1. FISH – Fluorescence in situ hybridization
in microbial ecology

2. What is FISH? Fluorescence in situ hybridization is a
method employing specific nucleic acid
probes labeled with fluorescent dyes for
tracking organisms or genes in their
environment. This method avoids the
need for cultivation.
FISH is a widespread
application in environmental
microbiology, for clinical diagnosis and
in food industry.
FISH can provide insight into microbial
community structure and spatial
arrangement.
FISH is a method where fluorescently tagged oligonucleotides are used to target specific organisms or genes in a sample. This is done in situ, and the technique avoids cultivation.
FISH is used widely in environmental microbiology, for clinical diagnosis, and in the food industry. In environmental microbiology, rRNA is usually targeted, but for eukaryotes, it is often used to target genes in chromosomes, called chromosome painting, where specific genes are located in the chromosome. mRNA can also be targeted.
In FISH microbes can be directly visualized in their environment, and this can give information about community structure and spatial arrangement, but further optimalization for assessing community spatial arrangement is needed, it is difficult to preserve the population architecture during the harsh conditions for embedding the cells.

3. Hopefully you remember this figure
Hopefully you remember this figure. I have stolen it from Olav’s lecture. The FISH method belong to this part of the scheme, so after extraction of rRNA, sequencing, it is possible to design probes that are universal, or specific for Bacteria or Archaea, or specific for a guild, e.g. nitrifying bacteria. It is even possible to distinguish between different species.

4. FISH in environmental microbiology
Usually with probes for 16S rRNA
Advantages with rRNA:
High copy number gives better signal
rRNA consists of regions of highly conserved and more variable regions. This makes it possible to design probes with variable specificity, from universal probes to probes for a specific class of related organisms (e.g. ammonium oxidizing bacteria) or even for one specific species.
Usually, probes targeting 16S rRNA are used. The advantages with targeting rRNA is that
-high copy number gives better signal. Logical because the more hybridized, fluorescently labeled probes, the more fluorescence is emitted.
-rRNA makes it possible to distinguish organisms on different levels, from distinguishing bacteria from archaea, or distinguishing metabolic groups, e.g. nitrifying bacteria, to single species. This is due to the fact that rRNA has different regions with different degree of conservation.

5. Method
Treat microbial sample with chemical fixatives. In some cases additional steps are needed for sufficient permeabilization of cells, for example treatment with lysozyme.
Immobilize cells. Dehydrate
Add hybridization buffer and probes. Probes are generally nt in length, and are labeled covalently at the 5’end with a fluorescent dye (e.g. DAPI, SYBRgreen). The right temperature is important for hybridization. Probes will penetrate the cells and hybridize to rRNA in the ribosomes. The target cells will be homogenously stained because the ribosomes are throughout the cytoplasm.
Wash to remove unhybridized probes that will otherwise create background noise.
Epifluorescence microscopy or flow cytometry. FISH can be combined with confocal laser scanning microscopy (CLSM) for accurate reconstruction of the spatial arrangement of microbial communities.
-chemical fixatives. Permeabilization
-immobilization, dehydration
-add probes, let hybridize -> cells stained. Different probes for different organisms labeled with different flyorescent dyes can be used to visualize several organsims in the same sample.
-wash
-observe. Different microscopic tecniques:
-Epifluorescens microscopy for observing fluorescently labeled cells.
-Flow cytometry, makes quanitfication easier. I will come back to
that
-Confocal scanning laser microscopy – possible to obeserv 3D structure of the community

6. Method

7. Epifluorescens microscopy
Epifluorescens microscopy is a
microscopic method where
excitatory light is passed onto
the sample. The light excites
the fluorescent probes, and
emitted light is focused to
the detector. Only
excitatory reflected light is
passed to the detector.
I’ll briefly go through the different microscopic techniques, just to give a better picture of the whole process.
-Epifluorecens microscopy
-exitatory light passed to sample
-leads to excitation of fluorophores on the probes
-exitatatory light passed to detector

8. Flow Cytometry Technique for counting, examining and sorting
suspended microscopic
particles. The stream of
particles flows through
an optical and/or electronic
detector where the
fluorescent particles are
exited, and the emitted and
scattered light is detected.
Fluorescence is measured for
every cell passing the
detector.
Flow cytometry
counting and sorting microscopic particles
The particles are suspended in liquid. When passing laser ray, the fluorophores are exited, and the resulting emitted and scattered light are passed to a detector.
In this way, the fluorescens of ideally every cell is measured.

