Imaging techniques in microbiology

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Imaging techniques in microbiology David C Fung and Julie A Theriot Recent advances in optical imaging have dramatically expanded the capabilities of the light microscope and its usefulness in microbiology research. Some of these advances include improved fluorescent probes, better cameras, new techniques such as confocal and deconvolution microscopy, and the use of computers in imaging and image analysis. These new technologies have now been applied to microbiological problems with resounding success.

Address Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5307, USA

Current Opinion in Microbiology 1998, 1:346-351 http://biomednet.com/elecref/1369527400100346 © Current Biology Ltd ISSN 1369-5274

Abbreviations 3D three dimensional CCD charged-coupleddevice DIC differentialinterference contrast GFP green fluorescent protein

Introduction

T h e past decade has seen major advances in optical imaging techniques. These have tremendously increased the scope of biological problems that can be addressed by microscopy. In this review we give a brief introduction to these techniques and give examples of some of their recent applications to microbiology. We limit ourselves to the discussion of methods that are currently available to researchers commercially, either as complete systems or as easily integrated components. For useful references on optical imaging and microscopy, see [1,2",3,4]. Technical developments Fluorescence

Much of the current power of microscopy can be attributed to the development of new and improved fluorescent probes. The old methods of producing contrast, namely phase contrast and differential interference contrast (DIe), remain essential tools for microscopy; however, developments in fluorescence, with its tremendous versatility, have opened up entirely new areas of research. Immunofluorescence has the advantage of biological specificity, and the use of green fluorescent protein (GFP) [5] allows the expression of a fluorophorc within living cells. Fluorescence has also been used extensively for the biological applications of confocal and deconvolution microscopy (see below). Along with these advances in probes have come improvements in optical components. For example, muhibandpass filters are now available that allow the detection of up to four different fluorophores without changing filter sets.

Cameras

Complementing the advances in fluorescent probes have been the advances in low-light-level detection [2°°]. Slow-scan charged-coupled device (CCD) cameras, which were first developed for use in astronomy, have become the detectors of choice for low-light-level fluorescence microscopy. These solid state devices are cooled to reduce thermal noise and, in addition to their high sensitivity, exhibit excellent spatial resolution, geometric linearity, and photometric linearity. As they are made up of arrays of pixels, they allow great flexibility in read-out. For example, smaller areas can be imaged with increased speed, or blocks of pixels can be grouped together for increased sensitivity (although at the expense of spatial resolution). The main drawback of slow-scan CCD cameras is their low temporal resolution. Full-frame acquisition and transfer times may range from 0.1-I0 seconds, depending on the brightness of the sample. For real-time low-light-level imaging, video rate (25-30 frames per second) CCD cameras equipped with intensifiers (ICCDs) provide speed at the expense of noise and loss of spatial resolution. Improvements in frame transfer rates, however, have made slow-scan CCD cameras viable alternatives to video-rate I C e D cameras for applications requiring imaging on the 100 ms time scale, such as for recording the movement of bacterial pathogens inside host ceils. Digital imaging

A major revolution in microscopy has occurred in the way that images are acquired and manipulated [6°]: digital images have now largely replaced film. This has major advantages beyond the convenience of image storage and reproduction. Digital images can be processed to improve image quality, for example through background subtraction, sharpening, and spatial filtering. They also permit quantitative computer analysis. Digital images, particularly three-dimensional (3D) images, can be easily displayed and manipulated (e.g. rotated) on computers. Finally, new imaging modalities such as dcconvolution microscopy (see below) require intensive computation that can only be performed on digital images. Three-dimensional imaging

T h e major problem with imaging thick samples in fluorescence microscopy is the contamination of the signal from the focal plane by light from out-of-focus regions. Two techniques, confocal microscopy and deconvolution microscopy, have been successfully developed to solve this problem. By removing out-of-focus glare, they provide much sharper 2D images. This also increases the axial (perpendicular to the focal plane) resolution, and enables optical sectioning and thus 3D imaging of thick specimens.

