Confocal laser.pdf

[9] CLSM ANALYSIS OF BIOFILMS 131

[9] Confocal Laser Scanning Microscopy for Analysis of Microbial Biofilms

By JOHN R. LAWRENCE and THOMAS R. NEU

Confoca l Laser Scann ing Microscopy: An In t roduct ion

Among the most versati le and effective of the nondestruct ive ap- proaches for studying biofi lms is confocal laser scanning microscopy (CLSM). CLSM reduces great ly the need for pret reatments such as disrup- t ion and fixation that reduce or e l iminate the evidence for microbia l rela- t ionships, complex structure, and organizat ion in biofilms. A l though confocal imaging has a relat ively long history of appl icat ion in the physical sciences and in medical research, its appl icat ions to biofi lms began in the early 1990s. 1 There has been increased appl icat ion of this tool in conjunct ion with an increasingly wide range of f luorescent probes and other novel techniques for biofi lm research. The reason for this increased interest is that CLSM allows optical thin sectioning of intact fully hydrated biofi lm mater ia l creat ing images with enhanced resolution, clarity, and informat ion content. As such, it is an ideal tool for studying spatial d istr ibut ion of a wide range of biofi lm propert ies. This capacity is evident in a series of CLSM-based research publications. 1-15 CLSM has also been used exten- sively in combinat ion with f luorescent in s itu hybr id izat ion techniques. 16-22

1 j. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell, J. Bacteriol. 173, 6558 (1991).

2 j. R. Lawrence, G. M. Wolfaardt, and D. R. Korber, AppL Environ. Microbiol. 60, 1166 (1994).

3 j. R. Lawrence, Y. T. J. Kwong, and G. D. W. Swerhone, Can. J. Microbiol. 43, 178 (1997). 4 j. R. Lawrence, T. R. Neu, and G. D. W. Swerhone, J. Microbiol. Methods 327 253 (1998). 5 j. R Lawrence, G. D. W. Swerhone, and Y. T. J. Kwong, Can. J. Microbiol., 44, 825 (1998). 6 T. L Bott, J. T. Brock, A. Battrup, P. A. Chambers, W. K. Dodds, K. Himbeault, J. R. Lawrence, D. Planas, E. Snyder, and G. M. Wolfaardt, Can. J. Fish. Aqu. Sci. 547 715 (1997).

7 T. R. Neu and J. R. Lawrence, FEMS Microbiol. Ecol. 247 11 (1997). 8 A. A. Massol-deya, J. Whallon, R. F. Hickey, and J. M. Tiedje, Appl. Environ. Microbiol. 61, 769 (1995).

9 G. M. Wolfaardt, J. R. Lawrence, R. D. Robarts, and D. E. Caldwell, Appl. Environ. Microbiol. 60, 434 (1994).

10 G. M. Wolfaardt, J. R. Lawrence, R. D. Robarts, and D. E. Caldwell, Appl. Environ. Microbiol. 617 152 (1995).

u G. M. Wolfaardt, J. R. Lawrence, R. D. Robarts, and D. E. Caldwell, Microbial. Ecol. 35, 213 (1998).

Copyright 1999 by Academic Press All rights of reproduction in any form reserved.

METHODS IN ENZYMOLOGY, VOL. 310 0076-6879/99 $30.00

132 BIOFILM FORMATION AND PHYSIOLOGY 191

With the advent of krypton-argon, commercial UV, and two-photon and mult iphoton laser microscopy systems, multiple parameter imaging of biofilms is practical, allowing the collection of multiple quantitative data sets at a single location within a biofilm. 5

CLSM provides a digital database that is amenable to image processing and analysis. Thus the user may obtain quantitative information on a wide variety of parameters, including cell numbers, cell area, object parameters such as min imum/maximum dimensions, orientation, and average gray value. Data sets may also be analyzed to determine diffusion coefficients within biofilms 2,23,24 and growth rates 25 of microorganisms. When combined with the variety of f luorescent antibodies, ol igonucleotide probes, or physio- logical probes (see other chapters in this volume), TM additional specific information may be derived.

