Green Fluorescent Protein Emission Research Articles

Thin and Scalable Hybrid Emission Filter via Plasma Etching for Low-Invasive Fluorescence Detection

Hybrid emission filters, comprising an interference filter and an absorption filter, exhibit high excitation light rejection performance and can act as lensless fluorescent devices. However, it has been challenging to produce them in large batches over a large area. In this study, we propose and demonstrate a method for transferring a Si substrate, on which the hybrid filter is deposited, onto an image sensor by attaching it to the sensor and removing the substrate via plasma etching. Through this method, we can transfer uniform filters onto fine micrometer-sized needle devices and millimeter-sized multisensor chips. Optical evaluation reveals that the hybrid filter emits light in the 500 to 560 nm range, close to the emission region of green fluorescent protein (GFP). Furthermore, by observing the fluorescence emission from the microbeads, a spatial resolution of 12.11 μm is calculated. In vitro experiments confirm that the fabricated device is able to discriminate GFP emission patterns from brain slices.

Green fluorescent protein emission obscures metabolic fluorescent lifetime imaging of NAD(P)H.

Autofluorescence of endogenous molecules can provide valuable insights in both basic research and clinical applications. One such technique is fluorescence lifetime imaging (FLIM) of NAD(P)H, which serves as a correlate of glycolysis and electron transport chain rates in metabolically active tissue. A powerful advantage of NAD(P)H-FLIM is the ability to measure cell-specific metabolism within heterogeneous tissues. Cell-type specific identification is most commonly achieved with directed green fluorescent protein (GFP) expression. However, we demonstrate that NAD(P)H-FLIM should not be analyzed in GFP-expressing cells, as GFP molecules themselves emit photons in the blue spectrum with short fluorescence lifetimes when imaged using two-photon excitation at 750 nm. This is substantially different from the reported GFP emission wavelength and lifetime after two-photon excitation at 910 nm. These blue GFP photons are indistinguishable from free NAD(P)H by both emission spectra and fluorescence lifetime. Therefore, NAD(P)H-FLIM in GFP-expressing cells will lead to incorrect interpretations of metabolic rates, and thus, these techniques should not be combined.

Quantifying bacterial cell lysis using GFP based fluorimetric assay

Quantitative measurement of cell lysis against a given microbial strain is essential to calculate the antimicrobial potency of protein/peptide/nanomaterial based formulations. Fluorescence spectroscopy based measurements offer precise quantification of a process via selected flurophore emission profile. In this context, we elucidate a reliable and robust green fluorescent protein (GFP) based fluorescence spectroscopy protocol to evaluate the antimicrobial activity of proteins. The technique is based on the fact that the intensity of the GFP emission released from cells correlates with cell lysis and henceforth the antimicrobial potential of the chosen agent. The technique was demonstrated with two different families of bacteriophage endolysins (T7 and T4 endolysins) using GFP expressing E. coli cells. The GFP based method allowed the absolute quantification of T4 and T7 endolysins cell lysis characteristics at different pH, salt concentrations, and metal ions. The results obtained from GFP based fluorimetric assay were substantiated with turbidimetric assay and fluorescence microscopy. This fluorimetric method in conjugation with different GFP expressing microbial strains and antimicrobial agents can be efficiently applied as a quantification technique to precisely measure cell lysis.

Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

The lineshape of the electronic spectrum of the green fluorescent protein chromophore, part I: gas phase.

In this work we present the vibrationally resolved optical absorption spectrum of p-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI), the green fluorescent protein (GFP) chromophore, computed at several levels of theory, including time-dependent DFT with various functionals and basis sets, CASSCF, CASPT2 and XMCQDPT2. We also investigated what happens to the spectrum if the ground- and excited-state geometries are optimized at different levels of theory (mixed approach), as has been used previously. The vibrationally resolved absorption spectra obtained by DFT, CASPT2 and XMCQDPT2 are very similar and consist of a main absorption peak and a shoulder that is ∼1500 cm(-1) higher in energy. The vibrational progression increases moderately with temperature. These spectra are in qualitative agreement with experimental action spectra, but much narrower and lack the long tail in the blue, even at high temperatures. Because our calculated emission spectra, which are equally narrow, are in good agreement with the emission of green fluorescent protein at 253 K, we argue that the action spectrum are too broad to be considered as the absorption spectrum. The CASSCF method and the mixed approaches overestimate the vibrational progressions with respect to CAM-B3LYP, CASPT2 and XMCQDPT2, due to inaccuracies in the geometric S0 →S1 displacements. Finally, we computed the vibronic spectra of four chromophore analogues with different substitutions on the rings and found that these substitutions hardly affect the lineshape in vacuum.

