two-photon polarization microscopy Dotaz Zobrazit nápovědu
Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study because of their requirement of a lipid membrane for function. Here we show that two-photon polarization microscopy can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. The technique allows sensitive imaging of G-protein activation, changes in intracellular calcium concentration and other processes, and is not limited to membrane proteins. Conveniently, many suitable probes for two-photon polarization microscopy already exist.
- MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- konformace proteinů MeSH
- membránové proteiny metabolismus ultrastruktura MeSH
- mikroskopie fluorescenční multifotonová metody MeSH
- polarizační mikroskopie metody MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- membránové proteiny MeSH
Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.
I have provided update to our two photon laser scanning microscope by adding new technique which enables us to simultaneously measured the second harmonic generation signals in the forward and backward directions; in the meantime, one can measure the two photon excitations fluorescence if the materials produce fluorescence. In the present work, the fascia muscles, muscles of pig and pig's skin were used. I found that these materials produced high second harmonic generation signal in both directions. These measurements show that the second harmonic generation strongly depends on the state of the polarization of the laser light and the orientation of the dipole moment in the molecules that interact with the laser light. It is therefore advantageous to control the laser's state of polarization, to maximize second harmonic generation. The novelty of this work is to establish new multi-functional technique by combing three platforms of laser scanning microscopy - the fluorescence microscopy, harmonic generation microscopy and polarizing microscopy in which one can use the second harmonic imaging to investigate the true architecture of the sensitive samples and the samples which do not produce auto-fluorescence. Moreover investigation of the new sample needs to look at all details of the true architecture of the sample. Thereby the sample will be exposed to the laser radiation more than the well-known sample, and that will cause photo-bleaching and photo-damage. Since the second harmonic generation does not undergo from photo-bleaching and photo-damage it will be the promising technique for investigating the sensitive and new samples. Then one can move to acquire fluorescence images after good investigation of the true architecture of the sample by the SH imaging.
- MeSH
- fascie chemie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- kůže chemie ultrastruktura MeSH
- mikroskopie fluorescenční multifotonová metody MeSH
- polarizační mikroskopie metody MeSH
- prasata MeSH
- svaly chemie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Multifunctional two-photon laser scanning microscopy provides attractive advantages over conventional two-photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. RESULTS: These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two-photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. CONCLUSIONS: With the multifunctional two-photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.
- MeSH
- chloroplasty chemie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- listy rostlin chemie ultrastruktura MeSH
- mikroskopie fluorescenční multifotonová metody MeSH
- počítačové zpracování obrazu metody MeSH
- Selaginellaceae chemie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: The effect of increasing the numerical aperture and changing the immersion imaging medium of the objective in the forward second harmonic imaging of the grana and intergranal parts of chloroplasts in the leaves of shade moss Plagiomnium affine plant were demonstrated. Also the enhancement of the forward second harmonic generation images and simultaneously records the forward and backward second harmonic signals were demonstrated. The second harmonic signals in the forward and backward directions are generated by a tightly focused linearly polarized femtosecond pulses laser. CONCLUSIONS: Forward second harmonic signals were successfully enhanced by capturing higher order of diffraction rays from the samples using high numerical aperture water immersion objective.
- MeSH
- čočky MeSH
- fluorescenční mikroskopie metody MeSH
- fotony MeSH
- konfokální mikroskopie metody MeSH
- lasery MeSH
- listy rostlin ultrastruktura MeSH
- mechy ultrastruktura MeSH
- polarizační mikroskopie metody MeSH
- tylakoidy ultrastruktura MeSH
- vylepšení obrazu MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: We provide an update of our two-photon laser scanning microscope by compressing or reducing the broadening of the pulse width of ultrafast laser pulses for dispersion precompensation, to enable the pulses to penetrate deeply inside the sample. FINDINGS: The broadening comes as the pulses pass through the optical elements. We enhanced and modified the quality and the sharpness of images by enhancing the resolution using special polarizer namely Glan Laser polarizer GL10. This polarizer consists of two prisms separated by air space. This air separation between the two prisms uses to delay the red wavelength when the light leaves the first prism to the air then to second prism. We note a considerable enhancing with using the GL polarizer, and we can see the details of the leaf structure in early stages when we trying to get focus through z-stacks of images in comparison to exactly the same measurements without using GL polarizer. Hence, with this modification we able to reduce the time of exposure the sample to the laser radiation thereby we will reduce the probability of photobleaching and phototoxicity. When the pulse width reduced, the average power of the laser pulses maintained at a constant level. Significant enhancement is found between the two kinds of images of the Two-Photon Excitation Fluorescence (TPEF). CONCLUSION: In summary reduction the laser pulse width allowed to collect more diffraction orders which will used to form the images. The more diffraction orders the higher resolution images.