An additional very significant aspect of confocal laser microscopy and optical sectioning is the creation of sets of images in perfect register allowing the product ion of three-dimensional (3D) reconstructions, renderings, and animations of data sets. 1'7'16 This provides additional power to scientific visualization of biofilm materials.

The primary goal of this article is to provide a primer for CLSM observation of biofilm materials.

12 S. MOiler, C. Sternberg, J. B. Anderson, B. B. Christensen, J. L. Ramos, M. Givskov, and S. Molin, Appl. Environ. Microbiol. 64, 721 (1998).

13 M. N. Mohamed, J. R. Lawrence, and R. D. Robarts, Microbiol. Ecol. 36, 121 (1998). 14 B. Assmus, P. Hutzler, G. Kirchhof, R. Amann, J. R. Lawrence, and A. Hartmann, Appl.

Environ. Microbiol. 61, 1013 (1995). 15 M. Wagner, G. Rath, H. P. Koops, J. Flood, and R. Amann, Wat. Sci. Technol. 34, (1/2),

237 (1996). 16 R. Amann, W. Ludwig, and K. H. Schleifer, Microbiol. Rev. 59, 143 (1995). 17 R. Amann, R. Snaid, M. Wagner, W. Ludwig, and K. H. Schleifer, J. Bacteriol. 178, 3496

(1996). 18 S. Miller, A. R. Pedersen, L. K. Poulsen, J. M. Carstensen, and S. Molin, Appl. Environ.

Microbiol. 62, 4632 (1996). 19 A. Neef, A. Zaglauer, A. H. Meier, R. Amann, H. Lemmer, and K. H. Schleifer, Appl.

Environ. Microbiol. 62, 4329 (1996). z0 W. C Ghiorse, D. N. Miller, R. L. Sandoli, and P. L. Siering, Microsc. Res. Technol. 33,

73 (1996). 21 M. Schuppler, M. Wagner, G. Shon, and U. B. Gobel, Microbiology 144, 249 (1998). 22 D. de Beer, P. Stoodley, F. Roe, and Z. Lewandowski, Biotechnol. Bioeng. 53, 151 (1997). 23 j. j. Birmingham, N. P. Hughes, and R. Treloar, Philos. Trans. Soc. Lond. B Biol. Sci. 350,

325 (1995). 24 L. K. Poulsen, G. Ballard, and D. A. Stahl, Appl. Environ. Microbiol. 59, 1354 (1993). 25 R. P. Haugland, "Handbook of Fluorescent Probes and Research Chemicals," 4th ed.

Molecular Probes, Eugene, OR, 1996. 25a Methods Enzymol. 310 [1-6] (1999) (this volume).

[91 CLSM ANALYSIS OF BIOFILMS 133

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 442- nm 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 in- stances the laser is connected to the scanning head via a fiber optic connec- tion. 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 Kr-Ar 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, differen- tial 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 valu- able for biofilm studies, including longer observation times with living

134 BIOFILM FORMATION AND PHYSIOLOGY [9]

Photomultiplh tube detector

Confocal aperture

Dichroic Mirror

i i '

i i I ! : )

i :! i

!~! XY scanning unit

microscope optics

FtG. 1. Schematic diagram of a confocal laser scanning microscope showing generalized construction and components.


specimens, increased fluorescence emission, increased depth for optical sectioning, and reductions in interference and photobleaching. These systems utilize a pulsed laser source such as titanium-sapphire with a major emission at 1047 nm. However, at the time of writing there have not been published applications of this technology in microbial ecology or biofilm studies (see further discussion later). All systems are operated using a combination host computer or workstation equipped with proprietary software specific to the manufacturer. These systems usually have two monitors, one displaying the control software and the other for the images. Linking the computer to additional workstations to separate image collection from image analysis and display is an important consideration. In addition, be- cause image data require an enormous amount of storage, a hard drive with a minimum of 5 GB capacity and equipment for production of CDs is essential (see later).

