OceanWiki - User contributions [en]

Single cell uptake (SIP-SIMS/nanoSIMS; CHIP-SIMS; CARD-FISH paired with nanoSIMS)

2026-02-10T10:22:15Z

Chreaa: Created page with " * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' single-cell isotope analysis |- | '''Context:''' incubation |- | '''Spatial scale:''' µm |- | '''Temporal scale:''' hours–days |- | '''Units:''' isotope enrichment, uptake rates |- | '''Communit..."

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User: Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' single-cell isotope analysis
|-
| '''Context:''' incubation
|-
| '''Spatial scale:''' µm
|-
| '''Temporal scale:''' hours–days
|-
| '''Units:''' isotope enrichment, uptake rates
|-
| '''Community captured:''' active single cells
|-
| '''Co-measurements:''' incubation time, tracer enrichment, temperature, nutrients
|}
</div>
<div style="clear:both"></div>

== Method Overview ==
Single-cell uptake methods combine stable isotope probing (SIP) with secondary ion mass spectrometry (SIMS) to quantify nutrient assimilation at the level of individual cells. In nitrogen fixation studies, samples are incubated with <sup>15</sup>N<sub>2</sub>, followed by analysis using nanoSIMS or related platforms. Taxonomic or functional identity can be resolved using complementary labeling techniques such as CARD-FISH or CHIP-SIMS, enabling direct linkage between nitrogen uptake and specific microbial taxa or functional groups. These approaches provide quantitative, cell-specific measurements of nitrogen assimilation and metabolic activity.

== Output ==
* Single-cell isotope enrichment values
* Cell-specific nutrient uptake rates
* Taxon-resolved nitrogen assimilation
* Spatial patterns of isotope incorporation

=== Scale of measurement ===
* Single-cell
* Quantitative
* Taxon-specific activity

=== Data generated ===
* NanoSIMS ion images
* Isotopic ratio measurements (e.g., <sup>15</sup>N/<sup>14</sup>N)
* Cell-specific uptake rate calculations
* Linked microscopy and hybridization images

=== Units & currency ===
* Atom percent <sup>15</sup>N
* Excess isotope enrichment
* fmol N cell<sup>−1</sup> h<sup>−1</sup>
* Relative enrichment above natural abundance

=== Sample size ===
* Typically:
** 10–100 analyzed cells per sample
** 3–10 samples per treatment
* Limited throughput relative to bulk methods

=== Repositories & databases ===

== Limitations ==
* Low throughput and high analytical cost
* Requires specialized instrumentation and expertise
* Incubation conditions may alter in situ activity
* Taxonomic identification depends on probe specificity and labeling efficiency

== Example Applications & Protocols ==

=== Classic examples ===

=== Recent applications ===

Protocols:
* <sup>15</sup>N<sub>2</sub> tracer incubation protocols
* CARD-FISH combined with nanoSIMS workflows
* CHIP-SIMS and SIP-SIMS sample preparation protocols
* Custom laboratory SOPs for fixation, hybridization, and nanoSIMS analysis

=== Common calculations/conversions ===
* Atom percent excess = measured atom % − natural abundance
* Single-cell uptake rate = (isotope enrichment × cellular N content) / incubation time
* Correction for tracer enrichment and dilution
* Scaling single-cell rates to community-level estimates (with caution)

== References ==

[[Category:Main Pages|Model types]]

NifH detection/quantification (qPCR, RT-qPCR; nifH database)

2026-02-10T10:17:17Z

Chreaa: Created page with " * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' gene quantification |- | '''Context:''' ''Molecular'' |- | '''Spatial scale:''' |- | '''Temporal scale:''' Days, Seasons |- | '''Units:''' gene copies, transcripts |- | '''Community captured:''' d..."

Nitrogenase quantification

2026-02-10T10:10:10Z

Chreaa: Created page with " * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' immunolabelling |- | '''Context:''' ''in situ or enviroment'', incubation |- | '''Spatial scale:''' |- | '''Temporal scale:''' days, seasons |- | '''Units:''' signal intensity, counts |- | '''Commu..."