Resistant ticks inhibit Metarhizium infection prior to haemocoel invasion by reducing fungal viability on the cuticle surface

We studied disease progression of, and host responses to, four species in the Metarhizium anisopliae complex expressing green fluorescent protein (GFP). We compared development and determined their relative levels of virulence against two susceptible arthropods, the cattle tick Rhipicephalus annulatus and the lepidopteran Galleria mellonella, and two resistant ticks, Hyalomma excavatum and Rhipicephalus sanguineus. Metarhizium brunneum Ma7 caused the greatest mortality of R. annulatus, Metarhizium robertsii ARSEF 2575 and Metarhizium pingshaense PPRC51 exhibited intermediate levels of virulence, and Metarhizium majus PPRC27 caused low mortality of cattle ticks. Conidia of all four species germinated on all hosts examined, but on resistant hosts, sustained hyphal growth was inhibited and GFP emission steadily and significantly decreased over time, suggesting a loss of fungal viability. Cuticle penetration was observed only for the three most virulent species infecting susceptible hosts. Cuticles of resistant and susceptible engorged female ticks showed significant increases in red autofluorescence at sites immediately under fungal hyphae. This is the first report (i) of tick mortality occurring after cuticle penetration but prior to haemocoel colonization and (ii) that resistant ticks do not support development of Metarhizium germlings on the outer surface of the cuticle. Whether reduced Metarhizium viability on resistant tick cuticles is due to antibiosis or limited nutrient availability is unknown.

Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.

Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.

Depolarization after resonance energy transfer (DARET): A sensitive fluorescence-based assay for botulinum neurotoxin protease activity

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)–fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134–206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants ( V max and k cat) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.

Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E (BoNT/A and BoNT/E). As detailed in that article, the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein (BFP) and green fluorescent protein (GFP) moieties linked via residues 134–206 of SNAP-25 (synaptosome-associated protein of 25 kDa), the protein substrate for BoNT/A and BoNT/E. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is very low due to depolarization following energy transfer from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance, the polarization is high due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows an assay, termed DARET (depolarization after resonance energy transfer), that is robust and sensitive. In this article, we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarizations, and lifetimes.

Circular Dichroism and Circular Polarized Luminescence from a Green Fluorescent Protein—Initial Research for Chiroptical Emission of Biological Materials

Optical properties of a green fluorescent protein (GFP) are examined using optical absorption, circular dichroism (CD), and circular polarized luminescence (CPL) spectroscopies. The GFP has chiroptical activity and exhibits green circular polarized emission, although the g em-factor is small. Poly(vinyl alcohol) (PVA)/GFP composite films are prepared to attempt long-term preservation of the GFP emission activity. After five years, the transparent PVA/GFP composite film still exhibits stable fluorescence that appears similar to the emission from the Aequorea jellyfish.

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein-protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of protein-protein interactions. A luminescent terbium complex, TMP-Lumi4, was introduced into cultured cells using two methods: (i) osmotic lysis of pinocytic vesicles; and (ii) reversible membrane permeabilization with streptolysin O. Upon intracellular delivery, the complex was observed to bind specifically and stably to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals from cellular autofluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 micros) between excitation and detection. Background elimination made it possible to detect interactions between the first PDZ domain of ZO-1 (fused to eDHFR) and the C-terminal YV motif of claudin-1 (fused to GFP) in single microscope images at subsecond time scales. We observed a highly significant (P<10(-6)), six-fold difference between the mean, donor-normalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing noninteracting mutants. The results show that time-resolved LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications.

Spectroscopic Characterization of Depolarization Via FRET in a BFP-GFP BoNT/A Assay

The assay is based on depolarization due to Forster Resonance Energy Transfer (FRET) between Blue Fluorescent Protein (BFP) and Green Fluorescent Protein (GFP) moieties linked by a peptide containing residues 134-206 of SNAP-25, the protein substrate for BoNT/A's proteolytic activity. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is −0.04 due to depolarization following FRET from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the FRET distance, the polarization increases significantly to ∼0.4, due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows for an assay (termed DARET for Depolarization After Resonance Energy Transfer) that is robust and sensitive. In this report we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarization and time-resolved methods. Our results suggest that the donor and acceptor dipoles are at a large angle (72°) with respect to one another but that the BFP and GFP are in direct contact in the intact substrate. Evidence is also provided to demonstrate the direct interaction of BFP and GFP after cleavage. We have used this assay to determine the enzyme kinetic parameters (Km, Kcat and Vmax) for BoNT/A proteolysis of the assay substrate. Our conclusions bear on the issue of the common choice of 2/3 for κ2 for FRET studies in general and on fluorescent proteins in particular. This work was supported by Allergan Inc.

Can Hg(II) be Determined via Quenching of the Emission of Green Fluorescent Protein from Anemonia sulcata var. smaragdina?