Imaging techniques in microbiology Fung and Theriot

Confocal microscopy Confocal microscopes solve the problem of out-of-focus light by placing an aperture in front of the detector that admits only light from the focal plane [3,7°]. In the most commonly used implementation, the specimen is illuminated by a narrowly focused spot of light. Fluorescence emissions from this small spot are collected by the objective and focused onto its focal plane. A pinhole aperture placed at this 'confocal' position allows the focused light through, to be collected by a detector on the other side. Fluorescence emanating from outside the focal plane, however, is defocused at the pinhole and is mostly blocked. As the specimen is only illuminated a point at a time, two-dimensional images are obtained by rapidly scanning the illumination over the sample, known as raster scanning. Three-dimensional images are obtained by repeating this process at different focal planes. Deconvolution microscopy Deconvolution microscopy uses conventional (wide-field) microscopy to obtain a series of images at different focal planes. T h e images are then computationally deconvolved, or deblurred, using iterative algorithms which remove out-of-focus photons and 'restore' them to within their correct focal plane [8,9,10"]. This technique has been made practical only by the availability of more powerful computers. Several different algorithms are available for deconvolution. Some require a knowledge of the imaging characteristics of the microscope (the point spread function), which can be obtained empirically, whereas 'blind'

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deconvolution is accomplished with only the images of the specimen using maximum-likelihood statistics. Confocal versus deconvolution

Despite their fundamentally different approaches, both confocal and deconvolution microscopy can produce similarly excellent images. Some of the pertinent characteristics of confocal scanning microscopy and deconvolution microscopy are summarized in Table 1. Each method has its advantages and disadvantages (and supporters and detractors). Deconvolution microscopy is more sensitive, and may be preferable for in vivo work if photodamage or phototoxicity are concerns, because it does not utilize the intense laser illumination required by confocal microscopes. On the other hand, the pinhole confers on the confocal microscope some advantage in lateral resolution [11], and confocal images can still be improved by computational deblurring. Which method will produce superior results depends on the nature of the specimen and the particular features of the instruments under consideration. A more detailed comparison of these two imaging methods can be found in [12]. Multiphoton microscopy

Recently, a new point scanning tcchnique has become available that rectifies some of the drawbacks of confocal microscopy [13,14]. In multiphoton microscopy, illumination at the exciting wavelength is replaced by intense infrared light of two or three times longer wavelength (and hence with photons of one half or one third the

Table 1 Comparison of laser scanning confocal microscopy and deconvolution microscopy. Properties

Laser confocal scanning microscopy

Deconvolution microscopy

Specimen illumination mode

Point scanning

Wide-field

Light source

Laser: useful chromophores limited by available laser frequencies

Arc lamp: can use all conventional chromophores

Sensitivity

Potentially less sensitive: out-of-focus photons not utilized photomultiplier tube dectectors have lower quantum efficiency

Potentially more sensitive: utilizes all collected photons slow-scan CCD cameras have higher quatum efficiency

Lateral resolution (within focal plane)

Pinhole provides some improvement over classic lateral resolution

Classic lateral resolution with out-of-focus blur removed

Specimen thickness

Better for thick specimens with uniform staining

Better for thick specimens with localized fluorescence; with uniform staining, out-of-focus signal may overwhelm in-focus signal

Specimen light exposure

Greater

Lesser

Single 2D images

OK

Requires several 2D sections for deconvolution

Data acquisition time

Slower: sample has to be raster scanned*

Faster: entire field imaged simultaneously

Computation

Confocal images can still be deblurred for highest image quality

Intensive computation required

*Other types of confocal microscopes provide faster imaging but compromise spatial resolution and sensitivity.

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energy). Fluorophore excitation occurs only when multiple photons are absorbed simultaneously, an improbable event except at the position where the photon energy density is the highest. This eliminates out-of-focus photobleaching by limiting the excitation to the focal volume, instead of to a cone-shaped volume above and below the focal point as in confocal microscopy. Furthermore, the problem of out-of-focus light is obviated and the higher axial resolution of the confocal microscope is maintained without the need for a confocal aperture. For living systems, however, the potential for photodamage from the intense infrared illumination may be a serious problem.