Getting Started

Turning on the system and checking alignment is usually done in advance of preparation of materials for observation, this allows the laser to warm up for 30 rain prior to initiating imaging. This is important with the Kr-Ar laser where production of the far-red line (640 nm) will vary during the warmup period. With Kr-Ar lasers the working lifetime may be rather short, in the range of 1000-1400 hr; problems that develop with age are instability and loss of far-red excitation wavelengths. With most of the current commercial CLSM systems there are few critical adjustments that can be made by the user. However, prisms and test samples may be provided to check alignment of mirrors, laser beam, and image quality. The user can make up slides consisting of fluorescent beads or Focal Check beads (Molecular Probes, Eugene, OR), which will allow them to routinely check whether image brightness and alignment are remaining within desired speci- fications. The use of Focal Check beads is particularly useful in ensuring that images obtained from the same location but with different excitation emission combinations (multiparameter imaging or colocalization studies) are in perfect register. A frequently overlooked adjustment is that of the gray scale and alignment of the computer screens themselves, which is vital to viewing the images collected correctly.

Sample Preparation

Biofilm sample materials range from prepared fixed materials, i.e., fluorescent in situ hybridization, which are covered by a glass coverslip, to biofilms grown on prepared substrata, to observations on natural irregular

136 BIOFILM FORMATION AND PHYSIOLOGY [91

surfaces recovered from various environments. All of these may be used for CLSM studies. The use of biofilm incubation systems such as the rotating annular bioreactor, continuous flow slide culture, Robbins devices, and others provides convenient surfaces for observations that are made for microscopic study. However, natural biofilms are usually not so conve- niently located or growing on optically perfect surfaces. Usually biofilm samples are associated with a solid interface. This so-called substratum covered with the biofilm must be mounted for CLSM observation. To examine the fully hydrated living features of a biofilm, the sample is prefera- bly used directly without any fixation or embedding procedures. In general, the preparation of the biofilm sample is dependent on the geometry of the substratum and the type of microscope available.

Upright versus Inverted Microscope

Dependent on the microscopic setup, upright or inverted, the following considerations are necessary. For the normal microscope the biofilm sample covered with the original liquid phase can be fixed to the bottom of a small petri dish (diameter 5 cm) with acid-free silicone glue, placed in a well slide, or a dam created with plasticine, wax, or silicone glue. All staining techniques are then applied in the small volume of the liquid layer covering the biofilm or within the larger volume of the whole container.

If an inverted microscope is employed, the sample has to be mounted upside down in a chamber having a coverslip bottom (Nunc, Roskilde, Denmark). Depending on the expected thickness of the biofilm and the working distance of the objective lens used, spacers with a thickness of 50-500/~m may be glued into the chamber to avoid damage to the biofilm. The staining is performed in the space in between the coverslip bottom of the chamber and the biofilm sample mounted upside down.

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

[9] CLSM ANALYSIS OF BIOFILMS 137

provides easy access to the observation surface using water-immersible lenses (see later) and upright microscopes. If an inverted microscope is used, the biofilm is immersed in the original liquid phase, but access to the sample is limited and the working distance of the objective lens may further limit examination of the biofilm sample.

Although care must be taken in handling biofilm samples, in real-world environments, biofilms are exposed to physical stress and are usually resil- ient enough to be manipulated and mounted for staining and observation as described.

Staining Options

After determination of sample type, one must select the type of staining or fluorescent or reflective probe to be used; this is based on the nature of the information desired. Important considerations when using combinations of fluors are relative intensity, narrowness of the emission band, photostabil- ity, potential for interference, additive effects, and quenching effects. The user has a wide range of stains and probes that may be used in conjunction with CLSM imaging to obtain information on cell position, identity, diffusion rates, chemistry, etc, 2'14'24'15'26 Fundamentally, staining in CLSM may be either positive or negative in nature. Negative staining of biofilms through the fooding of the sample with a fluor such as fluorescein was described in detail by Caldwell et aL 2v Positive staining encompasses the entire range of nucleic acid stains, i.e., the SYTO series (Molecular Probes, Eugene, OR), acridine orange (AO), 4',6-diamidino-2-phenylindole (DAPI), protein stains, fluorescein isothiocyanate (FITC), 5-(4,6-dichloroz- triazin-2-yl)aminofluorescein(DTAF), tetramethylrhodamine isothiocyanate (TRITC), other rhodamine stains, Texas Red, and lipophilic stains such as Nile Red. Other probes may be labeled using fluorescein (FLUOS, FITC), rhodamine (TRITC), cyanins (CY2, CY3, CY5), aminomethylcou- matin (AMCA), or phycoerythrin (PE). A new series of stains, the Alexa dyes, are also now available from Molecular Probes. In addition, autofluorescence can be a useful source of information, i.e., detecting and imaging algae and some bacteria. 4

