does not explicitly teach the method according to claim 1, wherein the substrate is placed on a TEM grid, with the TEM grid having a plurality of crossing grid bars defining grid windows between the crossing grid bars, and wherein, during step (b), embedding liquid is applied between grid bars in at least one grid window.
However, Wu teaches wherein the substrate is placed on a TEM grid, with the TEM grid having a plurality of crossing grid bars defining grid windows between the crossing grid bars, and wherein, during step (b), embedding liquid is applied between grid bars in at least one grid window (Fig. 3 e-h).
To be clear, both Wu and Gerardina teach graphene grids. Gerardian does not explicitly teach vars with grid windows. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Wu such that the substrate is placed on a TEM grid, with the TEM grid having a plurality of crossing grid bars defining grid windows between the crossing grid bars, and wherein, during step (b), embedding liquid is applied between grid bars in at least one grid window. The grid windows allow for the sample to be imaged with an electron beam.
Regarding claim 24, Gerardina fails to teach the method according to claim 1 wherein, in step (d), the at least one nanoscale biological specimen is exposed to an electron dose and a flux of electrons from the electron beam and wherein the flux of electrons is below a flux threshold equal to a flux level at which the structure of the at least one nanoscale biological specimen becomes distorted.
However, Wu teaches 1 wherein, in step (d), the at least one nanoscale biological specimen is exposed to an electron dose and a flux of electrons from the electron beam and wherein the flux of electrons is below a flux threshold equal to a flux level at which the structure of the at least one nanoscale biological specimen becomes distorted (Therefore, the application of low electron flux may be used to mitigate radiation damage in case of reversible processes, leaving time for the sample to equilibrate heat, charge pairs, and charge separation.[10] (section 2.1.1, para. [0001])) (For every experiment, a balance needs to be found between the minimum electron dose and electron flux on the one hand, and the required spatial and temporal resolution on the other hand (section 2.1.4, para. [0001])).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Wu such that the flux of the electrons is below a flux threshold equal to a flux level at which the structure of the at least one nanoscale biological specimen becomes distorted. Doing so mitigates radiation damage.
Regarding claim 25, Gerardina fails to teach the method according to claim 1, wherein, in step (d), the at least one nanoscale biological specimen is exposed to a preselected electron dose and a flux of electrons from the electron beam, wherein the flux of electrons is below a flux threshold, and wherein said flux threshold is determined by the following steps: α) investigating a first nanoscale biological specimen according to said method wherein, in step (d), the first nanoscale biological specimen is exposed to said preselected electron dose at a first flux of the electrons, and a structure of the first nanoscale biological specimen is detected; β) investigating a further nanoscale biological specimen according to said method wherein, in step (d), the further nanoscale biological specimen is exposed to said preselected electron dose at a further flux of the electrons higher than the first flux of the electrons, and a further structure of the further nanoscale biological specimen is detected, and γ) repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted, thereby determining said flux threshold of electrons.
However, Wu teaches wherein, in step (d), the at least one nanoscale biological specimen is exposed to a preselected electron dose and a flux of electrons from the electron beam, wherein the flux of electrons is below a flux threshold, and wherein said flux threshold is determined by the following steps: α) investigating a first nanoscale biological specimen according to said method wherein, in step (d), the first nanoscale biological specimen is exposed to said preselected electron dose at a first flux of the electrons, and a structure of the first nanoscale biological specimen is detected; β) investigating a further nanoscale biological specimen according to said method wherein, in step (d), the further nanoscale biological specimen is exposed to said preselected electron dose at a further flux of the electrons higher than the first flux of the electrons, and a further structure of the further nanoscale biological specimen is detected, and γ) repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted, thereby determining said flux threshold of electrons (Damage thresholds were determined by varying electron flux, accumulative electron flux, and using pulsed (1 s) or continuous exposure. The accumulative electron flux thresholds were ≈102–103 e− Å−2, but varied for the linear and cyclic versions, and increased for pulsed experiments. Moreover, it was shown that even when imaging at fluxes resulted in complete degradation of the peptide, self-assembled structures would appear in the liquid cell).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Gerardina by calculating a flux threshold as taught by Wu in order to optimize image resolution with minimum degradation to the sample.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Gerardina in view of Coon, and in further view of Srikanth Singamaneni (US 20200305416 A1), hereinafter referred to as Singamaneni.
Regarding claim 5, Gerardina fails to teach the method according to claim 1, wherein step (a) and step (b) are conducted at a preparation temperature PT, with PT > 0°C.