9. Confocal Laser Scanning Microscopy (CLSM)
Optical sectioning can provide pictures of thick specimens, or three-dimensional reconstructions of sample topology.
A laser beam is passed on to the sample
Resulting fluorescent light is detected. Out-of-focus light is suppressed by a pinhole, resulting in sharper pictures. One illuminated “volume element” of the sample represents one pixel.
The laser continues to scan over the sample. Information can be obtained from different focal planes by lowering or raising the microscopic stage.
Many pixels can be used to generate a three-dimensional image of the sample.
3D pictures provides information about sample topology.
Laser beam to sample
Detection of fluorescent light
One such illuminated volume of the sample represents one pixel.
By scanning over different focal planes, a 3D image can be obtained.

10. Case: FISH in pathogenomics
The picture to the right
shows the result from FISH
with CLSM where rRNA of
the pathogenic bacterium
Tropheryma whippelii was
targeted (blue). This
bacterium causes Whipple’s
disease. Nuclei of intestinal
cells are colored green,
intracellular cytoskeletal
protein are colored red.
The location of this
bacterium in intestinal tissue
from patients with Whipple’s
disease provided evidence
that the bacterium grows
outside the cells, and thus
suggested that T. whippelii is
not an obligate intracellular
pathogen.
This picture shows an example where FISH analysis with CLSM was used to detect the bacterium Tropheryma whippelii in human intestinal tissue. This bacterium causes Whipple’s disease (rare, systemic, infectious disease, which causes malabsorption in the intestine, diarrhea, weight loss, arthritis. and more). The bacterium was stained blue by targeting the rRNA. Combination of general staining methods and FISH.
The study showed that the bacterium mostly lived outside the cells, and this gives evidence that it is not an obligate intracellular pathogen.
Fredricks, Relman, Localization of Tropheryma
whippelii rRNA in Tissues from Patients with Whipple's Disease, The
Journal of Infectious Diseases 2001;183:1229–1237

11. Problems and limitations
This figure that I have stolen from Wagner et.al. shows different problems that are frequently encountered in FISH, and possible solutions. I will not go through all these techniques, only mention some of them briefly.

12. Problems and limitations
Permeabilization
Sensitivity when rRNA content is low
Little accessibility to target site due to folding of rRNA
Determination of accurate hybridization and washing conditions
Quantification
Differentiation between species
Gives no information about physiology
Nucleotides can act as quenchers and reduce the signal
Background noise from unspecific binding of probes
Not all bacterial and archaeal species can be permeabilized by standard fixation protocols. This means that the probes cannot enter the cell.
rRNA-targeted oligonucleotide probes that are marked with only one fluorescent dye molecule may give a to low signal to be detected if rRNA content is low. This may be the case for slow-growing cells, but this is not always true.
The higher order structure of the ribosome may prevent the probe form binding. The accessibility to target sites varies between species.
Difficult to determine washing and hybridization conditions when the microorganisms are not cultured.
Quantitative data on the abundance of microorganisms are mostly obtained by time-consuming manual counting with microscope. The accuracy from manual counting can be quite low, especially if population density is high (f.eks. in a biofilm)
rRNA are very conserved. There are generally no target sites for distinguishing between strains in one species. But there are sufficient diversity in some regions to distinguish between species.
FISH does not provide information about ecophysiology, because the rRNA, which is constitutively there, are targeted. rRNA does not even always reflect general physiological activity, although often slow-growing cells with low metabolic activity have low ribosome levels.
Nucleotides can act as quenchers by absorbing ecxitation energy (photoinduced electron transfer) from the fluorophore that the probe is marked with, and thereby reducing the signal. Nucleotides in the proximity of the fluorophore can reduce the signal up to 30 %. Guanosnie>adenosine>cytidine>thymidin. Some fluorophores are affected more by quenching than others.
Noise: caused by unspecific binding to sample material, leading to high background fluorescense.

