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C L S M ANALYSISOF BIOFILMS
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General Considerations
CLSM setups are available from most of the major microscopy companies, with a wide range of options (software and hardware) and peripheral
devices. CLSM is a combination of traditional epifluorescence microscope
hardware with a laser light source, specialized scanning equipment, and
computerized digital imaging. A general schematic diagram is shown in
Fig. 1. Lasers used include argon ion, helium-neon, krypton-argon, helium-cadmium, and UV excimer lasers. The most commonly used CLSM
systems are equipped with a helium-neon (543 or 633 nm) and mixed-gas
krypton-argon lasers (488-nm blue, 568-nm yellow, and 647-nm red lines).
Helium-cadmium lasers are seldom used but can provide a strong 442nm line. UV/VUV excimer lasers (157-351 nm) may be obtained with
commercially available CLSM systems. These lasers provide the advantage
of a wide range of usable fluorochromes and simultaneous excitation of up
to three fluorochromes with little spectral emission overlap. In most instances the laser is connected to the scanning head via a fiber optic connection. The scan head is a unit with galvanometric mirrors to scan the beam
onto the specimen and a system of mirrors and beam splitters that direct
the return signal to specific photomultiplier tubes (PMTs). The laser beam
is scanned point by point in a raster fashion to build a gray scale image of
the specimen under observation. The scanned areas may consist of 512 X
512, 512 x 768, or 1024 x 1024 pixels. The scan rate may usually be varied;
however, when the scan rate is increased, resolution is lost and when the
scan rate is low photobleaching is increased. Thus these factors must be
balanced by the user to obtain optimum results. The presence of a pinhole
or pinholes in the light path allows only those fluorescence signals that
arise from a focused XY plane to be detected by a PMT. These pinholes
are said to be confocal and thus prevent fluorescent signals originating from
above, below, or beside the point of focus from reaching the photodetector.
In addition, sets of wavelength specific filters are used to supply specific
excitation and emission wavelengths for the fluorescent probes used. For
example, when using a K r - A r laser, the following combinations of excitation and emission wavelengths are commonly available: 488 nm excitation,
522/32 nm emission (green); 568 nm excitation, 605/32 emission (red); and
640 nm excitation 680/32 nm emission (far-red). Most CLSM systems also
incorporate filter sets for reflection imaging and a separate system for
imaging nonconfocal-transmitted laser images using, for example, differential interference contrast (DIC) or phase-contrast optics.
Multiphoton excitation fluorescence imaging systems are a relatively
recent development and offer a number of features that may be very valuable for biofilm studies, including longer observation times with living
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Photomultiplh
tube
detector
Confocal aperture
Dichroic
Mirror
ii
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:!
!~!
X Y s c a n n i n g unit
microscopeoptics
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Flat~Irregular Substratum
Biofilm samples from flow-through devices, the Robbins device, rotating
annular biofilm reactors, or other sampling ports are usually flat. They can
be easily mounted and stained. In addition, this volume contains detailed
descriptions of several methods for biofilm cultivation that are suitable or
may be adapted for microscopic study.
If the biofilm sample to be examined is located on an irregular surface,
e.g., a piece of rock, some points need to be considered. With an upright
microscope, the sample should be placed in a small petri dish and kept
covered with the original liquid phase. We have found that mounting samples in petri dishes using wax, plasticine, or neutral chemistry silicone glues
or the creation of reservoirs on rock or wood surfaces using silicone dams
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extra long working distance lenses 20 ELWD or 40X ELWD or 40x 0.55
NA water-immersible lenses. Studies such as those by Neu and Lawrence]
Bott et aL, 6 and Lawrence et al.3-5 illustrate the application of water-immersible lenses to a variety of biofilms and substrata.
