Bioluminescence Microscopy Research Articles

Combining fluorescence and bioluminescence microscopy.

ABSTRACTBioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin—a substrate of luciferase in promoter assays of gene expression in cultured cells—confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492–506 nm and 524–578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol‐12‐myristate‐13‐acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells. Microsc. Res. Tech. 78:715–722, 2015. © 2015 Wiley Periodicals, Inc.

Bioluminescence microscopy using a short focal‐length imaging lens

Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.

Forkhead Box O1 (FOXO1) Protein, but Not p53, Contributes to Robust Induction of p21 Expression in Fasted Mice

Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes.

Rapid and High-Sensitivity Cell-Based Assays of Protein−Protein Interactions Using Split Click Beetle Luciferase Complementation: An Approach to the Study of G-Protein-Coupled Receptors

To identify biologically relevant compounds in basic biology and drug discovery processes, rapid quantitative methods for elucidating protein-protein interactions have become necessary. We describe a novel optical technique for monitoring protein-protein interactions in living cells based on complementation of split luciferase fragments from click beetle (Brazilian Pyrearinus termitilluminans). A new pair of amino-terminal and carboxy-terminal fragments of the luciferase was identified using semirational library screening, demonstrating achieved markedly higher sensitivity and signal-to-background ratio. The identified fragments were applied to the study of five G-protein coupled receptors (GPCR) that interact with beta-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and beta-arrestin connected with the luciferase fragments, we demonstrated rapid and sensitive screening of potential chemicals that act on GPCRs using a 96-well microtiter plate format. The screening time was reduced to 5-10 min after ligand stimulation. The maximum response became more than 15-fold higher than the background signal. This luciferase complementation method also enabled accurate spatial and temporal analyses of interactions in single living cells using bioluminescence microscopy. These GPCR assays will facilitate developments of high-throughput screening systems in a multiwell plate format. Furthermore, using specific proteins of interest, the novel fragments of luciferase will provide different assay methods for the study of many intracellular signals in living cells and animals.

Stem cell‐mediated accelerated bone healing observed with in vivo molecular and small animal imaging technologies in a model of skeletal injury

Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [(18)F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [(18)F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC's traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p < 0.05). Systemically administered ADMC's traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route.

Bioluminescence microscopy for cellular level circadian analysis in the suprachiasmatic nucleus

Traditionally, luminescence has not been able to provide sufficient spatial resolution at the microscopic level without the use of expensive imaging equipment. The new Olympus LV200 microscope has changed all this; its unique optical geometry and precise incubation capabilities enable acquisition of even the faintest signals with cellular-level detail. This has opened up new frontiers in many areas of research, especially in the study of circadian rhythm–related events.

Stably IntegratedluxCDABEfor Assessment ofSalmonellaInvasion Kinetics

Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC. The aim of this work was to characterize the invC dependence of invasion kinetics into disparate eukaryotic cells traditionally used as models of gut epithelium or neoplasms. Thus, a nondestructive real-time assay was developed to report eukaryotic cell invasion kinetics using lux+ Salmonella that contain chromosomally integrated luxCDABE genes. Bioluminescence-based invasion assays using lux+ Salmonella exhibited inoculum dose-response correlation, distinguished invasion-competent from invasion-incompetent Salmonella, and discriminated relative Salmonella invasiveness in accordance with environmental conditions that induce invasion gene expression. In standard gentamicin protection assays, bioluminescence from lux+ Salmonella correlated with recovery of colony-forming units of internalized bacteria and could be visualized by bioluminescence microscopy. Furthermore, this assay distinguished invasion-competent from invasion-incompetent bacteria independent of gentamicin treatment in real time. Bioluminescence reported Salmonella invasion of disparate eukaryotic cell lines, including neoplastic melanoma, colon adenocarcinoma, and glioma cell lines used in animal models of malignancy. In each case, Salmonella invasion of eukaryotic cells was invC dependent.

Lights, Camera, Action!

Understanding the vast array of diverse biochemical processes of the living cell is paramount to not only gaining an insight into the living organism as a whole, but also discovering when these cellular processes break down. Whilst innovative and specialised techniques have been instrumental for many advances in molecular biology, many questions concerning the structure and function of cells can only be answered by observing living cells. Live-cell imaging offers the power of capturing the dynamics of biological action in live cells and in real-time, something not previously available with biochemical approaches. Among the many optical tools that enable the capture of these cellular and molecular events, bio-luminescence imaging offers important opportunities. Although the concept of bio-luminescence microscopy (BLM) is not a novel one, recent advances introduced with new specialised imaging systems have made it a more realistic procedure.

Advanced imaging and detection R&amp;D in the Center for Biophotonics

I will describe some of the activities taking place in our National Science Foundation-sponsored Center for Biophotonics Science and Technology. The mission of this center is the application of photonics to the grand challenges of biosciences and medicine. Some examples of these challenges include imaging of single biomolecules within living cells and development of non-invasive diagnostic and therapeutic medical devices. Our Center currently sponsors 20 different research projects that involve interdisciplinary researchers from 10 different collaborating institutions. These institutions are UC Berkeley, UC San Francisco, Stanford University, Mills College, Lawrence Livermore National Laboratory, UT San Antonio, Hampton University, Alabama A&M University, Fisk University as well as UC Davis. Projects focus on developing new technologies for imaging from the whole organism to the single molecule levels. These technologies include bioluminescence, non-linear optical, X-ray laser and free-electron laser microscopy. We are also developing optical sensors that can be used for monitoring the biochemistry of living cells as well as detecting diseases at the level of single molecule sensitivity. Of particular emphasis is our work on portable monitors for infectious biologic agents. We likewise are developing new medical device technologies based on photo-activated materials, laser fluorescence as well as scattering and absorption spectroscopy. I will also discuss our industry partnership programs as well as opportunities for new collaborations and partnering.

The post-exposure response of Enterobacteriaceae to ceftibuten.

The responses of ten isolates of Enterobacteriaceae to ceftibuten exposure were monitored by measuring several parameters. Post-antibiotic effect (PAE), control-related effective regrowth time (CERT) and post-antibiotic sub-MIC effect (PA-SME) were determined by bacterial enumeration carried out either by impedance in combination with viable counting (IMP/VC) or by impedance in combination with bioluminescence (IMP/BIOL). Kill curves were carried out by bioluminescence, viable counting and direct microscopy and post-exposure morphology was established. Ceftibuten primarily provoked filamentation. Over 24 h, kill of up to 3.6 log10 was evident by viable counting and direct microscopy at and above the MIC. Minimal kill, of up to 0.26 log10, was shown by bioluminescence. PAE was found to be method dependent, with statistical differences established by Student's t-test. PAE values of up to 0.48 h and 1.47 h (by IMP/BIOL and IMP/VC respectively) were not concentration dependent above 1 x MIC. CERT values were not method dependent, with values of up to 1.71 h also showing a lack of concentration dependence above 1 x MIC. PA-SME may reflect the situation in vivo more accurately than either PAE or CERT. In PAE and CERT studies the antibiotic is eliminated almost immediately, whereas in vivo there is gradual decrease in antibiotic levels. These persisting levels are reflected more accurately by PA-SME. Compared with PAE and CERT, significantly longer values, of up to 7.27 h, were obtained by PA-SME, although this parameter was also found to be method dependent. The results of the PA-SME studies, which may be the most clinically relevant pharmacodynamic parameter, confirm the appropriateness of the current once- or twice-daily dosing schedules despite the lack of PAE.