Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 125-131 |
| Number of pages | 7 |
| Journal | Analytical Biochemistry |
| Volume | 356 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1 Sep 2006 |
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Keywords
- Attoliter
- Fluorescence microscope
- Minimized detection volume
- Surface-plasmon-coupled emission
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Minimization of detection volume by surface-plasmon-coupled emission. / Gryczynski, Z.; Borejdo, J.; Calander, N.; Matveeva, E. G.; Gryczynski, I.
In: Analytical Biochemistry, Vol. 356, No. 1, 01.09.2006, p. 125-131.Research output: Contribution to journal › Article
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.
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 -