Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.     

免疫磁珠分选人角朊干细胞的实验研究

目的:探索人角朊干细胞(keratinocyte stem cells,KSCs)的免疫磁珠分选(Magnetic Activated Cell Sorting,MACS)的方法。方法:从人包皮组织获得角朊细胞悬液,然后利用人角朊干细胞表面CD49f(整合素α6)和CD71的表达情况(CD49fbriCD71dim)通过MACS法将KSCs分选出来,在荧光显微镜下观察其荧光标记情况,同时将分选所得的CD49fbriCD71dim细胞进行体外无血清培养,观察其形态及克隆形成。结果:人角朊细胞经MACS法分选后所得的CD49fbriCD71dim细胞比率可达12．02(±6．92)%。CD49fbriCD71dim细胞经培养可见细胞为典型的上皮样特征,呈铺路石样形态,高核浆比例,细胞紧密排列,轮廓清楚,折光性好,并可在5-7天左右形成全克隆,符合KSCs的特点。结论:研究表明MACS分选法可以得到较高比例的人角朊干细胞,并能进行后续培养研究,其不失为一种较理想的人角朊干细胞的分选方法。     

Measuring and Sorting Cell Populations Expressing Isospectral Fluorescent Proteins with Different Fluorescence Lifetimes

Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs) fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP''s fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP) and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼1.5 ns vs ∼3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a “pseudophasor” that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.     

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis^1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA³. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment⁴. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion^5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages⁹. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment¹⁰. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture^11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)^13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs^9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting¹⁶. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.     

Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells

Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.     

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected ^1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) ³. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.Download video file.^{(80M, mov)}     

Host Cell Autophagy Modulates Early Stages of Adenovirus Infections in Airway Epithelial Cells

Human adenoviruses typically cause mild infections in the upper or lower respiratory tract, gastrointestinal tract, or ocular epithelium. However, adenoviruses may be life-threatening in patients with impaired immunity and some serotypes cause epidemic outbreaks. Attachment to host cell receptors activates cell signaling and virus uptake by endocytosis. At present, it is unclear how vital cellular homeostatic mechanisms affect these early steps in the adenovirus life cycle. Autophagy is a lysosomal degradation pathway for recycling intracellular components that is upregulated during periods of cell stress. Autophagic cargo is sequestered in double-membrane structures called autophagosomes that fuse with endosomes to form amphisomes which then deliver their content to lysosomes. Autophagy is an important adaptive response in airway epithelial cells targeted by many common adenovirus serotypes. Using two established tissue culture models, we demonstrate here that adaptive autophagy enhances expression of the early region 1 adenovirus protein, induction of mitogen-activated protein kinase signaling, and production of new viral progeny in airway epithelial cells infected with adenovirus type 2. We have also discovered that adenovirus infections are tightly regulated by endosome maturation, a process characterized by abrupt exchange of Rab5 and Rab7 GTPases, associated with early and late endosomes, respectively. Moreover, endosome maturation appears to control a pool of early endosomes capable of fusing with autophagosomes which enhance adenovirus infection. Many viruses have evolved mechanisms to induce autophagy in order to aid their own replication. Our studies reveal a novel role for host cell autophagy that could have a significant impact on the outcome of respiratory infections.     

The Acetyltransferase Activity of the Bacterial Toxin YopJ of Yersinia Is Activated by Eukaryotic Host Cell Inositol Hexakisphosphate

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFκB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP₆). We extend the applicability of our results to show that IP₆ also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP₆-induced conformational change in AvrA suggests that IP₆ acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP₆; once injected into mammalian cells by the pathogen these toxins bind host cell IP₆, are activated, and deregulate the MAPK and NFκB signaling pathways thereby subverting innate immunity.     

Viable Cell Detection by the Combined Use of Fluorescent Glucose and Fluorescent Glycine

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.     