However, Singamaneni teaches wherein step (a) and step (b) are conducted at a preparation temperature PT, with PT > 0°C (NGAL-spiked artificial urine was employed as a model biospecimen. To prepare a typical ZIF-8 preserved sample, NGAL-spiked artificial urine (50 μg/ml, 25 μl) was first mixed with 2-methylimidazole solution (640 mM, 12.5 μl) and then zinc acetate solution (160 mM, 12.5 μl), and incubated at room temperature for 1 hour. Subsequently, the mixture (50 μl) was air-dried on a 0.5×2 cm Whatman 903 paper strip (which usually takes 2 hours at room temperature) (para. [0076])).
Singamaneni teaches a method of preserving protein biomarkers in biospecimen. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Singamaneni by preparing the sample and thus transferring the sample at a temperature greater than 0°C. Doing so is an effective temperature for preserving the biospecimen.
Claims 9-11, 20-21, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Gerardina in view of Christoph Ollinger (US 12656230 B2), hereinafter referred to as Ollinger.
Regarding claim 9, Gerardina fails to explicitly teach the method according to claim 1 wherein, in step (b), an initial amount of the embedding liquid is placed on the substrate in the application zone by first dipping an application tip into a supply pool of the embedding liquid containing the at least one nanoscale biological specimen, and then touching the application zone with the application tip.
However, Ollinger teaches wherein, in step (b), an initial amount of the embedding liquid is placed on the substrate in the application zone by first dipping an application tip into a supply pool of the embedding liquid containing the at least one nanoscale biological specimen, and then touching the application zone with the application tip (An example of a nanopipetting and encapsulation method according to the present invention is shown schematically in FIGS. 2A-2D. A first graphene substrate 11 is located on a support grid 12, for which only several of the structural members are shown in cross-section. As shown in FIG. 2A, pipette 20 is used to dispense a droplet 22 of solution containing a protein molecule 24 onto a region of the graphene substrate between adjacent structural members of the grid (col. 4, lines 28-36)).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Ollinger such that the liquid is placed on the substrate zone by first dipping an application tip into a supply pool of the embedding liquid containing the at least one nanoscale biological specimen, and then touching the application zone with the application tip, as taught by Ollinger. Ollinger expresses the motivation for doing so being “the protein can be placed precisely into the center of a grid opening location, which is optimal for subsequent imaging (col. 4, lines 25-27)”
Regarding claim 10, Gerardina fails to explicitly teach the method according to claim 1 wherein, in step (b), an initial amount of the embedding liquid is placed on the substrate in the application zone with an application tip having a microchannel through which the initial amount of the embedding liquid is discharged.
However, Ollinger teaches in step (b), an initial amount of the embedding liquid is placed on the substrate in the application zone with an application tip having a microchannel through which the initial amount of the embedding liquid is discharged (In the exemplary embodiment of the invention, the pipette 20 is a hollow atomic force microscopy (AFM) tip with a sub-micron sized outlet port (col. 4, lines 37-39)).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Ollinger such in step (b), an initial amount of the embedding liquid is placed on the substrate in the application zone with an application tip having a microchannel through which the initial amount of the embedding liquid is discharged, as taught by Ollinger. Ollinger expresses the motivation for doing so being “the protein can be placed precisely into the center of a grid opening location, which is optimal for subsequent imaging (col. 4, lines 25-27)”
Regarding claim 11, Gerardina fails to explicitly teach the method according to claim 1 wherein, during step (b), an initial volume of the embedding liquid placed on the substrate is between 1 fL and 2 µL.
However, Ollinger teaches, an initial volume of the embedding liquid placed on the substrate is in the femtoliter range (In this case, a hollow AFM tip is used to deposit very small volumes of fluid (e.g., in the femtoliter range) containing protein onto the graphene (col. 4, lines 21-23)).
Optimizing initial volume of the embedding liquid is well within the bounds of normal experimentation. See MPEP 2144.05 II (A). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Furthermore, “[a] particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation.” In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977). In the case at hand, Gerardina teaches that “The thickness of the graphene liquid cell 100 can thus be adapted to the samples of interest through the use of the spherical nanoparticles 106 having an appropriate diameter (para. [0031])” and (The thinness of the graphene liquid cells allows high resolution imaging of the samples (para. [0007])). As such, Gerardina identifies the volume of the liquid as a variable which achieves a recognized result, i.e., optimizing image resolution for use in a TEM. Accordingly, it would have been obvious to one of ordinary skill in the art before the effective time of filing to optimize liquid volume in Gerardina to meet the that the an initial volume of the embedding liquid placed on the substrate is between 1 fL and 2 µL since it is not inventive to dis-cover the optimum or workable ranges by routine experimentation.
Regarding claim 20, Gerardina does not teach the method according to claim 1, wherein the substrate comprises at least one local coating defining the application zone, wherein the local coating provides that the substrate is wettable for the embedding liquid in the application zone.