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' immunolabelling
|-
| '''Context:''' ''in situ or enviroment'', incubation
|-
| '''Spatial scale:'''
|-
| '''Temporal scale:''' days, seasons
|-
| '''Units:''' signal intensity, counts
|-
| '''Community captured:''' nitrogenase-expressing diazotrophs
|-
| '''Co-measurements:'''
|}
</div>
<div style="clear:both"></div>

== Method Overview ==
Nitrogenase quantification by immunolabelling targets nitrogenase protein subunits using specific antibodies against the NifH protein. Cells are fixed and incubated with primary antibodies that bind nitrogenase, followed by enzyme-labeled secondary antibodies for detection. Immunolabelling can be combined with epifluorescence microscopy, confocal microscopy, or flow cytometry to quantify the presence, abundance, and spatial distribution of nitrogenase-expressing diazotrophs in environmental samples. This approach provides a direct measure of nitrogenase protein presence rather than gene abundance or enzymatic activity.

== Output ==
* Presence and abundance of nitrogenase-positive cells
* Spatial distribution of nitrogenase-expressing diazotrophs
* Relative protein expression levels (method-dependent)

=== Scale of measurement ===
* Protein / cellular level
* Semi-quantitative
* Single-cell to community-level inference

=== Data generated ===
* Fluorescence microscopy images
* Flow cytometry counts (if applicable)
* Signal intensity measurements
* Cell abundance estimates linked to nitrogenase expression

=== Units & currency ===
* Fluorescence intensity (relative units)
* Counts of nitrogenase-positive cells
* Proportion of labeled cells (%)

=== Sample size ===
* Varies by study design
* Includes biological and technical replicates

=== Repositories & databases ===
* No centralized database for nitrogenase immunolabelling data

== Limitations ==
* Requires highly specific and validated antibodies
* Signal intensity can vary with fixation and staining conditions
* Limited taxonomic resolution without complementary methods (e.g., FISH)
* Antibody availability is biased toward well-studied diazotrophs

== Example Applications & Protocols ==

=== Classic examples ===

=== Recent applications ===

Protocols:
* Immunofluorescence staining protocols targeting NifH
* Microscopy-based quantification workflows
* Custom laboratory SOPs optimized for fixation and antibody specificity

=== Common calculations/conversions ===
* Percentage of nitrogenase-positive cells = (labeled cells / total cells) × 100
* Normalization of signal intensity across samples
* Integration with cell counts for community-level estimates

== References ==

[[Category:Main Pages|Model types]]

Other diazotroph marker genes: nifD, nifK

2026-02-10T10:04:45Z

Chreaa: Created page with " * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' Sequencing |- | '''Context:''' ''Environmental'', incubation |- | '''Spatial scale:''' stations, across oceans |- | '''Temporal scale:''' days, seasons |- | '''Units:''' |- | '''Community capture..."

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' Sequencing
|-
| '''Context:''' ''Environmental'', incubation
|-
| '''Spatial scale:''' stations, across oceans
|-
| '''Temporal scale:''' days, seasons
|-
| '''Units:'''
|-
| '''Community captured:''' all, can be size-fractionated
|-
| '''Co-measurements:'''

|}
</div>
<div style="clear:both"></div>

== Method Overview ==
nifD and nifK amplicon sequencing targets genes encoding the α- and β-subunits of the molybdenum–iron (MoFe) protein of nitrogenase, respectively. These genes can be complementary to nifH sequencing and provide additional phylogenetic and functional resolution of diazotrophic communities. Environmental DNA is extracted, followed by PCR amplification of nifD and/or nifK using gene-specific primers. Amplicons are sequenced using high-throughput platforms, and resulting sequences are used to assess diazotroph diversity, composition, and relative abundance across environments.

== Output ==
* nifD and/or nifK amplicon sequence variants (ASVs) or operational taxonomic units (OTUs)
* Taxonomic and phylogenetic assignment of diazotrophs
* Relative abundance profiles of nifD/nifK variants

=== Scale of measurement ===
* Molecular / gene-level
* Semi-quantitative (relative abundance; absolute abundance requires complementary quantification)
* Community-level inference of nitrogen fixation potential

=== Data generated ===
* DNA sequence reads (FASTQ files)
* ASV or OTU tables
* Phylogenetic trees (often higher resolution than nifH)
* Metadata-linked abundance matrices

=== Units & currency ===
* Sequence counts
* Relative abundance (%)
* Reads per sample

