View source for Fluorescent in situ hybridization microscope counts

{{BreadcrumbsInteractions}}

* [[Page authors|Page authors]]: [[PRIMO]]
* [[Responsible curator|Responsible curator]]:  [[User:Hagi BucknWise|Hagen Buck-Wiese]]
----

__TOC__
<div class="model-box">
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Life cycle stage abundance (FISH)
|-
| '''Approach:''' fluorescence in situ hybridisation (FISH) with life-stage-specific or species-specific probes
|-
| '''Context:''' ''in situ'', lab
|-
| '''Spatial scale:''' point sample
|-
| '''Temporal scale:''' days
|-
| '''Units:''' cells L<sup>-1</sup>; % of population
|-
| '''Community captured:''' specific species (those with validated FISH probes)
|-
| '''Co-measurements:''' species-specific life stage duration
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

Fluorescence in situ hybridisation (FISH) uses rRNA-targeting probes labelled with fluorescent dyes to identify and enumerate specific life cycle stages or species in fixed water samples. For life cycle work, probes are designed to target rRNA transcripts that are expressed at different levels in different life stages, or probes are used in combination with morphological recognition to count distinct stages. Fixed samples are filtered onto polycarbonate membranes, hybridized with fluorescent probes, and cells are counted by epifluorescence microscopy. CARD-FISH (catalyzed reporter deposition) amplifies the signal for low-biomass samples<ref name="Frada2006">Frada, M., Not, F., de Vargas, C., & Probert, I. (2006). CaCO<sub>3</sub> optical detection with fluorescent in situ hybridization: a new method to identify and quantify calcifying microorganisms from the oceans. ''Journal of Phycology'', 42(6), 1162–1169. https://doi.org/10.1111/j.1529-8817.2006.00276.x</ref>.

=== Scale of measurement ===

Point samples; days between collection points.

=== Data generated ===

Species-specific and life-stage-specific cell abundances (cells L<sup>-1</sup>) or proportional abundances (%). When combined with total DAPI or SYBR Green counts, the fraction of the community in each life stage is calculated.

=== Units & currency ===

Units are cells L<sup>-1</sup> or % of cells in each life stage. The currency is measured abundance of each recognizable life stage.

=== Sample size ===

Typical samples are < 1 L in volume.

=== Repositories & databases ===

== Limitations ==

FISH is only possible for taxonomic groups for which validated probes exist. Cross-hybridization with non-target organisms can produce false positives; probe specificity must be validated. The method does not distinguish between life cycle stages unless probes specifically target stage-expressed transcripts. Low rRNA content in dormant cells (e.g., resting cysts) can lead to false negatives.

== Example Applications & Protocols ==

=== Classic examples ===
* Frada et al. (2006) ''CaCO<sub>3</sub> optical detection with fluorescent in situ hybridization: a new method to identify and quantify calcifying microorganisms from the oceans'' <ref name="Frada2006" />

=== Recent applications ===
*

=== Common calculations/conversions ===
* % of community in stage X = (FISH-positive cells for stage X) / (total DAPI-stained cells) × 100.

== References ==

[[Category:Main Pages|Model types]]