Multiparameter Microscopy and Spectroscopy for Single-Molecule Analytics

Prummer, Michael; Sick, Beate; Renn, A.; Wild, Urs P.

doi:10.1021/ac034976g

Cited by 34 publications

(39 citation statements)

References 32 publications

(37 reference statements)

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“…We believe that novel labeling strategies to target specific subpopulations of proteins (45,46), in combination with high-resolution microscopy and spectroscopy (47)(48)(49)(50), will become increasingly important for live-cell proteomics. Confocal Fluorescence Microscopy.…”

Section: Discussionmentioning

confidence: 99%

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Jacquier

Prummer

Segura

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Despite the importance of trafficking for regulating G proteincoupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulselabeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Singlemolecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of Ϸ190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.cell signaling ͉ G protein-coupled receptors ͉ single-particle tracking ͉ in vivo protein labeling ͉ fluorescence imaging T he sensation of smell is mediated by a specific family of olfactory G protein-coupled receptors (GPCRs), which recognize small volatile molecules (1). Although odorant receptors (ORs) account for the largest mammalian gene family, comprising up to 1,000 members, the mechanism of signal recognition and amplification in olfactory transduction remains elusive (2). This is partly because classical methods for OR detection, based on immunocytochemistry (3, 4) or genetic fusion to GFP (5), have not permitted the simultaneous live-cell imaging of olfactory processes at the cell membrane and in cytoplasmic compartments.Comprehensive functional studies on ORs in a native cellular environment, such as isolated olfactory sensory neurons, adenovirus-infected olfactory epithelia, or genetically engineered animals, are often hampered by practical limitations: (i) olfactory sensory neurons are difficult to maintain in primary culture (6), (ii) virus-mediated gene transfer does not consistently yield functional OR expression (7), and (iii) creating transgenic animals for each OR would be quite expensive. Functional expression of ORs in heterologous cells is, therefore, an important approach for elucidating the molecular mechanism of olfaction and helps to identify specific ligands ...

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Section: Discussionmentioning

confidence: 99%

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Jacquier

Prummer

Segura

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The photon data can either be used to build up photon distributions or to store the data of each individual photon. Thus, modern TCSPC modules can be configured to record either multiwavelength FLIM data (Becker et al, 2002, fast dynamic changes in the fluorescence decay behavior of a sample (Becker and Bergmann, 2005;Becker et al, 1999Becker et al, , 2004c, FCS and FIDA data combined with fluorescence lifetime data (Böhmer et al, 2002), and BIFL data (Eggeling et al, 2001;Prummer et al, 2004).…”

Section: Multidimensional Tcspcmentioning

confidence: 99%

“…Identification of the photon bursts of individual molecules and analysis within the bursts is the basis of the burst-integrated fluorescence lifetime (BIFL) technique (Eggeling et al, 2001;Prummer et al, 2004;Tinnefeld et al, 2000). The applicability of a large number of different techniques to one data set recorded in one spot of a sample is a considerable benefit of the TCSPC technique.…”

Section: Acquisition Of Fcs Fida or Bifl Datamentioning

confidence: 99%

“…The FIDA technique can be combined with spectral detection and fluorescence lifetime analysis (Kask et al, 2000;Palo et al, 2002). Burst-integrated fluorescence lifetime (BIFL) techniques (Eggeling et al, 2001;Prummer et al, 2004;Tinnefeld et al, 2000) identify the photon bursts of single molecules and analyze the lifetime, anisotropy, and spectral behavior of the fluorescence within the individual bursts.…”

Section: Introductionmentioning

confidence: 99%

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Fluorescence lifetime images and correlation spectra obtained by multidimensional time‐correlated single photon counting

Becker

Bergmann

Haustein

et al. 2006

Microscopy Res & Technique

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Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.

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“…Our recent efforts have focused on extracting the maximal amount of information from single trapped molecules. We summarize two parallel directions in this manuscript: first, we combined multi-parameter fluorescence detection [9][10][11] with the ABEL trap, where we measure different physical aspects of the fluorescent photons to yield mechanistic insight into the often-observed photodynamics; second, we developed a new strategy to estimate time-dependent transport properties (diffusion coefficient and electrokinetic mobility) of single trapped molecules in solution. …”

Section: Introductionmentioning

confidence: 99%

Spectroscopic and transport measurements of single molecules in solution using an electrokinetic trap

Wang

Moerner

2014

SPIE Proceedings

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In aqueous solution, diffusion generally limits the observation window of a nano-meter sized single molecule to milliseconds and prevents quantitative determination of spectroscopic and transport properties molecule-by-molecule. The anti-Brownian electrokinetic (ABEL) trap is a feedback-based microfluidic device that enables prolonged (multiseconds) observation of single molecules in solution. The amount of information that can be extracted from each molecule in solution is thus boosted by three orders of magnitude. We describe recent advances in extending the ABEL trap to conduct both spectroscopic and transport measurements of single trapped molecules. First, by combining the trap with multi-parameter fluorescence detection, synchronized dynamics in different observables can be visualized in solution. We use single molecules of Atto 633 as an example and show that this popular label switches between different emissive states under common imaging conditions. Next, we show how transport properties of trapped single molecules can be extracted in addition to spectroscopic readouts. Due to their direct sensitivity to molecular size and charge, measured transport coefficients can be used to distinguish different molecular species and trace biomolecular interactions in solution. We demonstrate this new paradigm by monitoring DNA hybridization/melting in real-time.

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Multiparameter Microscopy and Spectroscopy for Single-Molecule Analytics

Cited by 34 publications

References 32 publications

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Fluorescence lifetime images and correlation spectra obtained by multidimensional time‐correlated single photon counting

Spectroscopic and transport measurements of single molecules in solution using an electrokinetic trap

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