UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions

Schüttpelz M, Müller C, Neuweiler H, Sauer M (2006)
ANALYTICAL CHEMISTRY 78(3): 663-669.

Journal Article | Published | English

No fulltext has been uploaded

Author
; ; ;
Abstract
Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm. To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the pico-molar concentration range. The obtained affinity constant, K-a,of (1.4 +/- 0.6) x 10(9) M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.
Publishing Year
ISSN
PUB-ID

Cite this

Schüttpelz M, Müller C, Neuweiler H, Sauer M. UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions. ANALYTICAL CHEMISTRY. 2006;78(3):663-669.
Schüttpelz, M., Müller, C., Neuweiler, H., & Sauer, M. (2006). UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions. ANALYTICAL CHEMISTRY, 78(3), 663-669.
Schüttpelz, M., Müller, C., Neuweiler, H., and Sauer, M. (2006). UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions. ANALYTICAL CHEMISTRY 78, 663-669.
Schüttpelz, M., et al., 2006. UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions. ANALYTICAL CHEMISTRY, 78(3), p 663-669.
M. Schüttpelz, et al., “UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions”, ANALYTICAL CHEMISTRY, vol. 78, 2006, pp. 663-669.
Schüttpelz, M., Müller, C., Neuweiler, H., Sauer, M.: UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions. ANALYTICAL CHEMISTRY. 78, 663-669 (2006).
Schüttpelz, Mark, Müller, C, Neuweiler, H, and Sauer, Markus. “UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions”. ANALYTICAL CHEMISTRY 78.3 (2006): 663-669.
This data publication is cited in the following publications:
This publication cites the following data publications:

16 Citations in Europe PMC

Data provided by Europe PubMed Central.

A series of flexible design adaptations to the Nikon E-C1 and E-C2 confocal microscope systems for UV, multiphoton and FLIM imaging.
Botchway SW, Scherer KM, Hook S, Stubbs CD, Weston E, Bisby RH, Parker AW., J Microsc 258(1), 2015
PMID: 25664385
A method for rapid screening of interactions of pharmacologically active compounds with albumin.
Majcher A, Lewandrowska A, Herold F, Stefanowicz J, Slowinski T, Mazurek AP, Wieczorek SA, Holyst R., Anal. Chim. Acta 855(), 2015
PMID: 25542089
Monitoring on-chip Pictet-Spengler reactions by integrated analytical separation and label-free time-resolved fluorescence.
Ohla S, Beyreiss R, Fritzsche S, Glaser P, Nagl S, Stockhausen K, Schneider C, Belder D., Chemistry 18(4), 2012
PMID: 22179940
Asymmetric organocatalysis and analysis on a single microfluidic nanospray chip.
Fritzsche S, Ohla S, Glaser P, Giera DS, Sickert M, Schneider C, Belder D., Angew. Chem. Int. Ed. Engl. 50(40), 2011
PMID: 21948447
Lifetime imaging of FRET between red fluorescent proteins.
Rusanov AL, Ivashina TV, Vinokurov LM, Fiks II, Orlova AG, Turchin IV, Meerovich IG, Zherdeva VV, Savitsky AP., J Biophotonics 3(12), 2010
PMID: 20925107
Microarray analysis of protein-protein interactions based on FRET using subnanosecond-resolved fluorescence lifetime imaging.
Nagl S, Bauer R, Sauer U, Preininger C, Bogner U, Schaeferling M., Biosens Bioelectron 24(3), 2008
PMID: 18538558
Intravital two-photon microscopy: focus on speed and time resolved imaging modalities.
Niesner RA, Andresen V, Gunzer M., Immunol. Rev. 221(), 2008
PMID: 18275472
Molecular biosensing system based on intrinsically disordered proteins.
Cissell KA, Shrestha S, Purdie J, Kroodsma D, Deo SK., Anal Bioanal Chem 391(5), 2008
PMID: 18193204
Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation.
Botchway SW, Parker AW, Bisby RH, Crisostomo AG., Microsc. Res. Tech. 71(4), 2008
PMID: 18080329
Microfluidic glass chips with an integrated nanospray emitter for coupling to a mass spectrometer.
Hoffmann P, Hausig U, Schulze P, Belder D., Angew. Chem. Int. Ed. Engl. 46(26), 2007
PMID: 17516595
Time-resolved FRET and FLIM of four-way DNA junctions.
Mountford CP, Mount AR, Evans SA, Su TJ, Dickinson P, Buck AH, Campbell CJ, Terry JG, Beattie JS, Walton AJ, Ghazal P, Crain J., J Fluoresc 16(6), 2006
PMID: 17031573

Export

0 Marked Publications

Open Data PUB

Web of Science

View record in Web of Science®

Sources

PMID: 16448037
PubMed | Europe PMC

Search this title in

Google Scholar