Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule

被引:65
作者
Hassler, K [1 ]
Leutenegger, M
Rigler, P
Rao, R
Rigler, R
Gösch, M
Lasser, T
机构
[1] Ecole Polytech Fed Lausanne, Lab Opt Biomed, CH-1015 Lausanne, Switzerland
[2] Univ Basel, CH-4056 Basel, Switzerland
来源
OPTICS EXPRESS | 2005年 / 13卷 / 19期
关键词
D O I
10.1364/OPEX.13.007415
中图分类号
O43 [光学];
学科分类号
070207 ; 0803 ;
摘要
We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores. (c) 2005 Optical Society of America.
引用
收藏
页码:7415 / 7423
页数:9
相关论文
共 23 条
[1]   Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS [J].
Auer, M ;
Moore, KJ ;
Meyer-Almes, FJ ;
Guenther, R ;
Pope, AJ ;
Stoeckli, KA .
DRUG DISCOVERY TODAY, 1998, 3 (10) :457-465
[2]   Total internal reflection fluorescence microscopy in cell biology [J].
Axelrod, D .
TRAFFIC, 2001, 2 (11) :764-774
[3]  
AXELROD D, 1992, TOPICS FLUORESCENCE, V3
[4]  
Brock R, 1998, CELL MOL BIOL, V44, P847
[5]   Diffusion coefficient measurements in microfluidic devices [J].
Culbertson, CT ;
Jacobson, SC ;
Ramsey, JM .
TALANTA, 2002, 56 (02) :365-373
[6]   The fluctuating enzyme:: a single molecule approach [J].
Edman, L ;
Földes-Papp, Z ;
Wennmalm, S ;
Rigler, R .
CHEMICAL PHYSICS, 1999, 247 (01) :11-22
[7]   Fluorescence detection of single molecules near a solution/glass interface - an electrodynamic analysis [J].
Enderlein, J .
CHEMICAL PHYSICS LETTERS, 1999, 308 (3-4) :263-266
[8]   Total internal reflection fluorescence correlation spectroscopy for counting molecules at solid/liquid interfaces [J].
Hansen, RL ;
Harris, JM .
ANALYTICAL CHEMISTRY, 1998, 70 (13) :2565-2575
[9]   High count rates with total internal reflection fluorescence correlation spectroscopy [J].
Hassler, K ;
Anhut, T ;
Rigler, R ;
Gösch, M ;
Lasser, T .
BIOPHYSICAL JOURNAL, 2005, 88 (01) :L1-L3
[10]  
HASSLER K, 1015 EPFL