Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56150/1/20455_ftp.pd

Observation of calcium microdomains at the uropod of living morphologically polarized human neutrophils using flash lamp-based fluorescence microscopy

The present study outlines improved strategies for ratiometric imaging of cell calcium using a flash lamp-based excitation method and its application to neutrophil polarization. A brief (∼6 Μs) and intense flash was used to excite the Fluo-4 and Fura Red calcium dye combination in morphologically polarized human neutrophils. These illumination conditions do not allow the dye or calcium ions to diffuse significant distances during the exposure period. Buffer conditions such as pH, pyruvate concentration, and glucose levels were adjusted to more faithfully replicate these parameters in sepsis patients. Fluorescence images at both dyes' emission wavelengths were simultaneously collected using a Dual-View apparatus and an ICCD camera. The ratiometric images, when viewed as single frames or averaged image stacks, clearly demonstrated high calcium probe ratios at the uropod and comparatively low ratios at the cell body that were not evident using conventional imaging methods with longer exposure times. Calcium signaling at the uropod is likely associated with cytoskeletal remodeling during cell motility. © 2008 International Society for Advancement of CytometryPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60233/1/20580_ftp.pd

Bi-Directional Calcium Signaling Between Adjacent Leukocytes and Trophoblast-Like Cells

Citation Elfline M, Clark A, Petty HR, Romero R. Bi-directional calcium signaling between adjacent leukocytes and trophoblast-like cells. Am J Reprod Immunol 2010Trophoblasts are believed to play an important role in mitigating immunological responses against the fetus. To better understand the nature of trophoblast–leukocyte interactions, we have studied signal transduction during intercellular interactions.Using a highly sensitive microfluorometric ratioing method and Ca 2+ -sensitive dyes, we measured Ca 2+ signals in trophoblast-like cell lines (JEG-3 and JAR) or in leukocytes (neutrophils and monocytes) during intercellular contact.Trophoblast cell lines exhibit Ca 2+ signals during leukocyte contact. In contrast, leukocytes cannot elicit Ca 2+ signals in non-opsonized tumour cells, suggesting that Ca 2+ signaling is not a general feature of cell–cell encounters. Similarly, leukocytes demonstrate Ca 2+ signals during contact with trophoblast cell lines. Ca 2+ signals were confirmed using three dyes and with the Ca 2+ buffer BAPTA.We suggest that leukocyte-to-trophoblast interactions lead to mutual Ca 2+ signaling events in both cell types, which may contribute to immunoregulation at the materno–fetal interface.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79189/1/j.1600-0897.2010.00839.x.pd

Fluorescence spectroscopic detection of mitochondrial flavoprotein redox oscillations and transient reduction of the NADPH oxidase-associated flavoprotein in leukocytes

Steady-state and time-resolved fluorescence spectroscopy and fluorescence microscopy of leukocyte flavoproteins have been performed. Both living human peripheral blood monocytes and neutrophils have been utilized as experimental models, as the former relies much more heavily on mitochondrial metabolism for energy production than the latter. We confirm previous studies indicating that cellular flavoproteins absorb at 460 nm and emit at 530 nm, very similar to that of the FAD moiety. Furthermore, the emission properties of intracellular flavoproteins were altered by the metabolic inhibitors rotenone, antimycin A, azide, cyanide, DNP (2,4-dinitrophenol), and FCCP [carbonyl cyanide p -(trifluoromethoxy)phenylhydrazone]. Kinetic studies revealed flavoprotein emission oscillations in both monocytes and neutrophils. The flavoprotein intensity oscillations correlated with the physiological status of the cell and the nature of membrane receptor ligation. Microscopy revealed the presence of flavoprotein fluorescence in association with the plasma membrane, intracellular granules and distributed throughout the cytoplasm, presumably within mitochondria. Metabolic inhibitors such as cyanide suggest that the plasma membrane and granular components are cyanide-insensitive and therefore are likely associated with the flavoprotein component of the NADPH oxidase, which is located in these two compartments. This interpretation was found to be consistent with structural localization of the NADPH oxidase using an antibody molecule specific for this protein. Using peripheral blood neutrophils, which display less active mitochondria, and time-resolved emission spectroscopy, we show that the NADPH oxidase-associated flavoprotein undergoes a periodic transient reduction of about 54±2 ms in living cells. This finding is consistent with prior studies indicating that propagating substrate (NADPH) waves periodically promote electron transport across the NADPH oxidase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46725/1/249_2003_Article_361.pd

Tumor cell invasion of model 3‐dimensional matrices: demonstration of migratory pathways, collagen disruption, and intercellular cooperation