13. Solutions Limited accessibility to target site: Helper probes:
Accessibility to probe can be increased by using unlabeled “helper probes” that bind adjacent to the target rRNA site and induce conformational changes that increase accessibility to target.
Probe design is difficult: the helper probe must have an equal or broader specificity, and a Td at least as high as that for the probe.
Peptide nucleic acids probes:
PNA pseudopeptides have an uncharged polyamide backbone, without a charged phosphate group. Hybridization can occur at higher temperatures and low salt concentrations, which decrease the stability of the rRNA secondary structure and leads to higher accessibility to target site.
PNA probes must be shorter than nucleic acid probes because of the higher binding affinity. New probe design and hybridization conditions must be determined. Expensive
Helper probes
increasing accessibility
probe design is difficult
PNA
Peptide nucleic acid is an artificially synthesized polymer that can hybridize with DNA and RNA. Instead of a ribose or deoxyribose sugar backbone, peptide nucleic acids have repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Because PNA don’t have the charged phosphate group that DNA and RNA have, PNA bind more strongly to the target, which makes it possible for us to use higher hybridization temperatures, so that the secondary structure in rRNA is destabilized and target is exposed.
Unfortunately, probe design and hybridization conditions can not be directly adapted from previously published DNA probes. Because of the higher binding affinity of PNA probes, PNA probes must be shorter than DNA probes, or else the hybridization temperature necessary may destroy the fixed cells. PNA probes are also more expensive than oligonucleotide probes.

14. Solutions Noise Self-ligating probes
Probe pairs that target adjacent sites on rRNA, and are able to ligate to each other. Self-ligation leads to the loss of a quencher group from one of the probes. Only hybridized and autoligated probes can fluoresce.
Probe design is difficult
The use of self-ligating quenched probe pairs can reduce noise significantly. The probes target adjacent sequences on rRNA. When they hybridize close to eachother, they will self-ligate. This means that their backbones will be chemically linked by creation of a new phosphodiester bond. When this happens, the quencher on one of the probes will be removed, so that the fluorophore on the other probe can emit fluorescense.
With this technique, only hybridized and autoligated probes will emit fluorescence.
Unfortunately, probe design is, as you can imagine, difficult.

15. Solutions Low or missing signals due to low rRNA content
Multi-labeled polyribonucleotide probes
Enzymatic signal amplification, CARD-FISH, with horseradish peroxidase labeled probes.
Multi-labeled polynucleotide probes
Several fluorescent groups on one probe molecule increases the signal. A significantly higher percentage of prokaryotes can be visualized, even slow-growing or starving cells.
Increased length complicates probe design, and makes it more difficult to design probes that can discriminate between close relatives. ?
Enzymatic signal amplification
CARD=catalyzed reporter deposition.
The probes are labeled with horseradish peroxidase (HRP). When the preparation is treated with fluorescent tyramide, the peroxidase converts the tyramide into a highly reactive metabolite that binds to the nearby proteins. One HRP can convert many tyramides, so the signal is amplified.
For the large HRP-labeled oligonucleotides to penetrate the cells, rigorous pre-treatments are needed. This causes lysis of many of the cells, and are likely to change the microbial composition of the sample.

16. Solutions Quantification
Flow cytometry; only if the microorganisms occur as single cells.
Digital image analysis; With a confocal laser scanning microscope, the biovolume of probe-targeted cells is measured as a percentage of the total biovolume.
Quantitive analysis has been done manually. Both time consuming and inaccurate. Toward automation.

17. Inferring physiology and activity; MAR-FISH and ISRT-FISH
MAR-FISH is a combination of microautoradiography and FISH
Substrate uptake patterns are monitored by labeling substrate with a radioisotope
This enables us to identify organisms in the sample that are metabolizing the labeled substrate (MAR) and who they are (FISH)
ISRT-FISH (in situ reverse transcription – FISH)
Specific mRNA are amplified to cDNA by reverse transcription PCR, and then targeted by fluorescent probes.
MAR-FISH
Ribosome content is often assumed to reflect cellular activity. This is not always true. Physiological history also influence cellular ribosome content. Bacterial cells might be highly active despite low ribosome content
In MAR-FISH cells are exposed to a radiolabeled substrate, e.g, an organic compound. The raioactive decay from the incorporated substrate can then be visualized, and the cells metabolizing the compound can be identified.
ISRT-FISH
specific mRNA are amplified by RT-PCR to cDNA, and then targeted by fluorescent probes. Used to explore factors that control gene expression in populations. Also used to detect metabolically active groups of microorganisms.