TY - JOUR
T1 - Minimization of detection volume by surface-plasmon-coupled emission
AU - Gryczynski, Z.
AU - Borejdo, J.
AU - Calander, N.
AU - Matveeva, E. G.
AU - Gryczynski, I.
N1 - Funding Information:
This research was supported by NIH (NCI CA-114460, NIBIB EB-1690, AR-048622), BITC, and Philip Morris USA, Inc. The results were partially reported in SPIE Photonic West Conference, San Jose, January 2006. We dedicate this paper to Professor Enrico Gratton on the occasion of his 60th birthday.
PY - 2006/9/1
Y1 - 2006/9/1
N2 - We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a product of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.
AB - We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a product of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.
KW - Attoliter
KW - Fluorescence microscope
KW - Minimized detection volume
KW - Surface-plasmon-coupled emission
UR - http://www.scopus.com/inward/record.url?scp=33747197743&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2006.05.007
DO - 10.1016/j.ab.2006.05.007
M3 - Article
C2 - 16764813
AN - SCOPUS:33747197743
VL - 356
SP - 125
EP - 131
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -