Introduction

Fluorescent in situ hybridization (FISH) is a technique developed in the early ‘80s that allows the detection and localization of specific DNA or RNA sequences within prokaryotic and eukaryotic cells and tissues1,2. The basic principle of the method relies on the complementarity between a fluorescently-labelled molecular probe (MP), generally composed of DNA, and a specific nucleic acid sequence that is present inside the cell3. A standard FISH procedure includes five steps: fixation, permeabilization, hybridization, washing, and visualization or quantification of the fluorescent signal of the sample. The fixation/permeabilization of the cells is normally performed together, in order to facilitate the uptake of the MPs by destabilizing the cell membrane or envelope without lysis or extensive degradation. Hybridization takes place when a specific MP is fully complementary (or nearly fully complementary) to the target sequence. After washing the non-hybridized or loosely bound MPs, the hybridized MPs can be detected by fluorescence microscopy or quantified by flow-cytometry. FISH may be used both in vitro and in vivo . In the latter case the hybridization solutions are applied directly into the system, without a prior fixation or permeabilization step4.
The hybridization step is critical in FISH, as the efficiency of the hybridization is influenced by different factors that need to be taken into account for optimization. A low-fluorescence hybridization signal can result from cell-dependent limitations, such as low ribosomal content, inaccessibility of the target site, ribonucleases activity or difficulties in the permeabilization of the cellular envelope or membrane5. Optimization of the probe design by using nucleic acid mimics such as Peptide Nucleic Acid (PNA) or Locked Nucleic Acid (LNA) can overcome most of these limitations, improving probe specificity and sensitivity6. Also, in the case of FISH in fixed cells or tissues, the cellular permeabilization can be further enhanced by using different chemical fixatives to create pores in the cell membrane/envelope. Other parameters, such as the hybridization temperature and composition of the hybridization solution also compromise the success of the hybridization6,7.
Despite the extended use of FISH for several decades in different fields, a mechanistic approach of the process as a whole is still missing. In their seminal work, Yilmaz et al .8were able to develop a model which predicts the hybridization efficiency in FISH based on the affinity of the probe to the target rRNA. Although their approach quite accurately predicts the hybridization efficiency compared to standard prediction methods using the mid-transition temperature (Tm), it only takes into consideration the accessibility of the MPs to the rRNA as the limiting step in FISH, excluding diffusional barriers during the process.
In here, we approach FISH as a particular case of a diffusion-reaction kinetics, similar to other engineering problems that involve diffusion and mass transfer of chemical species within physical and biological systems and a subsequent chemical reaction. Based on literature models, we estimated the characteristic times taken by different DNA-MPs to cross several diffusional barriers and reach the molecular target. As different cell-types impose distinctive barriers for diffusion, in this approach both bacterial, including gram-negative and gram-positive bacteria, and animal cells are considered. The final goal is to assess which of the diffusional or reaction steps take longer and, hence, have a higher time contribution to the 30-90 minutes characteristic of a FISH procedure.