MICRO-CARD-FISH and Boncat

CAtalyzed Reporter Deposition Fluorescence In Situ Hybridization combined with MICROautoradiography is currently the method of choice to address the activity of selected prokaryotic groups. Fluorescence in situ hybridzation with cyanine 3 labeled probes already has a long history, however, this approach frequently suffered from low detection rates especially in open ocean settings with low ribosomal content of the prokayrotes.

Probes for CARD-FISH are labeled with a horseradish peroxidase (HRP) tag. After hybridization of the probe to the target site a signal amplification step is introduced where tyramides labeled with FITC or CY3 conjugates are deposited near the hybridized probe through the enzyme activity of the horseradish. This makes it possible to detect prokaryotes with very low ribosomal content and detection efficiencies of up to 80%.

Due to rather large HRP-probes the permeabilization of the prokaryotic cell wall is crucial. While for bacteria lysozyme treatment is usually sufficient the archaeal cell wall has to be treated with proteinase K to allow penetration of the probes. With microautoradiography it is possible to visualize the uptake of radiolabeled substrates such as leucine or glucose by the prokaryotes. After the hybridization step the samples are developed similar to photographic film resulting in silver grain halos around those cells which incorporated the radioactive molecules. Thus in combination with CARD-FISH it is possible to get information on who are the main microbial groups in a water sample and which ones are predominantly active. 

BioOrthogonal Non‑Canonical Amino acid Tagging is a non‑radioactive, fluorescence-based method to detect which microbes are actively making new proteins at the single‑cell level. In practice, seawater samples are incubated with a methionine analogue, most commonly L‑homopropargylglycine (HPG), that cells incorporate into newly synthesized proteins during translation. After incubation and fixation, the incorporated analogue is “clicked” to a fluorescent dye via a copper(I)‑catalyzed azide–alkyne cycloaddition (CuAAC), allowing active cells to be visualized and quantified by epifluorescence microscopy or flow cytometry. This targets methionine‑based protein synthesis as a proxy for heterotrophic activity in individual prokaryotic cells.

Two practical refinements enhance BONCAT’s sensitivity in environmental samples. First, transferring labeled cells from polycarbonate filters onto glass using the filter‑transfer‑freeze approach reduces background fluorescence roughly ten‑fold, improving the contrast between true cellular signal and background and increasing the fraction of cells detectable as active. Second, using picolyl‑azide fluorophores accelerates the copper‑catalyzed click reaction and strengthens labeling, which boosts the proportion of HPG‑positive cells and improves detection in low‑activity deep‑ocean communities.