TY - JOUR
T1 - Surface plasmon-assisted microscope
AU - Borejdo, Julian
AU - Gryczynski, Zygmunt
AU - Fudala, Rafal
AU - Joshi, Chaitanya R.
AU - Borgmann, Kathleen
AU - Ghorpade, Anuja
AU - Gryczynski, Ignacy
N1 - Funding Information:
We appreciate the assistance of the Laboratory of Developmental Biology at the University of Washington, Seattle, WA for providing human brain tissues; supported by NIH 5R24 HD0008836 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. We thank Satomi Stacey for technical assistance.
Publisher Copyright:
© 2018 The Authors.
PY - 2018/6/1
Y1 - 2018/6/1
N2 - Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100-to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.
AB - Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100-to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.
KW - fluorescence
KW - microscopy
KW - surface plasmon coupled emission
UR - http://www.scopus.com/inward/record.url?scp=85049201245&partnerID=8YFLogxK
U2 - 10.1117/1.JBO.23.6.060502
DO - 10.1117/1.JBO.23.6.060502
M3 - Article
C2 - 29935016
AN - SCOPUS:85049201245
SN - 1083-3668
VL - 23
JO - Journal of Biomedical Optics
JF - Journal of Biomedical Optics
IS - 6
M1 - 060502
ER -