=== Sample size ===
* Varies by study design
* Biological and technical replication recommended

=== Repositories & databases ===
* nifDdada2, github – focused on nifD
* NCBI GenBank – nifD and nifK reference sequences
* ENA / SRA – raw sequencing reads
* IMG/M – integrated microbial genomes and functional genes

== Limitations ==
* Lower primer coverage and fewer validated primer sets compared to nifH
* Larger gene size can complicate amplification and sequencing
* Lower reference database coverage relative to nifH
* Does not directly measure nitrogen fixation activity
* Horizontal gene transfer complicates evolutionary interpretation

== Example Applications & Protocols ==
* PCR amplification protocols adapted from specific primers
* Custom laboratory SOPs due to limited standardized protocols

=== Classic examples ===

=== Common calculations/conversions ===
* Relative abundance = (reads per nifD or nifK ASV) / (total nifD/nifK reads per sample)
* Rarefaction to standardize sequencing depth
* Phylogenetic clustering based on nifD/nifK sequences
* Comparative analyses with nifH-derived community profiles

== References ==

NifH amplicon sequence

2026-02-10T09:30:35Z

Chreaa:

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' Sequencing
|-
| '''Context:''' ''Environmental'', incubation
|-
| '''Spatial scale:''' stations, across oceans
|-
| '''Temporal scale:''' days, seasons
|-
| '''Units:'''
|-
| '''Community captured:''' all, can be size-fractionated
|-
| '''Co-measurements:'''

|}
</div>
<div style="clear:both"></div>

== Method Overview ==
nifH amplicon sequencing targets the nifH gene, which encodes the iron protein subunit of nitrogenase and is widely used as a functional marker for biological nitrogen fixation. Environmental DNA is extracted, the nifH gene is amplified a nested PCR approach with degenerate primers (outer primers: NifH3 and NifH4 and inner primers: NifH1 and NifH2), and amplicons are sequenced (typically via high-throughput sequencing). Resulting sequences are used to assess the diversity, composition, and relative abundance of diazotrophic microorganisms across environments.

== Output ==
• nifH amplicon sequence variants (ASVs) or operational taxonomic units (OTUs)
• Taxonomic or phylogenetic assignment of diazotrophs
• Relative abundance profiles of nifH variants

=== Scale of measurement ===
• Molecular / gene-level
• Semi-quantitative (relative abundance, not absolute gene copy number unless combined with qPCR)
• Community-level inference of functional potential

=== Data generated ===
• DNA sequence reads (FASTQ files)
• ASV or OTU tables
• Phylogenetic trees (commonly used for nifH)
• Metadata-linked abundance matrices

=== Units & currency ===
• Sequence counts or relative abundance (%)
• Reads per sample

=== Sample size ===
• Varies by study design
• Commonly:
o 10–100 environmental samples
o Thousands to millions of reads per sequencing run
• Replication often includes biological and technical replicates

=== Repositories & databases ===
• nFixDB – curated nifH sequences and annotations
• NCBI GenBank – raw nifH sequences
• ENA / SRA – raw sequencing reads
• IMG/M – integrated microbial genomes and marker genes

e.g. [https://github.com/raw-lab/NFixDB nFixDB]

== Limitations ==
• Primer bias due to high nifH sequence diversity
• Degenerate primers can amplify non-target genes
• Does not directly measure nitrogen fixation activity
• Limited taxonomic resolution for some diazotroph groups
• Horizontal gene transfer complicates phylogenetic interpretation

== Example Applications & Protocols ==

=== Classic examples ===
• Zehr & McReynolds (1989) – First use of nifH as a molecular marker for diazotrophs
• Zehr et al. (2003) – nifH diversity in marine environments
• Gaby & Buckley (2012) – Evaluation of nifH primers and phylogeny

=== Common calculations/conversions ===
• Relative abundance = (reads per nifH ASV) / (total nifH reads per sample)
• Rarefaction to standardize sequencing depth
• Conversion of read counts to proportions (%)
• Phylogenetic clustering (e.g., nifH clusters I–IV)

== References ==

• Zehr, J. P., & McReynolds, L. A. (1989). Use of degenerate oligonucleotides for amplification of the nifH gene. Applied and Environmental Microbiology.
• Zehr, J. P., et al. (2003). Global distribution and diversity of nitrogen-fixing microorganisms. Nature.
• Gaby, J. C., & Buckley, D. H. (2012). A comprehensive evaluation of PCR primers to amplify the nifH gene. PLOS ONE.