Anemonia sulcata var. smaragdina is a widely distributed Cnidarian species along Turkish coastlines. It is also a well-known example of a facultative symbiotic life form in sea ecosystems. Green fluorescent proteins (GFPs) in Anemonia sulcata var. smaragdina have vital roles in this symbiotic form. The fluorescence quenching by Hg(II) in the supernatants obtained from A. sulcata var. smaragdina was shown in this study. According to results, there was a statistical significant relationship (R(2) = 0.9913) between increased Hg(II) concentration and decreased fluorescence intensity of GFP supernatants obtained from A. sulcata var. smaragdina. Mn(II), Fe(II), and Al(II) showed no interference effect and did not change the fluorescence intensity of GFP supernatants obtained from A. sulcata var. smaragdina. In conclusion, the fluorescence quenching of GFPs by Hg(II) can be a novel method to determine the Hg(II) levels in aqueous solution. Therefore, further researches are strongly warranted because of the possible potential applications of the fluorescence quenching of GFPs by Hg(II).

Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens

Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or alpha-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, alphaB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of alphaB-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.

Monitoring of cell therapy and assessment of cardiac function using magnetic resonance imaging in a mouse model of myocardial infarction

We have developed a mouse severe combined immunodeficient (SCID) model of myocardial infarction based on permanent coronary artery occlusion that allows long-term functional analysis of engrafted human embryonic stem cell-derived cardiomyocytes, genetically marked with green fluorescent protein (GFP), in the mouse heart. We describe methods for delivery of dissociated cardiomyocytes to the left ventricle that minimize scar formation and visualization and validation of the identity of the engrafted cells using the GFP emission spectrum, and histological techniques compatible with GFP epifluorescence, for monitoring phenotypic changes in the grafts in vivo. In addition, we describe how magnetic resonance imaging can be adapted for use in mice to monitor cardiac function non-invasively and repeatedly. The model can be adapted to include multiple control or other cell populations. The procedure for a cohort of six mice can be completed in a maximum of 13 weeks, depending on follow-up, with 30 h of hands-on time.

HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.

Improved in Vivo Whole-Animal Detection Limits of Green Fluorescent Protein–Expressing Tumor Lines by Spectral Fluorescence Imaging

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 x 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.

Optical Manipulation Reveals Strong Attracting Forces at Membrane Contact Sites between Endoplasmic Reticulum and Chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs

The power and simplicity of genetically encoded fluorophores (fluorescent proteins, FPs) have drawn many molecular biologists to light microscopy. First generation FPs suffered from overlapping excitation and emission spectra, which limited their use together in pairs (Patterson et al., J Cell Sci 2001;114 (Part 5):837-838). Image acquisition and processing techniques, collectively known as linear unmixing, have been developed to separate overlapping fluorescence signals encountered in the imaging of FP pairs and also in FRET. These specialized techniques are not without their potential drawbacks, including limitations on sensitivity and time-resolution for live cell imaging, and the risk of artifact in the hands of nonspecialists. With the advent of a new generation of red-shifted FPs (Shaner et al., Nat Biotechnol 2004;22:1567-1572; Verkhusha and Lukyanov, Nat Biotechnol 2004;22:289-296) careful selection of excitation sources and emission filters obviate the need for linear unmixing when simple two channel imaging of FPs is required. Here we introduce a new configuration of the Zeiss LSM 510 laser scanning confocal microscope, optimized for live cell imaging of green fluorescent protein (GFP) together with spectral variants such as mRFP1 and mCherry using standard photo-multipliers. A 2 mW, 594 nm HeNe laser was chosen as the excitation source for the red FP. This wavelength efficiently excites the aforementioned red variants without limiting the detection range of GFP emission during simultaneous two-channel imaging. Compared to excitation of GFP and mCherry at 488 and 543 nm, excitation at 488 and 594 nm approximately doubles the sensitivity of GFP detection and eliminates bleed-through of GFP into the mCherry channel. However, sensitivity of mCherry detection is decreased by 30%, suggesting the need for red FPs having longer emission peaks. Practical advantages to the simultaneous optical separation of FPs with nonoverlapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging.

Intrinsic Photoluminescence from Gels Containing Amine or Amide Chemical Groups

Gels obtained through the sol-gel procedure by mixing an alkoxide with a carboxylic acid emit intense intrinsic photoluminescence when the alkoxide contains an amine or amide group. However, also simple polyether chains with two end amino groups but no alkoxysilane groups gel by interaction with carboxylic acids and also emit photoluminescence. Luminescence emission in these gels could be justified by a model similar to the emission of Green Fluorescent Protein. According to this model, amide groups, abundant in these gels, go through multiple reactions with other amide or amino groups so that they could yield conjugated species that are capable to emit photoluminescence. This model could open the possibility to synthesize a great variety of photoluminescent species based on amine-carboxylate interactions.