However, Ollinger teaches wherein the substrate comprises at least one local coating defining the application zone, wherein the local coating provides that the substrate is wettable for the embedding liquid in the application zone (In an alternative embodiment, a linker molecule may be used to bond to the molecule of interest and to the graphene substrate. In this embodiment, the linker molecule is first deposited on the graphene substrate (col. 2, lines 42-45)).
Ollinger teaches a graphene liquid cell for imaging a protein molecule. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Ollinger by depositing a linker molecule as a coating on the substrate. The linker bonds to the graphene as well as to the protein of interest such that the proteins of interest adhere to the substrate.
Regarding claim 21, Gerardina fails to teach the method according to claim 1, wherein the substrate is at least partially coated with linker molecules for linking the at least one nanoscale biological specimen to the substrate, wherein the linker molecules attach on a first side to the substrate via a non-polar chemical group or groups, and wherein the linker molecules on a side other than the first side have a polar chemical group or groups.
However, Ollinger teaches wherein the substrate is at least partially coated with linker molecules for linking the at least one nanoscale biological specimen to the substrate, wherein the linker molecules attach on a first side to the substrate via a non-polar chemical group or groups, and wherein the linker molecules on a side other than the first side have a polar chemical group or groups (In an alternative embodiment, a linker molecule may be used to bond to the molecule of interest and to the graphene substrate. In this embodiment, the linker molecule is first deposited on the graphene substrate (col. 2, lines 42-45)).
The specifications explain “[a]n advantageous variant provides that the substrate is at least partially coated with linker molecules for linking the nanoscale biological specimens to the substrate,
in particular wherein the nanoscale biological specimens are proteins and the substrate comprises a graphene foil, that the linker molecules attach on one side to the substrate via a non-polar chemical group or groups, in particular wherein the non-polar chemical group or groups are based on pyrene, pyridine, porphyrin, or amine,and that the linker molecules on their other side have a polar group or polar groups, in particular wherein the polar group or groups comprise butanoic acid-succinimidyl ester, pyrenebutyric acid (PBA) or carboxyl.” Ollinger teaches the nanoscale biological specimen are proteins and the substrate comprises graphene foil. Therefore, as evidenced by the specifications of the present disclosure, Ollinger teaches wherein the linker molecules attach on a first side to the substrate via a non-polar chemical group or groups, and wherein the linker molecules on a side other than the first side have a polar chemical group or groups.
Ollinger teaches a graphene liquid cell for imaging a protein molecule. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Ollinger by depositing a linker molecule as a coating on the substrate. The linker bonds to the graphene as well as to the protein of interest such that the proteins of interest adhere to the substrate.
Regarding claim 23, Gerardina fails to teach the method according to claim 21, wherein the linker molecules comprise molecules for specially binding to the at least one nanoscale biological specimen to be investigated.
However, Ollinger teaches teach the method according to claim 21, wherein the linker molecules comprise molecules for specially binding to the at least one nanoscale biological specimen to be investigated (In an alternative embodiment, a linker molecule may be used to bond to the molecule of interest and to the graphene substrate. In this embodiment, the linker molecule is first deposited on the graphene substrate (col. 2, lines 42-45)).
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Gerardina in view of Coon, and in further view of Neel Satish Joshi (US 20180334483 A1), hereinafter referred to as Joshi.
Regarding claim 16, Gerardina fails to teach the method according to claim 1, wherein the embedding liquid contains electrically conductive polymers and/or electrically conductive proteins.
However, Joshi teaches wherein the embedding liquid contains electrically conductive proteins (As such, the protein fibers are electrically conductive (para. [0058])).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Joshi such that the protein is electrically conductive. These proteins are capable of mediating long-range electron transport.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Akira Tanaka (US 4447374 A), hereinafter referred to as Tanaka.
Regarding claim 18, Gerardina fails to teach the method according to claim 1 wherein, in step (c), the thin film on the substrate is first cooled down to a low temperature LT, with LT ≤ -196°C, and then warmed up again to the measuring temperature MT.
However, Tanaka teaches wherein, in step (c), the thin film on the substrate is first cooled down to a low temperature LT, with LT ≤ -196°C, and then warmed up again to the measuring temperature MT (The set of the negative electrode blocks (14, 15, 20, 21) were taken out of the vacuum vessel 10 and cooled to a temperature of -190.degree. C. with liquid nitrogen. The cooled negative electrode was then set in the vacuum vessel 10 and the rock-salt specimen was placed on the specimen support 14. The vacuum vessel 10 was evacuated and the temperature of the specimen was controlled at -100.degree).