NifH amplicon sequence

2026-02-10T09:30:01Z

Chreaa:

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' Sequencing
|-
| '''Context:''' ''Environmental'', incubation
|-
| '''Spatial scale:''' stations, across oceans
|-
| '''Temporal scale:''' days, seasons
|-
| '''Units:'''
|-
| '''Community captured:''' all, can be size-fractionated
|-
| '''Co-measurements:''' Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history
|}
</div>
<div style="clear:both"></div>

== Method Overview ==
nifH amplicon sequencing targets the nifH gene, which encodes the iron protein subunit of nitrogenase and is widely used as a functional marker for biological nitrogen fixation. Environmental DNA is extracted, the nifH gene is amplified a nested PCR approach with degenerate primers (outer primers: NifH3 and NifH4 and inner primers: NifH1 and NifH2), and amplicons are sequenced (typically via high-throughput sequencing). Resulting sequences are used to assess the diversity, composition, and relative abundance of diazotrophic microorganisms across environments.

== Output ==
• nifH amplicon sequence variants (ASVs) or operational taxonomic units (OTUs)
• Taxonomic or phylogenetic assignment of diazotrophs
• Relative abundance profiles of nifH variants

=== Scale of measurement ===
• Molecular / gene-level
• Semi-quantitative (relative abundance, not absolute gene copy number unless combined with qPCR)
• Community-level inference of functional potential

=== Data generated ===
• DNA sequence reads (FASTQ files)
• ASV or OTU tables
• Phylogenetic trees (commonly used for nifH)
• Metadata-linked abundance matrices

=== Units & currency ===
• Sequence counts or relative abundance (%)
• Reads per sample

=== Sample size ===
• Varies by study design
• Commonly:
o 10–100 environmental samples
o Thousands to millions of reads per sequencing run
• Replication often includes biological and technical replicates

=== Repositories & databases ===
• nFixDB – curated nifH sequences and annotations
• NCBI GenBank – raw nifH sequences
• ENA / SRA – raw sequencing reads
• IMG/M – integrated microbial genomes and marker genes

e.g. [https://github.com/raw-lab/NFixDB nFixDB]

== Limitations ==
• Primer bias due to high nifH sequence diversity
• Degenerate primers can amplify non-target genes
• Does not directly measure nitrogen fixation activity
• Limited taxonomic resolution for some diazotroph groups
• Horizontal gene transfer complicates phylogenetic interpretation

== Example Applications & Protocols ==

=== Classic examples ===
• Zehr & McReynolds (1989) – First use of nifH as a molecular marker for diazotrophs
• Zehr et al. (2003) – nifH diversity in marine environments
• Gaby & Buckley (2012) – Evaluation of nifH primers and phylogeny

=== Common calculations/conversions ===
• Relative abundance = (reads per nifH ASV) / (total nifH reads per sample)
• Rarefaction to standardize sequencing depth
• Conversion of read counts to proportions (%)
• Phylogenetic clustering (e.g., nifH clusters I–IV)

== References ==

• Zehr, J. P., & McReynolds, L. A. (1989). Use of degenerate oligonucleotides for amplification of the nifH gene. Applied and Environmental Microbiology.
• Zehr, J. P., et al. (2003). Global distribution and diversity of nitrogen-fixing microorganisms. Nature.
• Gaby, J. C., & Buckley, D. H. (2012). A comprehensive evaluation of PCR primers to amplify the nifH gene. PLOS ONE.