Rapid Multiplex Detection and Differentiation of Listeria Cells by Use of Fluorescent Phage Endolysin Cell Wall Binding Domains

The genus Listeria comprises food-borne pathogens associated with severe infections and a high mortality rate. Endolysins from bacteriophages infecting Listeria are promising tools for both their detection and control. These proteins feature a modular organization, consisting of an N-terminal enzymatically active domain (EAD), which contributes lytic activity, and a C-terminal cell wall binding domain (CBD), which targets the lysin to its substrate. Sequence comparison among 12 different endolysins revealed high diversity among the enzyme''s functional domains and allowed classification of their CBDs into two major groups and five subclasses. This diversity is reflected in various binding properties, as determined by cell wall binding assays using CBDs fused to fluorescent marker proteins. Although some proteins exhibited a broad binding range and recognize Listeria strains representing all serovars, others target specific serovars only. The CBDs also differed with respect to the number and distribution of ligands recognized on the cells, as well as their binding affinities. Surface plasmon resonance analysis revealed equilibrium affinities in the pico- to nanomolar ranges for all proteins except CBD006, which is due to an internal truncation. Rapid multiplexed detection and differentiation of Listeria strains in mixed bacterial cultures was possible by combining CBDs of different binding specificities with fluorescent markers of various colors. In addition, cells of different Listeria strains could be recovered from artificially contaminated milk or cheese by CBD-based magnetic separation by using broad-range CBDP40 and subsequently identified after incubation with two differently colored CBD fusion proteins of higher specificity.Listeria belong to the low G+C Gram-positive bacteria; are ubiquitously present in nature; and can be isolated from many sources such as soil, water, sewage effluents, and the feces of humans and animals (52). Within the genus Listeria, six species are recognized (Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi), in addition to the recently proposed new species L. marthii (12) and L. rocourtiae (24). Five major serovar (sv.) groups exist (1/2, 3, 4, 6, and 7), and at least 16 subserovars can be distinguished. Variations are mainly due to the structure and composition of cell wall-associated carbohydrates, wall teichoic acid (WTA), and lipoteichoic acid (LTA). However, serotyping does not necessarily correlate with the species (7, 8). Listeria monocytogenes is an opportunistic, intracellular pathogen causing an infection termed listeriosis and is exclusively transmitted via contaminated food such as raw meat, milk products, fish products, and vegetables. Listeriosis is a serious infection primarily affecting immunocompromised patients, pregnant women, the elderly, and newborns and is characterized by a high mortality rate up to >40% (5). Strains belonging to sv. groups 1/2 and 4b have been responsible for the majority of Listeria infections in humans (52).Based on their host specificity, bacteriophages are useful tools for bacterial detection and differentiation (reviewed in references 40 and 43). To date, many phages infecting Listeria cells have been isolated. All of them are strictly genus specific. The few known virulent (obligately lytic) phages have a very broad host range (19, 29), whereas the majority are temperate and are restricted to a limited number of host strains within the individual serovar groups (32). Various applications based on Listeria phages have been developed, including phage typing (27) and the detection of viable Listeria cells by a recombinant luciferase reporter phage (31).Bacteriophage endolysins are peptidoglycan hydrolases that mediate lysis of the host cell at the end of the lytic multiplication cycle. These enzymes represent powerful tools with many applications in molecular biology, biotechnology, and medicine (26). Listeria phage endolysins show a domain organization and belong to category 1 of modular enzymes, in which catalysis and substrate specificity are clearly separated (18). They feature an N-terminal enzymatically active domain (EAD) and a C-terminal cell wall binding domain (CBD) (30, 33). The EAD determines the catalytic activity of the enzyme, and the CBD is responsible for targeting the protein to the bacterial cell wall. Surprisingly, the CBDs feature high binding specificity; although they are able to lyse all Listeria cell walls, the individual endolysins display highest activity against cells or cell walls from specific serovars. Besides recognition specificity, the CBDs feature very strong, saturation-dependent binding to listerial cell walls, with equilibrium constants in the nanomolar ranges (30). Because of the absence of an outer membrane in Gram-positive bacteria, the cell wall can also be accessed from outside, enabling the CBD to attach to its ligand. These properties can be harnessed for rapid and efficient labeling and immobilization of bacterial cells (23, 30). While CBD118 recognizes cells of Listeria sv. groups 1/2 and 3 and predominantly binds at the polar and septal regions of these cells, CBD500 and CBD PSA (21, 30) exhibit binding over the entire cell surfaces of strains, belonging to sv. groups 4, 5, and 6. Although the ligands recognized by the various CBDs have not been conclusively identified, we have strong evidence for an involvement of cell wall-associated carbohydrates in recognition and binding. Furthermore, the proteins retain their lectinlike binding function in complex matrices and environments, such as infected eukaryotic cells (15) and homogenized food and enrichment cultures (23).Fluorescent proteins (FP) such as the green fluorescent protein are very popular tools in molecular biology, medicine, and cell biology, based on their wide compatibility, lack of toxicity, incredible stability, and the fact that they do not require any cofactors other than oxygen for chromophore formation. Several FP derivatives with shifted spectral characteristics such as blue, cyan, and yellow fluorescent proteins have been developed (reviewed in references 45 and 49), which are useful for simultaneous staining applications. In addition to the green fluorescent protein (GFP) variants, several red fluorescent proteins have been described (14, 34). The RedStar protein (RS) (20) was derived from dsRed and features rapid development of high fluorescence intensity and a reduced tendency for oligomerization.The aim of the present study was to develop a comprehensive toolbox consisting of different combinations of Listeria phage CBDs and FPs. Toward this goal, we first established a classification system for CBDs from all known Listeria phage endolysins, and characterized representative CBDs from each class regarding their binding range binding affinity, and spatial distribution and density of ligands on the cell surface. We then used fluorescent proteins for the construction of differently tagged reporter-CBDs and demonstrate the suitability of using these proteins in a single and simple assay for simultaneous detection and differentiation of Listeria strains in mixed cultures. We also provide proof of concept for application of this technique for differential staining and identification of different Listeria strains after recovery from contaminated food by magnetic separation with CBD-coated paramagnetic beads (CBD-MS).     