We report a novel 3‐dimensional model for visualizing tumor cell migration across a nylon mesh‐supported gelatin matrix. To visualize migration across these model barriers, cell proteolytic activity of the pericellular matrix was detected using Bodipy‐BSA (fluorescent upon proteolysis) and DQ™ collagen (fluorescent upon collagenase activity). For 3‐dimensional image reconstruction, multiple optical images at sequential z axis positions were deconvoluted by computer analysis. Specificity was indicated using well‐known inhibitors. Using these fluorescent proteolysis markers and imaging methods, we have directly demonstrated proteolytic and collagenolytic activity during tumor cell invasion. Moreover, it is possible to visualize migratory pathways followed by tumor cells during matrix invasion. Using cells of differing invasive potentials (uPAR‐negative T‐47D wild‐type and uPAR‐positive T‐47D A2–1 cells), we show that the presence of the T‐47D‐A2–1 cells facilitates the entry of T‐47D wild‐type cells into the matrix. In some cases, wild‐type cells follow T‐47D A2–1 cells into the matrix whereas other T‐47D‐wild‐type cells appear to enter without the direct intervention of T‐47D A2–1 cells. Thus, we have developed a new 3‐dimensional model of tumor cell invasion, demonstrated protein and collagen disruption, mapped the pathways followed by tumor cells during migration through an extracellular matrix, and illustrated cross‐talk among tumor cell populations during invasion.—Horino, K., Kindezelskii, A. L., Elner, V. M., Hughes, B. A., Petty, H. R. Tumor cell invasion of model 3‐dimensional matrices: demonstration of migratory pathways, collagen disruption, and intercellular cooperation. FASEB J. 15, 932–939 (2001)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154275/1/fsb2fj000392com.pd

Leukocyte Pyruvate Kinase Expression is Reduced in Normal Human Pregnancy but not in Pre-eclampsia

Citation Xu Y, Madsen-Bouterse SA, Romero R, Hassan S, Mittal P, Elfline M, Zhu A, Petty HR. Leukocyte pyruvate kinase expression is reduced in normal human pregnancy but not in pre-eclampsia. Am J Reprod Immunol 2010; 64: 137–151Emerging evidence suggests that metabolism influences immune cell signaling and immunoregulation. To examine the immunoregulatory role of glycolysis in pregnancy, we evaluated the properties of pyruvate kinase in leukocytes from non-pregnant women and those with normal pregnancy and pre-eclampsia.We evaluated pyruvate kinase expression in lymphocytes and neutrophils from non-pregnant, pregnant, and pre-eclampsia patients using fluorescence microscopy and flow cytometry. Leukocyte pyruvate kinase activity and pyruvate concentrations were also evaluated. To study pyruvate’s effect on signaling, we labeled Jurkat T cells with Ca 2+ dyes and measured cell responses in the presence of agents influencing intracellular pyruvate.The expression of pyruvate kinase is reduced in lymphocytes and neutrophils from normal pregnant women in comparison with those of non-pregnant women and pre-eclampsia patients. Similarly, the activity of pyruvate kinase and the intracellular pyruvate concentration are reduced in leukocytes of normal pregnant women in comparison with non-pregnant women and women with pre-eclampsia. Using Jurkat cells as a model of leukocyte signaling, we have shown that perturbations of intracellular pyruvate influence Ca 2+ signals.Normal pregnancy is characterized by reduced pyruvate kinase expression within lymphocytes and neutrophils. We speculate that reduced pyruvate kinase expression modifies immune cell responses due to reduced pyruvate concentrations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79222/1/j.1600-0897.2010.00881.x.pd

Toll-Like Receptor 4 (TLR4) of Retinal Pigment Epithelial Cells Participates in Transmembrane Signaling in Response to Photoreceptor Outer Segments

