Exploring fluorescence antibunching in solution to determine the stoichiometry of molecular complexes.
Fluorescence antibunching is a well-known technique for determining the number of independent emitters per molecule or molecular complex. It was rarely applied to autofluorescent proteins due to the necessity of collecting large numbers of fluorescence photons from a single molecule, which is usually impossible to achieve with rather photolabile autofluorescent proteins. Here, we measure fluorescence antibunching on molecules in solution, allowing us to accumulate data over a large number of molecules. We use that method for determining an average stoichiometry of molecular complexes. The proposed method is absolute in the sense that it does not need any calibration or referencing. We develop the necessary theoretical background and check the method on pure dye solutions and on molecular complexes with known stoichiometry.
Sýkora
Jan
J
Institute for Neuroscience and Biophysics 1, Forschungszentrum Jülich, D 52425 Jülich, Germany.
Kaiser
Karin
K
Gregor
Ingo
I
Bönigk
Wolfgang
W
Schmalzing
Günther
G
Enderlein
Jörg
J
D000818:Animals; D017930:Genes, Reporter / Q000235:genetics; D005960:Glucosides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D018009:Receptors, Glycine / Q000737:chemistry; D018048:Receptors, Purinergic P2 / Q000737:chemistry; D058469:Receptors, Purinergic P2X; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014982:Xenopus laevis
D005960:Glucosides; D009841:Oligonucleotides; D018009:Receptors, Glycine; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D012996:Solutions; C040358:dodecyl maltoside