Tissue Optics And Spectroscopy

The depth that light penetrates tissue is wavelength dependent and is affected largely by hemoglobin absorption.13,14 Shorter wavelengths of light (ultraviolet to blue) are absorbed by hemoglobin in mammalian tissue to a greater extent than are longer wavelengths of light (red to near infrared). At wavelengths of 700 nm or longer, absorption is minimal, yet scattering continues to affect the path that a given photon travels in tissue. Wavelengths of bioluminescent light have been described from 490 to 700 nm,11,15 although the best-characterized bioluminescent reporters, those most often used in biological assays (bacterial, jellyfish, and firefly luciferases), are blue to yellow-green (490 to 560 nm). The ability to detect light through tissues is influenced by tissue depth and optical features, or opacity of the tissues. Therefore, detecting light emitted from tissues deep within a mammal or through relatively opaque organs, such as liver, is less efficient than detection through translucent structures or at more superficial sites (such as skin and bone).

Some knowledge of the depth of the signal and nature of the tissue between the signal and the detector is useful in the analysis of optically based reporters in living tissues. In addition, the consistency and intensity (as determined, in part, by the promoter strength) of the bioluminescent signal contribute to its detection and quantitation. Quantification of an internal signal is possible provided that the parameters of depth, intensity, and opacity are held constant in the model system. Thus, signals at specific tissue sites with a relatively constant physiology can be reliably assessed.1,3,7 There are a number of advantages to using an internal biological source of light over other optical signatures. A significant advantage of monitoring bioluminescence in vivo over fluorescence is that there are few, if any, sources of light in mammalian systems, and, therefore, background is minimal if not completely absent. The use of luminescent tags permits an integrated approach whereby the same label can be used in vitro and in correlative cell culture assays, and then used in vivo to test the predictions made in vitro. Moreover, once studies in the animal model are completed, the bioluminescent tag can be used to quantify the tagged organism or process in ex vivo biochemical assays. The results of the animal studies can thus be verified using the standard assays that measure protein levels, enzymatic activity, or amounts of nucleic acid. In these bacterial infectious disease models, the infecting pathogen can be readily distinguished from normal flora using the bioluminescent tag. For gastrointestinal models,1 this feature is a tremendous asset, given the number of microorganisms that comprise the normal flora of the gut. In addition, since pathogens tend to adhere to tissues, in vivo monitoring of bioluminescence may provide more accurate quantitation than aspirates, washes, or homogenized tissues where adherent organisms pellet with debris or are not released from the tissues because the inhomogeniety of bacterial-tissue suspensions skews recovery and quantitation of pathogens.


lux operons from bioluminescent bacteria are ideal for labeling bacterial pathogens since they not only encode the luciferase genes but also the genes for the biosynthetic enzymes for synthesis of the substrate.16,17 Use of these operons negates the need for an exogenous supply of substrate. Moreover, no deleterious effects of substrate biosynthesis on bacterial metabolism or virulence have been observed.18 Among the lux operons from bioluminescent bacteria, those from Photorhabdus luminescens (previously known as Xenorhabdus luminescens)19'20 appears to be ideally suited for use in mammalian animal models, given that mammalian body temperatures lie within the optimum temperature range for this enzyme.17,21 This is in contrast to the low optimal temperature range for beetle luciferases (Luc) and other characterized bacterial luciferases (those from Vibrio spp.).

Five essential genes that are organized in an operon as luxCDABE are necessary for the synthesis of light in naturally occurring bioluminescent bacteria. Blue-green light is emitted from these bacteria with a peak at 490 nm as a result of a heterodimeric luciferase (encoded by luxAB) catalyzing the oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain fatty aldehyde (synthesized by a fatty acid reductase complex, encoded by luxCDE). Although a number of additional lux genes have been identified in bioluminescent bacteria, only luxA-E are essential for the biosynthesis of light.17


Well-characterized vectors have been used for the expression of the lux operons in Gram-negative organisms. The original pUC-based plasmid, used to clone the lux operon from P. luminescens including the promoter region (vector designated pCGLS1)16 is well suited for expressing luciferase from Gram-negative organisms such as Escherichia coli and Salmonella. Introduction of the lux-encoding vector into cells of these bacterial species can be carried out using standard methods of bacterial cell transformation for Gram-negative organisms. Alternatively, the lux operon can be stably integrated into the chromosome of a wide range of Gram-negative bacteria using lux transposons, such as Tn5 luxCDABE.22

Recently, using both lux transposons and homologous recombination, we have generated a wide range of stable, highly bioluminescent Gram-negative bacteria, including several strains of pathogenic E. coli, Haemophilus influenza, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and different species of Salmonella, Shigella, and Yersinia. These bioluminescent bacteria are currently being tested in a number of different animal models of infection.