Retinal pigment epithelial (RPE) cells mediate the recognition and clearance of effete photoreceptor outer segments (POS), a process central to the maintenance of normal vision. Given the emerging importance of Toll-like receptors (TLRs) in transmembrane signaling in response to invading pathogens as well as endogenous substances, we hypothesized that TLRs are associated with RPE cell management of POS. TLR4 clusters on human RPE cells in response to human, but not bovine, POS. However, TLR4 clustering could be inhibited by saturating concentrations of an inhibitory anti-TLR4 mAb. Furthermore, human POS binding to human RPE cells elicited transmembrane metabolic and calcium signals within RPE cells, which could be blocked by saturating doses of an inhibitory anti-TLR4 mAb. However, the heterologous combination of bovine POS and human RPE did not trigger these signals. The pattern recognition receptor CD36 collected at the POS–RPE cell interface for both homologous and heterologous samples, but human TLR4 only collected at the human POS–human RPE cell interface. Kinetic experiments of human POS binding to human RPE cells revealed that CD36 arrives at the POS–RPE interface followed by TLR4 accumulation within 2 min. Metabolic and calcium signals immediately follow. Similarly, the production of reactive oxygen metabolites (ROMs) was observed for the homologous human system, but not the heterologous bovine POS–human RPE cell system. As (a) the bovine POS/human RPE combination did not elicit TLR4 accumulation, RPE signaling, or ROM release, (b) TLR4 arrives at the POS–RPE cell interface just before signaling, (c) TLR4 blockade with an inhibitory anti-TLR4 mAb inhibited TLR4 clustering, signaling, and ROM release in the human POS–human RPE system, and (d) TLR4 demonstrates similar clustering and signaling responses to POS in confluent RPE monolayers, we suggest that TLR4 of RPE cells participates in transmembrane signaling events that contribute to the management of human POS

Evidence for chronic, peripheral activation of neutrophils in polyarticular juvenile rheumatoid arthritis

Although strong epidemiologic evidence suggests an important role for adaptive immunity in the pathogenesis of polyarticular juvenile rheumatoid arthritis (JRA), there remain many aspects of the disease that suggest equally important contributions of the innate immune system. We used gene expression arrays and computer modeling to examine the function in neutrophils of 25 children with polyarticular JRA. Computer analysis identified 712 genes that were differentially expressed between patients and healthy controls. Computer-assisted analysis of the differentially expressed genes demonstrated functional connections linked to both interleukin (IL)-8- and interferon-γ (IFN-γ)-regulated processes. Of special note is that the gene expression fingerprint of children with active JRA remained essentially unchanged even after they had responded to therapy. This result differed markedly from our previously reported work, in which gene expression profiles in buffy coats of children with polyarticular JRA reverted to normal after disease control was achieved pharmacologically. These findings suggest that JRA neutrophils remain in an activated state even during disease quiescence. Computer modeling of array data further demonstrated disruption of gene regulatory networks in clusters of genes modulated by IFN-γ and IL-8. These cytokines have previously been shown to independently regulate the frequency (IFN-γ) and amplitude (IL-8) of the oscillations of key metabolites in neutrophils, including nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and superoxide ion. Using real-time, high-speed, single-cell photoimaging, we observed that 6/6 JRA patients displayed a characteristic defect in 12% to 23% of the neutrophils tested. Reagents known to induce only frequency fluctuations of NAD(P)H and superoxide ion induced both frequency and amplitude fluctuations in JRA neutrophils. This is a novel finding that was observed in children with both active (n = 4) and inactive (n = 2) JRA. A subpopulation of polyarticular JRA neutrophils are in a chronic, activated state, a state that persists when the disease is well controlled pharmacologically. Furthermore, polyarticular JRA neutrophils exhibit an intrinsic defect in the regulation of metabolic oscillations and superoxide ion production. Our data are consistent with the hypothesis that neutrophils play an essential role in the pathogenesis of polyarticular JRA

Nonlinear gyrokinetic simulations of the I-mode high confinement regime and comparisons with experimenta)

For the first time, nonlinear gyrokinetic simulations of I-mode plasmas are performed and compared with experiment. I-mode is a high confinement regime, featuring energy confinement similar to H-mode, but without enhanced particle and impurity particle confinement [D. G. Whyte et al., Nucl. Fusion 50, 105005 (2010)]. As a consequence of the separation between heat and particle transport, I-mode exhibits several favorable characteristics compared to H-mode. The nonlinear gyrokinetic code GYRO [J. Candy and R. E. Waltz, J Comput. Phys. 186, 545 (2003)] is used to explore the effects of E × B shear and profile stiffness in I-mode and compare with L-mode. The nonlinear GYRO simulations show that I-mode core ion temperature and electron temperature profiles are more stiff than L-mode core plasmas. Scans of the input E × B shear in GYRO simulations show that E × B shearing of turbulence is a stronger effect in the core of I-mode than L-mode. The nonlinear simulations match the observed reductions in long wavelength density fluctuation levels across the L-I transition but underestimate the reduction of long wavelength electron temperature fluctuation levels. The comparisons between experiment and gyrokinetic simulations for I-mode suggest that increased E × B shearing of turbulence combined with increased profile stiffness are responsible for the reductions in core turbulence observed in the experiment, and that I-mode resembles H-mode plasmas more than L-mode plasmas with regards to marginal stability and temperature profile stiffness.United States. Department of Energy (Contract No. DE-FC02-99ER54512-CMOD)United States. Department of Energy. Office of Science (Contract No. DE-AC02- 05CH11231