The Center for Genomics Sciences Imaging Facility
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The new imaging facility
consists of a state of the art Leica TCS SP2 AOBS filter free spectral
confocal system with both inverted and upright microscopy capabilities. The
system is equipped with tissue culturing apparatus for the study of mucosal
biofilms.
The planned purchase of the Leica Fixed Stage
Automated (LFSA) microscope will facilitate the observation of biofilm
infections in whole animals.
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The new confocal imaging facility at the Center for Genomics Sciences
(CGS) is dedicated primarily to the study of bacterial biofilms and human
disease. The facility combines confocal scanning laser microscopy and
real-time time lapse imaging of bacterial biofilms growing in situ
without having to fix or dehydrate the sample significantly reducing
artifacts. The new CGS Imaging Facility will improve our ability to
investigate dynamic, living biofilms in situ in three dimensions and in real
time.
Imaging is crucial to the investigation of biofilms. Such studies lead to
a better understanding of the complex nature of biofilms, their relationship
to the surface substratum or tissue, microbial physiology and species
composition.
What is confocal microscopy?
Advantages of confocal microscopy for imaging biofilms and tissues
- 3D real-time imaging of live
samples.
- Red, orange, green and blue
lasers can target stains specific for cellular components with
excitation wavelengths ranging from 458 to 633 nm.
- Detectors can be tuned to
'look for' specific signal emissions and autofluorescence between 400 an
800 nm and tune out background autofluorescence.
- Short exposure times
significantly reduce photobleaching and UV killing associated with
conventional epifluorescence microscopy allowing long-term continuous
monitoring.
- SP2 quadruple channel detector
allows monitoring of 4 stains simultaneously.
- Confocal microscopy integrates
extremely well with molecular techniques such as Fluorescence In-Situ
Hybridization (FISH) for studying the localization of specific types of
bacteria within complex communities and Fluorescent Protein Expression
(FP) for visualization of live cells without staining (the cells are
genetically engineered to produce their own stain) and temporal gene
expression by linking FP to the transcription of a specific gene.
Click here to read more
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3D image of a Streptococcus mutans bacterial biofilm. S.
mutans is an early colonizer of tooth surfaces and by turning the sugars
and starches we eat into acid can cause caries (cavities). Staining with
MolecularProbes live/dead kit stained the individual bacterial cells green
indicating viability. In biofilms the bacteria often form complex cell
cluster colonies consisting of 'tower', 'mushroom', and 'mound' shaped
structures.
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This image appears in Post, C.J., Stoodley, P., Hall-Stoodley, L., and
Ehrlich, G.D. 2004. The role of biofilms in otolaryngologic infections. Current
Opinion in Otolaryngolog & Head & Neck Surgery. 12(3):185-190.
For further reading on how this biofilm was grown refer to:
Heersink, J., Costerton, W.J. and Stoodley, P. 2003. Influence of the
Sonicare® toothbrush on the structure and thickness of laboratory grown Streptococcus
mutans biofilms assessed by digital time-lapse and confocal microscopy. American
Journal of Dentistry. 16(2): 79-83.
Adams, H., Winston, M.T., Heersink, J., Buckingham-Meyer, K.A, Costerton,
W.J., and Stoodley, P. 2002. Development of a laboratory model to assess the
removal of biofilm from interproximal spaces by powered tooth brushing. American
Journal of Dentistry. 15(Special issue):12B-17B.
Fluorescence Protein Technology
Expression of multiple genes and species can be monitored for involvement
in biofilm formation over time using a growing family of differently colored
fluorescent proteins Blue (BFP), Cyan (CFP), Green (GFP), Yellow (YFP) and
Red (RFP).
GFP for tracking Quorum Sensing in Biofilms
Pseudomonas aeruginosa PAO1 genetically engineered to produce GFP
when the lasB gene is expressed. LasB is a protein involved in the production
of elastase, a virulence factor under control of the las-rhl QS system which
is also associated with biofilm maturation. Biofilms were grown under low
shear, laminar flow (left) and high shear turbulent flow (right). Under
laminar flow (A) the microcolonies were mound shaped (white arrow) while
under turbulent flow (B) they formed filamentous streamers (arrow). The green
color indicates that QS was active in both biofilms. The construct, PAO-MH454
(lasB-gfp), was made and gifted by Morten Hentzer (DTU) and the images
were taken by Laura Purevdorj-Gage in collaboration with Matt Parsek and
Mary-Jo Kirisits (Northwestern).
Digital-Time Lapse Microscopy
In addition to confocal microscopy the imaging facility will house 3
epifluorescence and bright-field digital time-lapse microscopy stations. The main
components of the system are the microscope, the digital camera, and computer
with framestore board. Biofilms can be grown in flow cells for tracking
development and behavior in real time.
What are biofilm flow cells?
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What are
Biofilms?
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What
are Biofilms?
Biofilms are adherent communities of bacteria
surrounded by extracellular polymeric material that are found everywhere in
nature, as well as in device-related and chronic human infections, such as
cystic fibrosis, otitis media, endocarditis and osteomyelitis.
The ability of bacteria to attach to surfaces and
organize into complex aggregates of cells reflects a fundamental survival
strategy of bacteria. Bacteria within biofilms are more resistant to
sub-optimal growing conditions such as poor nutrients, desiccation and the
effects of antimicrobial agents and host defenses.
Studying bacteria in the environment in which they
naturally grow is essential to understanding their multifaceted
developmental responses including their responses to antibiotics.
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Research overview:
The imaging of biofilms is fundamental to biofilm research.
High-resolution confocal images provided the first evidence for the
structural heterogeneity of biofilm architecture. This evidence was used to
establish models of biofilm growth and ultrastructure. Imaging studies also
showed that flow conditions, nutrient conditions and concentrations of
signaling molecules were important in biofilm development. Such studies are
crucial to a better understanding of the complex nature of biofilms, their
relationship to the substratum, microbial physiology and species composition.
Technical progress in microbial genetics in the last several years has led
to the development and widespread use of molecular reporters to analyze gene
regulation and function. These reporter molecules function only in viable
cells. Therefore analysis of fluorescent protein constructs must be
consistent with the in situ analysis of viable cells.
Confocal microscopy is an essential component of imaging bacteria in biofilms
because other high-resolution microscopic techniques require fixation that
disrupts the three dimensional structure and kills the bacteria being
analyzed. Therefore reporter gene constructs cannot be viewed after fixation.
Furthermore, both conventional fluorescent microscopy and electron microscopy
preclude the analysis of thick specimens such as biofilms because they fail
to resolve thick samples.
Cross section made up from a composite of individual confocal images of a Streptococcus
mutans biofilm grown on a glass slide. The biofilm was too thick to
visualize the interior, which has been subsequently false colored in yellow.
The cells on the outer edge of the biofilm were stained with MolecularProbes
live/dead™ kit.
Integration of Confocal Microscopy with CGS Research
The synergy from high resolution 4D microscopy, molecular techniques,
collaborations with surgeons and Carnegie Mellon MEMS engineers promises
significant advancements in our understanding and treatment of interactions
between the biofilm, the host and medical devices and the role of biofilms in
infectious disease.
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