1 May 2007 Parallel two-channel near- and far-field fluorescence microscopy
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Abstract
We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the water/glass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.
© (2007) Society of Photo-Optical Instrumentation Engineers (SPIE)
Doriel Verdes, Doriel Verdes, Thomas Ruckstuhl, Thomas Ruckstuhl, Stefan Seeger, Stefan Seeger, } "Parallel two-channel near- and far-field fluorescence microscopy," Journal of Biomedical Optics 12(3), 034012 (1 May 2007). https://doi.org/10.1117/1.2747627 . Submission:
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