Although a light-encoding lux operon can easily be introduced into a variety of Gram-negative bacteria to confer a bioluminescent phenotype, because all identified species of naturally occurring marine and terrestrial bioluminescent bacteria are Gram-negative, the transformation of Grampositive bacteria to a light phenotype has been problematic because of the differing genetics of these two bacterial groups. Bioluminescent Gram-positive bacteria such as Staphylococcus aureus and Mycobacterium tuberculosis have been constructed. However, these bacteria contain the firefly luciferase gene (luc) or variations of isolated bacterial luxAB luciferase genes, each of which require the addition of an exogenous substrate to allow bioluminescence. Moreover, most bioluminescent Gram-positive bacteria have been generated using bacterial luciferase genes that encode enzymes that are unstable at temperatures above 30°C. Although such bacteria are useful for environmental studies (e.g., the assessment of food products for contamination by such bacteria), luxAB constructs that only permit bioluminescence to occur below 30°C are of limited use for experimentation on pathogenicity carried out at 35°C and above in vivo. To address this problem, we have recently reengineered the entire Photorhabdus luminescens luxCDABE operon, allowing Gram-positive infections to be monitored in vivo in live animal.


Use of bioluminescent reporters in bacteria provides a rapid means of quantitation that is also amenable to use in high-throughput antibacterial drug screening. Assays for minimal inhibitory concentrations (MIC) of drugs can be rapidly performed on cultures of living bacteria and are particularly useful in the case of slow-growing bacteria such as Mycobacterium. More elaborate correlative cell culture assays, involving the interaction of bacterial cells with those of a mammalian host, can also be conducted to provide a rapid means for evaluating biological events for patho-genesis and gene expression prior to introduction into living animals. Contag et al.1 described functional assays, Salmonella adherence and entry, that correlate cell-cell interactions to in vivo pathogenesis, where bioluminescence was used as a marker in these live cell assays. Similar assays have been described for many organisms, and the imaging can be performed on populations of cells using macroscopic detection, or on single living cells with microscopic detection.23 Tagging bacterial pathogens with luciferase enables in vitro, in vivo, and ex vivo assays to be optimally integrated such that an analysis can be run full circle with a single reporter gene at its hub.

Rapidly evolving paradigms in drug development, including combinatorial chemistries, genom-ics, high-throughput in vitro screening, and chip technologies are generating hundreds or even thousands of potential lead compounds for a given disease that require efficacy testing in animal models. Drug screening in animals remains a significant bottleneck in the development of therapeutics and can be severely limiting. Noninvasive in vivo assays would accelerate the data acquisition, require fewer animals for drug testing, and produce significantly more information per protocol. The increased depth of understanding that could be obtained through noninvasive assays for pathogenic mechanisms and potential interventions would yield improved preclinical data and ultimately preservation of time, effort, and investments.


In the initial study by Contag et al.,1 patterns of disease caused by three strains of bioluminescently labeled Salmonella were monitored over an 8-day time course in mice. Groups of mice were infected orally, the natural route of infection for mice or humans, with three strains of Salmonella: the wildtype, SL1344lux, the less invasive mutant, BJ66lux, or the less virulent strain, LB5000lux. Images were obtained daily using an intensified charge-coupled device (CCD) camera. At 1 to 2 days, the bioluminescent signal localized to a single focus in all infected animals. The distribution of bioluminescence did not spread in mice inoculated with the BJ66lux. This was in contrast to mice infected with the less virulent LB5000lux, where bioluminescence was not detected in any animal at 7 days. In the mice infected with the wild-type SL1344lux, bioluminescence was detected throughout the study period, with multiple foci of transmitted photons at 8 days. In one third of the animals infected with SL1344lux, transmitted photons were apparent over much of the abdominal

Unilateral Bilateral

FIGURE 40.1 (Color figure folllows p. 266.) Murine models of bacterial pneumonia. Mice were inoculated by intranasal inoculation using a labeled strain of P. aeruginosa. Immediately after infection, the mice were imaged using an intensified CCD camera (model C2400-32, Hamamatsu, Japan).

Unilateral Bilateral

FIGURE 40.1 (Color figure folllows p. 266.) Murine models of bacterial pneumonia. Mice were inoculated by intranasal inoculation using a labeled strain of P. aeruginosa. Immediately after infection, the mice were imaged using an intensified CCD camera (model C2400-32, Hamamatsu, Japan).

area at 8 days, resembling the distribution of photons in a systemic infection following an intraperitoneal inoculation.1

The lux operon has also been used to generate labeled Pseudomonas aeruginosa and these organisms have been utilized in mouse lung infection models (Figure 40.1). In these models, variation in infection was observed within groups of mice that were inoculated in exactly the same manner. This illustrates how imaging can improve the data sets, in that the animals with bilateral infections may present with a different disease course than animals with unilateral infections. Also, if tissue samples are to be obtained from these animals, the images can serve as a guide for what is to be expected, and directed sampling may improve the data analyses.

In vivo imaging has been adapted for drug screening and drug development assays.1'3'24 Recently, Rocchetta et al.24 generated a highly bioluminescent strain of an E. coli clinical isolate, EC 14, using a multicopy plasmid carrying the full luxCDABE operon. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidine, tetracycline, or ciprofloxacin), a strong correlation (0.98) existed between bioluminescence levels and viable cell counts in vitro and in vivo. Moreover, this study showed that the ability to measure the same animals repeatedly reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy.

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