- Publikační typ
- časopisecké články MeSH
A laser-scanning microscope using second harmonic generation (SHG) as a probe is shown to produce high-resolution images of duckweed Lemna minuta leaves. These leaves are multi-cell layer thick. Second harmonic light is generated by a tightly focused short pulse laser beam and is collected by two objectives yielding forward and backward second harmonic digital images. This measurement shows that the signal of the second harmonic imaging in the forward and backward directions depends on the thickness of the chloroplast and that the forward-SH image was brighter than the backward-SH image. The image intensity also depended on the orientation of the chloroplast in relation to the illuminating polarization direction. Their light-induced re-orientation which was affected by the intensity of the illumination could be observed during the experiments. The novelty of this work is to establish new compact technique in which one can use the SH imaging to investigate the true architecture of the sensitive samples, the unknown samples and the samples which is not producing auto-fluorescence. Moreover, investigation of new or unknown samples needs a long time for looking at details of the sample. Thereby the sample will be exposed for long time to the laser radiation that will cause photobleaching and photodamage. Since the SHG does not undergo photobleaching and photodamage this will be the promising technique for investigating the sensitive and new unknown samples. Then one can move to acquire fluorescence images after complete investigation of the true architecture of the sample. The other advantage of SHG is that it has the ability to image highly ordered structural proteins without any exogenous labels. The SHG is an intrinsic and a coherent process. Imaging of intrinsic compounds avoids the complications of slicing and labeling, and samples can be investigated under physiological conditions.
Fermi surfaces of transition metals, which describe all thermodynamical and transport quantities of solids, often fail to be modeled by one-electron mean-field theory due to strong correlations among the valence electrons. In addition, relativistic spin-orbit coupling pronounced in heavier elements lifts the degeneracy of the energy bands and further modifies the Fermi surface. Palladium and rhodium, two 4d metals attributed to show significant spin-orbit coupling and electron correlations, are ideal for a systematic and fundamental study of the two fundamental physical phenomena and their interplay in the electronic structure. In this study, we explored the Fermi surface of the 4d noble metals palladium and rhodium obtained via high-resolution constant initial state momentum microscopy. The complete 3D-Fermi surfaces of palladium and rhodium were tomographically mapped using soft X-ray photon energies from 34 eV up to 660 eV. To fully capture the orbital angular momentum of states across the Fermi surface, the Fermi surface tomography was performed using p- and s- polarized light. Applicability and limitations of the nearly-free electron final state model in photoemission are discussed using a complex band structure model supported by experimental evidence. The significance of spin-orbit coupling and electron correlations across the Fermi surfaces will be discussed within the context of the photoemission results. State-of-the-art fully relativistic Korringa-Kohn-Rostoker (KKR) calculations within the one-step model of photoemission are used to support the experimental results.
- Klíčová slova
- Complex band structure, Fermi surface tomography, Final-state self-energy, Momentum microscopy, Photoelectron final-state,
- Publikační typ
- časopisecké články MeSH
Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.
- Klíčová slova
- Adrenergic Receptor, Cell Signaling, Heterotrimeric G Proteins, Membrane Proteins, Microscopic Imaging, Plasma Membrane, Potassium Channels, Two-photon Polarization Microscopy,
- MeSH
- aktivace enzymů fyziologie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- protein GAP-43 genetika metabolismus MeSH
- proteiny vázající GTP - alfa-podjednotky Gi-Go genetika metabolismus MeSH
- proteiny vázající GTP - beta-podjednotky genetika metabolismus MeSH
- proteiny vázající GTP - gama-podjednotky genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- protein GAP-43 MeSH
- proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
- proteiny vázající GTP - beta-podjednotky MeSH
- proteiny vázající GTP - gama-podjednotky MeSH
The Gi/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of Gi/o-GPCR coupling and the high rate of Gi/o signal transduction have been hypothesized to be enabled by the existence of stable associates between Gi/o proteins and their cognate GPCRs in the inactive state (Gi/o-GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gαi1 subunits labeled with fluorescent proteins and four GPCRs: the α2A-adrenergic receptor, GABAB, cannabinoid receptor type 1 (CB1R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gαi1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gαi1 subunits and α2A-adrenergic receptor, GABAB, or dopamine receptor type 2 receptors did not reveal any interaction between the Gi1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB1R exhibited an interaction with the Gi1 protein. Further experiments revealed that this interaction is caused solely by CB1R basal activity; no preassembly between CB1R and the Gi1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active Gi1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and Gi1 (basal coupling). These findings suggest that Gi1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs.
- Klíčová slova
- G protein, G protein-coupled receptor (GPCR), GABA receptor, adrenergic receptor, basal coupling, cannabinoid receptor type 1 (CB1), dopamine receptor type 2 (D2R), preassembly, signal transduction, two-photon polarization microscopy,
- MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mutace MeSH
- proteiny vázající GTP - alfa-podjednotky Gi-Go genetika metabolismus MeSH
- receptory spřažené s G-proteiny genetika metabolismus MeSH
- signální transdukce fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- GNAI1 protein, human MeSH Prohlížeč
- proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
- receptory spřažené s G-proteiny MeSH