建立以EGFP为报告基因检测HSV感染的细胞株

通过PCR扩增HSV-1ICP6启动子目的基因片段,克隆至真核表达载体pEGFP-1构建重组质粒pICP6-EGFP,转染Vero细胞,经G418筛选获得稳定转染细胞株Vero-ICP6-EGFP细胞。以不同量的HSV-1感染Vero-ICP6-EGFP细胞,分别以倒置荧光显微镜和流式细胞术检测EGFP荧光的量和强度。结果表明,转染细胞株Vero-ICP6-EGFP感染HSV-16h后,倒置荧光显微镜下即可检测绿色荧光;流式细胞术检测结果表明,EGFP荧光的量及强度随HSV-1病毒的滴度增加而增加。已建立的以EGFP为报告基因的Vero-ICP6-EGFP细胞株具有早期、快速、灵敏等优点,特异性较高,可望用于HSV感染的快速诊断及抗病毒药物的检测。     

Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE⁺ cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.     

Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.     

基于荧光激活细胞分选技术的超高通量酶活性筛选方法及其应用

基于荧光激活细胞分选(FACS)技术的超高通量酶活性筛选方法是新出现的一类高通量筛选技术.它利用流式细胞仪高灵敏度、高通量的特点,能以极高的速度(108/天)对大容量酶基因文库进行筛选.FACS筛选技术的出现突破了常规筛选方法低效、耗时、费力等瓶颈问题,极大地提升了人类对大容量基因文库的探索能力,因此在新酶基因筛选、酶活性检测、酶定向进化等领域有广泛的应用潜力.综述了FACS超高通量酶活性筛选方法的最新研究进展,着重介绍了其在酶定向进化中的应用.     

Localization and Identification of Populations of Phosphatase-Active Bacterial Cells Associated with Activated Sludge Flocs

Abstract The majority of phosphatase (PO₄ase) activity detected in fresh aerobic activated sludge from a municipal wastewater treatment plant was associated with suspended floc material. PO₄ase activity appeared to be localized in discrete bacteria-containing areas of the floc matrix based on the distribution of nucleic acid–stained cells and precipitated fluorescent crystals produced as a result of reaction of the enzyme(s) with the artificial substrate ELF™-PO₄. Of the total floc-associated bacterial cells that stained positive with the nucleic acid–binding fluorochrome acridine orange (AO), 8.8 ± 1.2% displayed PO₄ase activity based on the proximity of AO-stained cells to precipitated ELF crystals. Using a 16S rRNA oligonucleotide probe specific for the cytophaga–flavobacteria group, it was determined that 17–20% of the floc-associated bacteria that probed positive also displayed PO₄ase activity. Furthermore, 35–45% of the ELF fluorescence was associated with bacterial cells that probed positive for the cytophaga–flavobacteria group. The results suggest that the cytophaga–flavobacteria, as a group, is important in mediating the liberation of inorganic orthophosphate (P_i) from phosphomonoesters of detrital organic phosphate (organic-P) in the aerobic activated sludge process of wastewater treatment. Received: 17 March 1999; Accepted: 9 June 1999     

Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.     

Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pH_i) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pH_i, allowing fast and noninvasive determination of pH_i that is ideally suited for direct analysis of individual bacterial cells present in complex environments.