Developments with GFP Although GFP has already provided a powerful tool in biological research, improvements and new applications are continually being devised [32]. Of particular interest to microbiologists are mutant forms of GFP that are brighter and more efficiently folded than the wild-type protein [33]. These show detectable fluorescence eight minutes after induction of transcription in E. co/i, compared with 1-2 hours for wild-type GFP. T h e y have been used in conjunction with a fluorescence-activated cell sorter to identify genes of Salmonella typhimu~um that are expressed inside macrophage phagosomes [34], and should be useful for measuring gene expression in real time.

Applications to m i c r o b i o l o g y research Fluorescence microscopy for subcellular localization In the past few years, fluorescence microscopy has become a widely used tool for visualizing the subcellular localization of proteins within bacteria. It is much more sensitive and easier to use than immunoelectron microscopy, although it cannot match the spatial resolution of electron microscopy. Two labeling methods have been employed: indirect immunofluorescence requires that the bacteria be fixed and treated with lysozyme [15,16], whereas the use of GFP fusion proteins allows live bacteria to be observed [17]. These methods have been used to study the proteins required for cytokinesis in Escherichia co/i, several of which have now been shown to localize to the site of cell division at various times during the cell cycle (reviewed in [18]; see also [19",20,21]). By conventional fluorescence microscopy, these proteins arc usually seen localized as transverse bands. Only when seen end-on or at an angle does a ring-like distribution becomes apparent [22]. By deconvolution microscopy, with serial sections taken 0.1-0.2 ~tm apart, the bands are sharper than by conventional microscopy, and there is the further advantage that the ring-like distribution can be demonstrated by rotating the images by computer [17]. Other proteins that have been localized by fluorescence microscopy include chromosome partitioning proteins in Bacillus subtilis [23,24] and Caulobacter crescentus [25].

As an example of how GFP continues to surprise, it has recently been discovered that GFP can be photoactivated by blue light to produce a stable red-emitting form under conditions of low oxygen concentration. This has been demonstrated in Schizosaccharomyces pombe, in E. coli, and in vitro for the wild-type GFP as well as for several variants [35°,36"*]. By photoactivating GFP at one pole of a bacterium and following the spatial distribution of the red fluorescence, the diffusion coefficient of a protein in the bacterial cytoplasm has now been measured for the first time [36"'].

Fluorescence microscopy has also been used to observe DNA segregation during the bacterial cell cycle. This has been done by inserting multiple copies of the lac operator into the chromosome and expressing G F P - L a c I [26,27], or by fluorescence in situ hybridization of the F plasmid [28]. Overall, fluorescence microscopy has the capability to be very sensitive. For example, the spatial distribution of a protein present at a level of only about 100 molecules per cell has been reported using indirect immunofluorescence [19"]. Greater sensitivity is still possible as single fluorescent molecules have been imaged in aqueous solution using specialized equipment [29]. Fluorescence has also been used to detect single copies of mRNA within individual bacteria using in situ polymerase chain reaction (PCR) [30,31].

Interactions of pathogens with hosts Confocal and deconvolution microscopy are most useful for examining relatively thick specimens, which many microbes are not. One area where these technologies are proving invaluable, however, is in studying the interactions between microbial pathogens and host cells. T h e malaria parasite Plasmodium falciparum induces the formation of membrane structures in host erythrocytes. These structures were first observed by transmission electron microscopy, but their topology remained unknown until confocal microscopy was used to perform 3D sectioning. This demonstrated that, instead of consisting of discrete vesicles, the membrane structures form an interconnected network (Figure 1) [37]. Subsequently, deconvolution microscopy has shown that exogenously added Lucifer yellow dye is found in both this network and in the parasite. If the formation of the network is blocked, however, no dye is found in the parasite, providing evidence that the network is involved in transporting nutrients to the parasite [38]. In the study of bacterial pathogencsis, confocal microscopy permits the sensitive detection of bacteria in much thicker sections than is possible by conventional immunohistochemistry. This permits the use of much smaller, more realistic, infectious doses to study pathogenesis in vivo; artificially high inocula are no longer necessary to facilitate bacterial visualization [39"']. As further examples, confocal microscopy has been used to study the permeability properties of the Chlamydia trachomatis parasitophorous vacuolar membrane [40], and the interaction of S. typhimurium with the host-cell actin cytoskeleton [41].

