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.