NifH amplicon sequence

2026-02-10T09:28:51Z

Chreaa:

{{NifH amplicon sequence}}

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' Sequencing
|-
| '''Context:''' ''Environmental'', incubation
|-
| '''Spatial scale:''' stations, across oceans
|-
| '''Temporal scale:''' days, seasons
|-
| '''Units:'''
|-
| '''Community captured:''' all, can be size-fractionated
|-
| '''Co-measurements:''' Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history
|}
</div>
<div style="clear:both"></div>

== Method Overview ==
nifH amplicon sequencing targets the nifH gene, which encodes the iron protein subunit of nitrogenase and is widely used as a functional marker for biological nitrogen fixation. Environmental DNA is extracted, the nifH gene is amplified a nested PCR approach with degenerate primers (outer primers: NifH3 and NifH4 and inner primers: NifH1 and NifH2), and amplicons are sequenced (typically via high-throughput sequencing). Resulting sequences are used to assess the diversity, composition, and relative abundance of diazotrophic microorganisms across environments.

== Output ==
• nifH amplicon sequence variants (ASVs) or operational taxonomic units (OTUs)
• Taxonomic or phylogenetic assignment of diazotrophs
• Relative abundance profiles of nifH variants

=== Scale of measurement ===
• Molecular / gene-level
• Semi-quantitative (relative abundance, not absolute gene copy number unless combined with qPCR)
• Community-level inference of functional potential

=== Data generated ===
• DNA sequence reads (FASTQ files)
• ASV or OTU tables
• Phylogenetic trees (commonly used for nifH)
• Metadata-linked abundance matrices

=== Units & currency ===
• Sequence counts or relative abundance (%)
• Reads per sample

=== Sample size ===
• Varies by study design
• Commonly:
o 10–100 environmental samples
o Thousands to millions of reads per sequencing run
• Replication often includes biological and technical replicates

=== Repositories & databases ===
• nFixDB – curated nifH sequences and annotations
• NCBI GenBank – raw nifH sequences
• ENA / SRA – raw sequencing reads
• IMG/M – integrated microbial genomes and marker genes

e.g. [https://github.com/raw-lab/NFixDB nFixDB]

== Limitations ==
• Primer bias due to high nifH sequence diversity
• Degenerate primers can amplify non-target genes
• Does not directly measure nitrogen fixation activity
• Limited taxonomic resolution for some diazotroph groups
• Horizontal gene transfer complicates phylogenetic interpretation

== Example Applications & Protocols ==

=== Classic examples ===
• Zehr & McReynolds (1989) – First use of nifH as a molecular marker for diazotrophs
• Zehr et al. (2003) – nifH diversity in marine environments
• Gaby & Buckley (2012) – Evaluation of nifH primers and phylogeny

=== Common calculations/conversions ===
• Relative abundance = (reads per nifH ASV) / (total nifH reads per sample)
• Rarefaction to standardize sequencing depth
• Conversion of read counts to proportions (%)
• Phylogenetic clustering (e.g., nifH clusters I–IV)

== References ==

• Zehr, J. P., & McReynolds, L. A. (1989). Use of degenerate oligonucleotides for amplification of the nifH gene. Applied and Environmental Microbiology.
• Zehr, J. P., et al. (2003). Global distribution and diversity of nitrogen-fixing microorganisms. Nature.
• Gaby, J. C., & Buckley, D. H. (2012). A comprehensive evaluation of PCR primers to amplify the nifH gene. PLOS ONE.

NifH amplicon sequence

2026-02-10T09:22:50Z

Chreaa: Created page with "{{BreadcrumbsPhotoautotrophy}} * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' Sequencing |- | '''Context:''' ''Environmental'', incubation |- | '''Spatial scale:''' stations, across oceans |- | '''Temporal scale:''' days, seasons |- | '''Units:..."

{{BreadcrumbsPhotoautotrophy}}

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' Sequencing
|-
| '''Context:''' ''Environmental'', incubation
|-
| '''Spatial scale:''' stations, across oceans
|-
| '''Temporal scale:''' days, seasons
|-
| '''Units:'''
|-
| '''Community captured:''' all, can be size-fractionated
|-
| '''Co-measurements:''' Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

== Output ==

=== Scale of measurement ===

=== Data generated ===

=== Units & currency ===

=== Sample size ===

=== Repositories & databases ===

e.g. [https://github.com/raw-lab/NFixDB nFixDB]

== Limitations ==

== Example Applications & Protocols ==

=== Classic examples ===
* Classic/original papers

=== Recent applications ===
* New papers + protocol links (e.g. word doc used in lab) if applicable

=== Common calculations/conversions ===
* E.g. conversion to standard units

== References ==

[[Category:Main Pages|Model types]]