Sampling Considerations
The essential question in any analysis is how many or how often is
enough. Few authors have considered this question in detail for biofilm
studies. However, it is essential to the advancement of biofilm research
that each study considers how to achieve a statistically valid impression of
samples or treatment effects. For example, the study of Korber et al. 3 used
the combination of a computer-controlled microscope stage and CLSM
imaging to create large-scale montages of biofilm materials and used a
representative elements analysis to determine that analysis areas exceeding
105/xm2 were required for statistically valid comparisons of the biofilms
examined in their study. We have adopted a procedure of using five replicate
microscope fields per treatment replicate, allowing application of analysis
of variance to determine significant effects at p < 0.05.4
Collecting Images
After the sample is secure (and unlikely to leak fluid on the microscope),
it is customary to use phase-contrast or epi-fluorescence microscopy to
examine the sample and find suitable microscope fields for further observation using CLSM. With experience, or by necessity due to the nature of
the sample, this preliminary step may be omitted. The microscope should
be set up with the correct excitation and emission filters in place for the
fluor that was selected by the user. Then, with the gain (white level) set
to a low sensitivity, the laser intensity at its lowest level, and the pinhole
at its smallest aperture, the operator scans the sample and adjusts the
pinhole, the gain, and laser intensity to produce a well-defined image of
the biofilm material under observation. These optimum settings should
correspond to the smallest pinhole aperture, lowest laser intensity, and
lowest PMT sensitivity. The image should be illuminated evenly and contain
relatively few saturated pixels (i.e., white with a value of 255). Optimal
settings must also be established with reference to any autofluorescence
signals emitted by the sample being scanned. This is particularly important
for interpretation of the distribution of fluorescence or reflective probes in
the sample and later use of images for quantitative analyses. After the
30D. R. Korber,J. R. Lawrence,M. J. Hendry,and D. E. Caldwell,Biofouling 7, 339 (1993).
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BIOFILMFORMATIONAND PHYSIOLOGY
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Image Processing
Flow Chart
Actual
Example
Acquire image
Collect image
series with SCLM
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Threshold
f.~J
"--f" l_
Erode
"
--
Dilate
t
Count number of
white pixels
Measure
I
Process data
Determine
volume of
biofilm component
per biofilm area
FIG. 2. A flow chart showing a sequence of image processing and analysis steps carried
out on CLSM images or image stacks to define objects for measurement of cell area, including
application of erode and dilate functions to reduce noise.
sured. A typical series of steps is shown in the flow chart in Fig. 2. Deconvolution may also be applied to CLSM images to sharpen the image through
mathematical removal of out-of-focus information.33'34 All of these functions are applied to smooth the image, reduce noise, and thus more accurately define the objects to be measured. Manual editing of digital images
may also be performed.
There are many options for the visual presentation of CLSM images,
gallery display showing each section, stereo pairs, 1 red-green anaglyph
p r o j e c t i o n s , 7'16'35 three-color s t e r e o p a i r s . 4'5 Figure 3 (see color insert) shows
a (3D) red-green anaglyph projection of a river biofilm. Stereo projections
may also be color coded by depth so that materials present at the same
depth appear the same color. This approach can be very useful for the
33 D. A. Agard, Biophys. Bioeng. 13, 191 (1984).
34 G. L. Gorby, J. Histochem. Cytochem. 42, 297 (1994).
35 D. E. Caldwell, D. R. Korber, and J. R. Lawrence, Adv. Microb. Ecol. 12, 1 (1992).
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36j. Bloem, M. Veninga,and J. Sheperd, Appl. Environ. MicrobioL 61, 926 (1995).
37S. Miller, C. S. Kristensen,L. K. Poulsen,J. M. Carstensen, and S. Molin,Appl. Environ.
Microbiol. 61, 741 (1995).
38N. Blackburn, A. Hagstrom,J. Wikner, R. Cuadros-Hansson,and R. K. Bjornsen,AppL
Environ. Microbiol. 64, 3246 (1998).
FIG. 3. (A) A series of confocal laser images of a river biofilm stained with the nucleic
acid probe SYTO 9 showing the distribution of bacterial cells and general biofilm structure.
(B) A three-color rendering of a confocal image series using a stacked height fields approach
and the rendering package POV Ray. The image shows the distribution of exopolymeric
substances, with Limulus polyphemus-FITC lectin (green), Ulex europeaus-TRITC lectin
(red), and Arachis hypogaea CY5 (blue) in a river biofilm. The gridlines are 25 /zm apart.
The application of rendering allows the viewer to observe the data set from a variety of
perspectives, including this one, which places the observer within the biofilm looking up.
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TABLE I
CONVENTIONALCONEOCALLASERSCANNINGMICROSCOPY(CLSM)VERSUSTwo-PHOTON
LASERSCANNINGMICROSCOPY(2-PLSM)
Feature
CLSM
Laser
Excitationvolume
Ar-Kr and UV
Whole sample
Out of focusbleaching
Out of focus
Optics
Yes
Yes
Chromatic aberration due to
UV laser
Yes, pinhole throughputloss
Small (50-100/~m)
Pinhole
Light penetration
2-PLSM
TiSph
Extremelysmall
(femtoliter)
No
No
No UV optic
necessary
Not necessary
High (200-1000 ~m)
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