Single Molecule Spectroscopy of the Green Fluorescent Protein: A Critical Assessment

Two-Photon Thermal Bleaching of Single Fluorescent Molecules

“…The lifetime of the triplet state varies from few ms to tens of ms (Veerman et al, 1999), while the length of the dark periods observed by us is on the order of seconds. On the other hand the blinking due to the triplet-singlet interconversion (Dickson et al, 1997;Zumbusch and Jung, 2000;Song et al, 1996;Haupts et al, 1998;Schwille et al, 2000) is masked in our ffi250 ms acquisition time. Instead, our finding of emission intervals separated by dark periods suggests the existence of an additional (nonemissive) state B in the usual three-levels electronic system, S 0 (singlet ground state), S 1 (singlet excited state) and T 1 (triplet ground state).…”

Two-Photon Thermal Bleaching of Single Fluorescent Molecules

“…The lifetime of the triplet state varies from few ms to tens of ms (Veerman et al, 1999), while the length of the dark periods observed by us is on the order of seconds. On the other hand the blinking due to the triplet-singlet interconversion (Dickson et al, 1997;Zumbusch and Jung, 2000;Song et al, 1996;Haupts et al, 1998;Schwille et al, 2000) is masked in our ffi250 ms acquisition time. Instead, our finding of emission intervals separated by dark periods suggests the existence of an additional (nonemissive) state B in the usual three-levels electronic system, S 0 (singlet ground state), S 1 (singlet excited state) and T 1 (triplet ground state).…”

Dynamics of green fluorescent protein mutant2 in solution, on spin‐coated glasses, and encapsulated in wet silica gels

“…Similar results were interpreted as a light‐driven flickering of GFP (Schwille et al 2000). At higher excitation powers, the relaxation time drops to ≅5 μs, a typical value for the thermally activated delayed fluorescence (Zumbusch and Jung 2000; Schwille et al 2000).…”

“…Representative auto‐correlation functions (ACFs) are shown in Figure 3. For GFPmut2, the ACFs were analyzed according to a simple diffusion model for longer lag times plus an exponential relaxation that corresponds to protein flickering or triplet state interconversion (Song et al 1996; Haupts et al 1998; Zumbusch and Jung 2000):…”

Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

“…Number of collected photons before bleaching versus the excitation intensity. A (2) outside attached to PSS layer ( ); A (1) family (•) and B family (O) in the layers; silica gel (S (3) in table 1) (◊); agarose gel (▼) as in [9].…”

Two-Photon Thermal Bleaching of Single Fluorescent Molecules

“…The lifetime of the triplet state varies from few ms to tens of ms (Veerman et al, 1999), while the length of the dark periods observed by us is on the order of seconds. On the other hand the blinking due to the triplet-singlet interconversion (Dickson et al, 1997;Zumbusch and Jung, 2000;Song et al, 1996;Haupts et al, 1998;Schwille et al, 2000) is masked in our ffi250 ms acquisition time. Instead, our finding of emission intervals separated by dark periods suggests the existence of an additional (nonemissive) state B in the usual three-levels electronic system, S 0 (singlet ground state), S 1 (singlet excited state) and T 1 (triplet ground state).…”

Dynamics of green fluorescent protein mutant2 in solution, on spin‐coated glasses, and encapsulated in wet silica gels

“…Similar results were interpreted as a light‐driven flickering of GFP (Schwille et al 2000). At higher excitation powers, the relaxation time drops to ≅5 μs, a typical value for the thermally activated delayed fluorescence (Zumbusch and Jung 2000; Schwille et al 2000).…”

“…Representative auto‐correlation functions (ACFs) are shown in Figure 3. For GFPmut2, the ACFs were analyzed according to a simple diffusion model for longer lag times plus an exponential relaxation that corresponds to protein flickering or triplet state interconversion (Song et al 1996; Haupts et al 1998; Zumbusch and Jung 2000):…”

Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

“…Number of collected photons before bleaching versus the excitation intensity. A (2) outside attached to PSS layer ( ); A (1) family (•) and B family (O) in the layers; silica gel (S (3) in table 1) (◊); agarose gel (▼) as in [9].…”

Two-Photon Thermal Bleaching of Single Fluorescent Molecules

“…The lifetime of the triplet state varies from few ms to tens of ms (Veerman et al, 1999), while the length of the dark periods observed by us is on the order of seconds. On the other hand the blinking due to the triplet-singlet interconversion (Dickson et al, 1997;Zumbusch and Jung, 2000;Song et al, 1996;Haupts et al, 1998;Schwille et al, 2000) is masked in our ffi250 ms acquisition time. Instead, our finding of emission intervals separated by dark periods suggests the existence of an additional (nonemissive) state B in the usual three-levels electronic system, S 0 (singlet ground state), S 1 (singlet excited state) and T 1 (triplet ground state).…”

Dynamics of green fluorescent protein mutant2 in solution, on spin‐coated glasses, and encapsulated in wet silica gels

“…Similar results were interpreted as a light‐driven flickering of GFP (Schwille et al 2000). At higher excitation powers, the relaxation time drops to ≅5 μs, a typical value for the thermally activated delayed fluorescence (Zumbusch and Jung 2000; Schwille et al 2000).…”

“…Representative auto‐correlation functions (ACFs) are shown in Figure 3. For GFPmut2, the ACFs were analyzed according to a simple diffusion model for longer lag times plus an exponential relaxation that corresponds to protein flickering or triplet state interconversion (Song et al 1996; Haupts et al 1998; Zumbusch and Jung 2000):…”

Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

“…Number of collected photons before bleaching versus the excitation intensity. A (2) outside attached to PSS layer ( ); A (1) family (•) and B family (O) in the layers; silica gel (S (3) in table 1) (◊); agarose gel (▼) as in [9].…”