Tanaka teaches a film of a biological specimen suitable for electron microscopy. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Tanaka by cooling the specimen to -196°C and then warming the specimen to the MT. Doing so optimizes image resolution in electron microscopy.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Gerardina in view of Coon and Ollinger, and in further view of Jesper Lohse (WO 2010094284 A1), hereinafter referred to as Lohse.
Regarding claim 22, Gerardina fails to explicitly teach the method according to claim 21, wherein the linker molecules contain an electrically conductive chain.
However Lohse teaches wherein the linker molecules contain an electrically conductive chain (In some embodiments, a linker molecule comprises a linear chain of atoms wherein every two connected carbon atoms are followed by an atom of oxygen or nitrogen).
The specifications of the present disclosure explain “in particular wherein the electrically conductive chain is based on carbon atoms with unsaturated carbon bonds or a carotenoid or a chain of carbon atoms with oxygen atoms” (para. [0055]). Therefore, as evidenced by the specifications, Lohe teaches linker molecules with an electrically conductive chain.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Louse such that the linker molecule contains a conductive chain. Carbon atoms followed by an oxygen atom are known in the art as effective linker molecules.
Claims 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Gerardina in view of Coon, and in further view of Keskin.
Regarding claim 24, Gerardina fails to teach the method according to claim 1 wherein, in step (d), the at least one nanoscale biological specimen is exposed to an electron dose and a flux of electrons from the electron beam and wherein the flux of electrons is below a flux threshold equal to a flux level at which the structure of the at least one nanoscale biological specimen becomes distorted (The electron flux was Df =16± 2e−/A2s, and after the first image, each image was collected at a time interval of 4.0 ± 0.4 s of continuous exposure so that D increased by 64 ± 13 e−/A2 going from one to the next image (Fig. 2 caption)) (Spatial features of f = 0.20 nm−1 are thus preserved up to Dmax as indicated by the vertical dotted line representing Dmax. Damaged structure is included as gray data points (Fig. 2 caption)).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Keskin such that the flux of electrons is below a flux threshold equal to a flux level at which the structure of the at least one nanoscale biological specimen becomes distorted. Doing so mitigates radiation damage.
Regarding claim 25, Gerardina fails to teach The method according to claim 1, wherein, in step (d), the at least one nanoscale biological specimen is exposed to a preselected electron dose and a flux of electrons from the electron beam, wherein the flux of electrons is below a flux threshold, and wherein said flux threshold is determined by the following steps: α) investigating a first nanoscale biological specimen according to said method wherein, in step (d), the first nanoscale biological specimen is exposed to said preselected electron dose at a first flux of the electrons, and a structure of the first nanoscale biological specimen is detected; β) investigating a further nanoscale biological specimen according to said method wherein, in step (d), the further nanoscale biological specimen is exposed to said preselected electron dose at a further flux of the electrons higher than the first flux of the electrons, and a further structure of the further nanoscale biological specimen is detected, and γ) repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted, thereby determining said flux threshold of electrons.
However, Keskin teaches the method according to claim 1, wherein, in step (d), the at least one nanoscale biological specimen is exposed to a preselected electron dose and a flux of electrons from the electron beam, wherein the flux of electrons is below a flux threshold, and wherein said flux threshold is determined by the following steps: α) investigating a first nanoscale biological specimen according to said method wherein, in step (d), the first nanoscale biological specimen is exposed to said preselected electron dose at a first flux of the electrons, and a structure of the first nanoscale biological specimen is detected (Fig. 2 a); β) investigating a further nanoscale biological specimen according to said method wherein, in step (d), the further nanoscale biological specimen is exposed to said preselected electron dose at a further flux of the electrons higher than the first flux of the electrons, and a further structure of the further nanoscale biological specimen is detected (Fig. 2 b), and γ) repeating the procedure of step (β) until the structure of the nanoscale biological specimen becomes distorted, thereby determining said flux threshold of electrons (The electron flux was Df =16± 2e−/A2s, and after the first image, each image was collected at a time interval of 4.0 ± 0.4 s of continuous exposure so that D increased by 64 ± 13 e−/A2 going from one to the next image (Fig. 2 caption)) (Spatial features of f = 0.20 nm−1 are thus preserved up to Dmax as indicated by the vertical dotted line representing Dmax. Damaged structure is included as gray data points (Fig. 2 caption)) (Fig. 2 above).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device described in Gerardina to include the teachings of Keskin by using the method of Fig. 2 of Keskin to determine a flux threshold. Doing so mitigates radiation damage.
Conclusion
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/MICA JILLIAN EINHORN/ Examiner, Art Unit 2881
/WYATT A STOFFA/ Primary Examiner, Art Unit 2881
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