It should also be noted that unstained controls should be imaged with all samples using the same settings as for the stained materials to ensure that autofluorescence artifacts are not present in the resulting images.

When staining for observation using water-immersible lenses, the sample of the substratum with the attached biofilm should be covered with

26 T. R. Neu and J. R. Lawrence, Methods Enzymol. 310 [10] (1999) (this volume). 27 D. E. Caldwell, D. R. Korber, and J. R. Lawrence, J. Microbiol. Methods 15, 249 (1992b).


original water. Alternately, the biofilm sample may be kept in a moist chamber such as a petri dish with a wet tissue. In general, many staining procedures for CLSM study do not require fixation of the biofilm sample. The complete staining procedure may be carried out in the liquid droplet covering the biofilm while it is still attached to the substratum. An example of a specifc staining procedure is given by Neu and Lawrence 26 for use with fluor-conjugated lectins.

Consideration should also be given to application of fade retardants 28 such as Citifluor (UKC Chemlab, Canterbury, UK) or Slow-Fade prepara- tions (Molecular Probes). Although these are generally used with fixed stained samples, their use should not be restricted to this type of sample preparation.

Objective Lenses

Establishment of the sample type leads to clear decisions regarding the primary imaging tool, the objective lens. The major limitation of all objective lenses, particularly when applied for CLSM imaging, is that the axial or Z dimension resolution is poor relative to the lateral resolution. Additional concerns arise from the fact that some objective lenses are not corrected for imaging in the far-red. 29 Similarly, when excitation wavelengths are extended into the ultraviolet there may be loss of transmission and serious image aberration. However, in general, the use of high numerical aperture (NA) oil or water-immersion lenses (i.e., NA 1.2-1.4) is recommended. Some of the water-immersion lenses are designed with confocal microscopy applications in mind and may allow imaging through up to 200 /zm of biological materials. This, however, presupposes the use of fixed stained materials, optically appropriate mounting media, and high-quality cov- erslips. However, the real power of CLSM in biofilm studies comes from the enhanced ability to observe and analyze fresh, undisturbed materials in real time. Thus for many studies of biofilms the ideal lenses for the examination of fluorescently stained biofilm samples are water immersible. These include relatively high NA water-immersible objectives supplied by Leica, Nikon, and Zeiss, such as the Zeiss 0.90 NA 63 x water-immersible lens. Advantages for biofilm research are long working distance, high numerical aperture, and superior brightness. Furthermore, they can be employed for direct observation without the need to use a coverslip. We have found that we can effectively section through several hundred microns with 63x 0.9 NA water-immersible lenses and up to 1 mm and more when using

28 R. J. Florijn, J. Slats, H. J. Tanke, and A. K. Raap, Cytometry 19, 177 (1995). 29 C. Cullander, J. Microsc. 176, 281 (1994).


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/xm 2 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

30 D. R. Korber, J. R. Lawrence, M. J. Hendry, and D. E. Caldwell, Biofouling 7, 339 (1993).


settings are optimized (a procedure that may have to be repeated many times) for the sample material under observation, the user must then determine which imaging options will be useful. The choices include a single XY optical thin section, a series of XY optical thin sections through the material, or a single or a series of XZ sections through the specimen. CLSM systems may also be programmed to collect images through time allowing the user to capture 4D data sets. If additional magnification is desired, various zoom functions that reduce the area scanned and thereby increase magnification may also be selected. Images may also be collected using mathematical filters such as Kalman or running average filters to reduce noise in the primary image. For low signal samples, options such as photon counting, summation, or cumulative collection of the image may be applied.