Imaging techniques in microbiology Fung and Theriot

Figure 1

'0 Three-dimensional reconstruction of the surface of Plasmodium falciparum and associated membrane structures inside an infected erythocyte to which the dye Lucifer yellow has been added, demonstrating that the membrane structures form an interconnected network. Sequential images (left to right, top to bottom), obtained from confocal microscopy, are rotated around the x-axis at 40" intervals. Warm colors (reds) are closest to the reader, cooler colors (blue) are furthest away. Reproduced with permission from [3?].

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[44°°]. Microtubules were labeled with a dynein-GFP fusion protein expressed at very low levels to prevent interference with the cell cycle. In order to prevent photobleaching and phototoxicity, low excitation light levels were used. The challenge was to image this weak signal (barely detectable by eye) in a 3D volume of approximately 6 g m dimension over the duration of the cell cycle (up to 120min). Conventional wide field microscopy and a slow-scan CCD camera were optimized for optical efficiency, and digital background subtraction and image processing were utilized. Multimode imaging, which allowed 4',6-diamidino-2-phenlindole (DAPI)stained chromosomes and DIC images to be taken as well, was made possible by a multibandpass filter and computer-controlled filter wheels and shutters. Normally, the yeast spindle is not visible by DIC, but embedding the cells in 25% gelatin improved the contrast to the extent that spindle position could be easily monitored. Five fluorescent GFP images taken 1 btm apart in the axial direction provided a 3D data set. For this application, true 3D imaging was not necessary, and image deconvolution was not utilized. Instead, noise was digitally removed and the 3D data sets were projected onto two dimensions and overlaid onto the DIC images. The resulting time-lapse sequences beautifully show how dynamic instabilities of the astral microtubules push the nucleus around in the G1 stage of the cell cycle, and how astral microtubule penetration into the bud is required for nuclear movement to the neck of budding yeast ([45"]; these sequences may be seen at www.unc.edu/depts/biology/bloomlab/gfp.htm).

Conclusions Time-lapse imaging Time is one dimension in imaging, and for this aspect video microscopy remains essential. For example, video microscopy has recently been used to observe the interactions between Candida albicans and macrophages, and to compare these interactions for the wild-type pathogen and nonfilamentous mutants [42]. Video data can now be digitally processed and stored. Here, 'video' refers to the rate of image acquisition (25-30 frames per second), and not to storage on video tape. Digitized time-lapse sequences permit much more precise quantitative analysis of movement than tape recordings, and have been used to study the relationship between movement and actin dynamics in the intracellular movement of ListeHa m onocytogenes [43]. Although video microscopy is limited to two spatial dimensions, it is now also possible to obtain 3D time-lapse imaging. An excellent example, which also demonstrates what can be achieved by integrating current imaging technology, is provided by the recent work on astral microtubule dynamics and nuclear migration in yeast

Twenty years ago, optical microscopy may have been seen as a static technology. That is clearly not the case today. Improvements are constantly being made in all aspects of imaging technology, from lasers to cameras to probes. Confocal and deconvolution microscopy are now established techniques, yet continue to be refined. The third generation of commercial confocal microscopes is now available on the market, while ever more powerful computers allow more sophisticated deconvolution algorithms to be implemented. Although we have focused on currently available technology, new imaging modalities are also being developed. Among these is near-field scanning microscopy, which promises significantly better resolution than the methods we have discussed here but whose applicability remains to be proven [46]. We have shown how these advances in imaging have contributed to microbiology research and how they will continue to do so as microbiologists become increasingly aware of their power.

Acknowledgements We thank K Haldar for the figure.

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