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51.
We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidin-actin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.Abbreviations 1PEF one-photon excited fluorescence - 2PEF two-photon excited fluorescence - APD avalanche photo diode - CHO Chinese hamster ovary - DMEM Dulbecco's modified Eagle's medium - EGFP enhanced green fluorescent protein - EW evanescent wave - FCS fetal calf serum - GPI glycosylphosphatidylinositol - TIR total internal reflectionThis paper is dedicated to the memory of Prof. Horst Harreis (1940–2002) 相似文献
52.
D. A. Brown 《Neurophysiology》2007,39(4-5):243-247
Vladimir Skok and his colleagues did much of the pioneering work on fast excitatory synaptic transmission in sympathetic ganglia
and on nicotinic acetylcholine receptors that mediate fast transmission. I and my colleagues (including Alex Selyanko, one
of Vladimir’s protégés) have studied the additional process of slow synaptic excitation that is mediated by the action of
acetylcholine on muscarinic receptors. This results primarily from the closure of “M-channels,” a subset of voltage-gated
potassium channels composed of Kv7.2 and Kv7.3 channel subunits. These channels require membrane phosphatidylinositol-4,5-bisphosphate
(PIP2) for their opening, and their closure by muscarinic receptor activation is now thought to result from the reduction in PIP2 levels that follows receptor-induced PIP2 hydrolysis. The dynamics of these two forms of synaptic excitation are compared.
Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 284–289, July–October, 2007. 相似文献
53.
Legall H Stiel H Beck M Leupold D Gruszecki WI Lokstein H 《Journal of biochemical and biophysical methods》2007,70(3):369-376
Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR). 相似文献
54.
Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor QA of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F
o towards a maximum F
mSTF when QA becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first
twin-partner is followed by a 20–30% increase when the second partner is applied within 20–100 μs after the first one. The
amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok
cycle (S states) and originates from two different trapped states with a life time of 0.2–0.4 and 2–5 ms, respectively. The
oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor
side. The F(t) response in high frequency flash trains (1–4 kHz) shows (i) in the first 3–4 flashes a transient overshoot 20–30% above
the F
mSTF = 3*F
o level reached in the 1st flash with a partial decline towards a dip D in the next 2–3 ms, independent of the flash frequency,
and (ii) a frequency independent rise to F
m = 5*F
o in the 3–60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S0 fraction with QB-nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair
in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate
a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and
electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport
in single reduced QB-nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency
in these centers. 相似文献
55.
Devaki A. Kelkar 《生物化学与生物物理学报:生物膜》2007,1768(9):2011-2025
The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid-protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media. 相似文献
56.
Amanda Gatesman Ammer Karen E. Hayes Karen H. Martin Lingqing Zhang George A. Spirou Scott A. Weed 《Journal of visualized experiments : JoVE》2011,(53)
Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting. 相似文献
57.
Mark Pottek Gabriel C. Knop Reto Weiler Karin Dedek 《Journal of visualized experiments : JoVE》2011,(57)
Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6.In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels. 相似文献
58.
J.?KvetonEmail author M.?Durchan K.?Rohacek J.?Santrucek F.?Vacha Z.?Sestak 《Photosynthetica》2005,43(3):323-328
Life and research results of Pavel Siffel, a talented but untimely deceased Czech scientist in photosynthesis, are reviewed.
He studied biophysics and physiology of chlorophyll, its complexes with proteins, their absorption and fluorescence spectra,
activities in mutants and transformants, dealt with chlorophyll biosynthesis and protochlorophyllide photoreduction, pigments
in plants grown at CO2 deficiency and under simulated acid rain, with changes accompanying leaf and plant development, photobleaching, etc. He participated in construction of specialised spectrofluorometers, finally he built the kinetic spectrophotometer SpeKin. 相似文献
59.
S.?E.?Jorge?Villar H.?G.?M.?EdwardsEmail author M.?R.?Worland 《Origins of life and evolution of the biosphere》2005,35(5):489-506
Raman spectra have been obtained for extremophiles from several geological environments; selected examples have been taken
from hot and cold deserts comprising psychrophiles, thermophiles and halophiles. The purpose of this study is the assessment
of the effect of the wavelength of the laser excitation on the ability to determine unique information from the Raman spectra
about the specificity of detection of biomolecules produced as a result of the survival strategies adopted by organisms in
extreme terrestrial environments. It was concluded that whereas FT-Raman spectroscopy at 1064 nm gave good quality results
the time required to record the data was relatively large compared with other wavelengths of excitation but that better access
to the CH stretching region for organic molecules was given. Shorter wavelength excitation of biomolecules in the blue-green
regions of the visible spectrum using a conventional dispersive spectrometer was more rapid but very dependent upon the type
of chemical compound being studied; most relevant biomolecules fluoresced at these wavelengths but carotenoids exhibited a
resonance effect which resulted in an improved detection capability. Minerals and geological materials, in contrast, were
best studied at these visible wavelengths. In general, the best compromise system for the excitation of the Raman spectra
of both geological and biological materials was provided using a 785 nm laser coupled with a dispersive spectrometer, especially
for accessing the 1800–200 cm−1 wavenumber shift region where much of the definitive analytical information resides. This work will have conclusions relevant
to the use of miniaturised Raman spectrometers for the detection of biomolecules in extraterrestrial planetary exploration. 相似文献
60.
Lead (Pb2+) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence
microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb2+ binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease
of the fluorescence emission, which occurred even in resting condition. In the presence of 20 μM Pb2+, uptake reached a plateau level (≈45% of initial fluorescence) in 4 min and was partially antagonized by 25 μM lanthanum.
Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous
saturation was due to inactivation of the transport system. Intracellular Pb2+ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free
Pb2+ sufficient to inactivate the transport system is close to 50 pM. 相似文献