Euromedim 2006 :1st European Conference on Molecular Imaging Technology

Shrunken detection volumes in fluorescence 4Pi-microscopy

11 May 2006, 14:00

1h

Palais du Pharo, Marseille

poster Molecular Imaging needs for biologists and physicians Poster session : Imaging systems, Molecular Imaging

Speaker

Dr Nicolas Sandeau (Institut Fresnel)

Description

Keywords: Fluorescence Confocal Microscopy, 4Pi-microscopy, Molecular Detection Efficiency Function, Lateral Resolution, Axial Resolution. Improving the spatial resolution in optical microscopes is a challenging task for many applications. In fluorescence confocal microscopy the resolution is given by the Detection Efficiency Function (DEF) resulting from the product of the Excitation Efficiency Function (EEF) by the Collection Efficiency Function (CEF). The focalisation of the pump laser beam by a high numerical aperture objective lens defines the excitation efficiency volume whereas the image of the pinhole represents the collection efficiency volume. The axial dimension of the detection efficiency volume of fluorescence microscopes can be considerably improved by superposing two coherent illumination beams [1] and by adding coherently the two fluorescence wave- fronts emitted on the both sides of the luminescent sample [2]. This solution implemented in 4Pi-microscopes [3] must be coupled with a two-photon excitation mode [4] to be really efficient along the optical axis. Unfortunately, this method increases the lateral dimensions of Detection Efficiency Volume because of the infrared wavelength used for the illumination. Within this context, we propose an arrangement of the 4Pi-microscope [5] using the “spatial incoherence” of fluorescent samples to shrink the collection efficiency volumes by a factor of two [6]. In this case, the lateral extent of the Detection Efficiency Function is smaller with a two- photon [7] excitation mode than the one obtained in the classic case, with a one- photon excitation mode. Moreover the amplitudes of the side lobes are strongly reduced. References: 1. C. J. R. Sheppard and Yunrui Gong, “Improvement in Axial Resolution by Interference Confocal Microscopy,” Optik 87, 129-132 (1991). 2. N. Sandeau, H. Giovannini, P.-F. Lenne and H. Rigneault, "Observation of the interferences between the emitted beams in a 4Pi microscope by Partial Coherence Interferometry" Applied Physics Letters 87 (18103), 2005. 3. S. Hell and E. H. K. Stelzer, “Properties of a 4pi Confocal Fluorescence Microscope,” J. Opt. Soc. Am. A 9, 2159-2166 (1992). 4. S. Hell and E. H. K. Stelzer, "Fundamental improvement of resolution with 4Pi- confocal fluorescence microscope using two-photon excitation," Opt. Comm. 93, 277- 282 (1992). 5. S. Hell, European Patent Application EP0491289 (filed 18. dec. 1990) published 1992. 6. N. Sandeau and H. Giovannini, “Increasing the lateral resolution of 4Pi fluorescence microscopes” accepted for publication in J. Opt. Soc. Am. A (Nov. 2005) 7. N. Sandeau and H. Giovannini, “Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation” submitted to Optics Communications (Nov. 2005)

Presentation materials

Paper

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