In confocal fluorescence correlation microscopy (FCM) it is important to ensure that the correlation measurement is actually performed at the chosen location of the three-dimensional image of the specimen. We present a confocal FCM design that provides an automatic real-time readout of the location in the confocal microscopic image, which is aligned with the detector of the fluorescence correlation spectrometer. The design accomplishes this without using any special positioning device. The design is based on an apertured fluorescence detector placed close to the back aperture of the objective lens and can be easily incorporated into virtually any confocal microscope. We demonstrate the method by performing FCM measurements of a dye diffusing on a cell membrane.
© 2005 Optical Society of America
(170.1530) Medical optics and biotechnology : Cell analysis
(170.1790) Medical optics and biotechnology : Confocal microscopy
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(180.1790) Microscopy : Confocal microscopy
Sanjeev Kumar Kaushalya, Jayaprakash Balaji, Kanchan Garai, and Sudipta Maiti, "Fluorescence correlation microscopy with real-time alignment readout," Appl. Opt. 44, 3262-3265 (2005)