The optical sections may be collected at the same location using the full range of excitation and emission options provided by the particular system, thereby collecting information on several variables within a single microscope field. Lawrence et al. 4 demonstrated the application of multiple parameter imaging to determine the abundance of algae, bacteria, and exopolymers in river biofilms. Although some systems allow the simultaneous collection of images, sequential collection in general results in less photobleaching and optimal image quality. For example, in the Kr-Ar laser the user may collect images in the green, red, and far-red with the option of also collecting a reflection image of the biofilm materials. 5 If the system is provided with a UV laser, an additional channel is available for staining and observation. The introduction of two photon and multiphoton systems may expand the range of imaging options; however, as noted earlier, these have not been used in practice on biofilm materials at this writing.

Image Processing and Presentation

The crisp high-quality CLSM image may be improved for presentation purposes through the application of basic image processing techniques. Readers should consult Russ 3t and Gonzalez and Wintz 3a for extensive detail on image processing. Although every effort is made to obtain the highest quality primary image, some processing or enhancement may be required before analysis. Common processing steps include histogram analysis, gray level transformation, normalization, contrast enhancement, application of median, lowpass, Gaussian, Laplacian filters, image subtraction, addition, multiplication, and erosion and/or dilation of objects to be mea-

31 j. C. Russ, "The Image Processing Handboook," 2nd ed. CRC Press, Boca Raton, FL, 1995. 32 R. C. Gonzalez and P. Wintz, "Digital Image Processing." Addison-Wesley, Reading,

MA, 1977.


Image Processing Flow Chart

Acquire image

Process to enhance features

Measure

"-- f" l _ f.~J

" - - i

t

Process data

Actual Example

Collect image series with SCLM

and then for each slice:

Threshold

Erode

Dilate

Count number of white pixels

I D e t e r m i n e 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. Deconvo- lution 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 accu- rately 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 pro jec t ions , 7'16'35 three-color s tereo pairs. 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).


interpretation of 3D information. Another option for display of 3D data sets is "simulated fluorescence," whereby the material is viewed as though it were illuminated from an oblique angle and the surface layer was fluorescent. Projections may be made as a solid body or surface projection and animated to show the entire data set. The application of 3D rendering through ray tracing or surface contour-based programs may also provide a useful presentation of three-dimensional data sets, allowing the reader to examine the data set from various perspectives (Fig. 3B, see color insert).

Image Analysis Options

Having obtained a high-quality primary image from the CLSM, the user then has various options for extracting as much information as possible from the digital data set. Although the images are striking and visually pleasing, it is through the application of digital image analysis that the user can extract and present quantitative data. Image analysis is a critical tool for use in conjunction with the 2D, 3D, and even 4D data sets that can be created by CLSM imaging. The tools available range from relatively straightforward image analysis, including object recognition, counting, and gray level measurements, to increasingly sophisticated dedicated programs allowing 2D and 3D image analysis. The essential tools may be found in a number of analytical packages such as the Quantimet system36; MOiler et aL 37 used Cellstat, which is available for UNIX workstations (see http://www.lm.dtu.dk/cellstat/index.html). NIH image, a versatile analysis package for a Macintosh platform, is available as freeware over the internet at http://rsb.info.nih.gov/nih-image/ and is compiled for Windows 95 or NT-based systems (Scion ImagePC at www.scioncorp.com). Neural network systems have been proposed for the analysis of fluorescence images. 38 Sili- con Graphics-based software is also offered by Molecular Dynamics; this is a versatile package that also allows three-dimensional image analysis of CLSM XY image series. To date, studies have been limited to the analysis of serial 2D images rather than the application of true 3D analyses. Al- though all of the CLSM manufacturers offer supplementary analytical packages that work with their operating systems and image formats, none offer all the required options for image processing.

36 j. Bloem, M. Veninga, and J. Sheperd, Appl. Environ. MicrobioL 61, 926 (1995). 37 S. Miller, C. S. Kristensen, L. K. Poulsen, J. M. Carstensen, and S. Molin, Appl. Environ.

Microbiol. 61, 741 (1995). 38 N. 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.


TABLE I CONVENTIONAL CONEOCAL LASER SCANNING MICROSCOPY (CLSM) VERSUS Two-PHOTON

LASER SCANNING MICROSCOPY (2-PLSM)

Feature CLSM 2-PLSM

Laser Ar-Kr and UV TiSph Excitation volume Whole sample Extremely small

(femtoliter) Out of focus bleaching Yes No Out of focus Yes No Optics Chromatic aberration due to No UV optic

UV laser necessary Pinhole Yes, pinhole throughput loss Not necessary Light penetration Small (50-100/~m) High (200-1000 ~m)

Image Archiving / Printing

Application of CLSM techniques results in the creation of vast image and data sets; our facilities can produce several gigabytes per day. Thus, the fnal consideration is how one archives all this information. First, it is critical to have the largest hard drive available for the operating computer. Second, many options exist for long-term storage, including optical drives (write once and rewriteable formats), Bernoulli drives, Syquest, ZIP, and CDs. For cost-effective, secure, portable, relatively universal storage media, CDs remain the best recommendation. However, it is likely that digital video disks may replace CD technology. Images may be stored in a variety of formats, such as tagged image formats (TIFF), GIFF, RAW, PICT, EPS, JPEG, and BioRadTIFF. Each of these has advantages and disadvantages, such as degrees of image fidelity and their ability to compress images (i.e., JPEG). In general, TIFF are used the most universally and will be opened by most software such as NIH Image or Adobe. Archiving represents another major hurdle that should be considered early in the process of developing a CLSM-based research program.

Images may be printed for publication using a variety of means, including video printers, dye sublimation printers and slide printers.

Perspectives

Since the first application of CLSM for studying biofilms in 1991, it has become the key technique for the microscopic study of interfacial microbial communities. CLSM offers the only means for real-time, in-depth analysis of undisturbed biofilms. However, rapid advancement in the field is both occurring and required in some areas. Fluor creation is extensive and the


commercially available selection increases monthly; this is an area in which the user must take particular care to stay current. Future software needs lie in the areas of 3D image processing and analysis. On the hardware side, considerable work is required to improve the axial resolution of objective lenses. Current research in this field is investigating so-called 4Pi and Theta microscopy to improve axial resolution. There are several combinations possible to set up a hybrid microscope with elements from confocal, 4Pi, and Theta microscopes. With this approach, the axial resolution may be enhanced by a factor of 7.6 if, for example, a two-photon/4Pi-confocal Theta microscope is employed. 39 Additional rethinking of standard corrections for objective lenses is also required.

In the meantime, however, new developments have created significant potential advantages over conventional CLSM. One of the new techniques is called two-photon laser scanning microscopy (2-PLSM). 4,41 Several CLSM companies already offer this option within their product line. The major advantage of 2-PLSM over normal CLSM is an extremely small excitation volume and thus dramatically reduced photodamage to the sample. Furthermore, there is no need to use a UV laser, thus reducing chromatic aberration and cell damage. A summary of CLSM versus 2-PLSM is given in Table I. More recently, even three-photon excitation has been reported for UV fluorochromes. 42 Thus multiphoton laser scanning microscopy will become part of a new generation of laser scanning microscopes for three-dimensional imaging of interracial microbial communities. In con- clusion, future progress in three-dimensional imaging will further reduce the observation volume in all three dimensions, thereby leading to the ultimate resolution possible in light/laser microscopy. As a consequence for biofilm research, the freedom of imaging in four dimensions without significant disadvantages will become a reality.

Acknowledgments

The authors acknowledge the financial support of the Canada-Germany Agreement on Science and Technology and Environment Canada. The technical support of George D. W. Swerhone and Ute Kuhlicke is gratefully acknowledged.

39 S. Lindek, E. H. K. Stelzer, and S. Hell, in "Handbook of Confocal Microscopy" (J. B. Pawley, ed.), p. 417. Plenum Press, New York, 1995.

40 W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990). 41 W. Denk, D. W. Piston, and W. W. Webb, in "Handbook of Confocal Microscopy" (J. B.

Pawley, ed.), p. 445. Plenum Press, New York, 1995. 42 C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci.

U.S.A. 93, 10763 (1996).

Documents

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