High‐resolution confocal microscopy using synchrotron radiation

Abstract

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2 optics allows for confocal imaging over the wavelength range 200-700 nm. This includes UV light, which is particularly suitable for high-resolution imaging. The results of test measurements using 290-nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real-time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9-dihydroxy-6H-benzofurol[3,2-c][1]benzo-pyran-6-one (Chem. Abstr. Reg. No. 479-13-0). The trivial name will be used throughout this paper.

Heterodimeric interaction and interfaces of S100A1 and S100P

“…The fluorescence lifetime of ECFP-S100A1 was collected using a purpose-built scanning confocal microscope [26] from HeLa cells that co-expressed (i) ECFP-S100A1 and S100P-EYFP, (ii) ECFP-S100A1 and EYFP vectors (negative control), (iii) ECFP-S100A1 (negative control) or (iv) ECFP-S100A1 and S100A1-EYFP (positive control). The data were recorded at room temperature (20 • C), and FLIM (fluorescence lifetime imaging microscopy) images in the time domain from single cells were collected using a time-correlated single-photon counting [27] fluorescence lifetime imaging module (SPC-730; Becker & Hickl, Berlin, Germany) and a photomultiplier tube (PMH-100-1; Hamamatsu Photonics, Bridgewater, NJ, U.S.A.).…”

Confocal laser scanning microscopy of calcium dynamics in living cells

“…With microscopical refinements and additional processing of the confocal data, it is theoretically possible to increase the lateral resolution in CLSM to below 100 nm Schrader et al, 1996;Vanderoord et al, 1996;Vanoijen et al, 1998). However, the lateral and axial resolutions that are actually obtained by CLSM in analyses of fluorescent biological samples can be negatively affected by such factors as: (1) the alignment and cleanliness of the optical components (Centonze and Pawley, 1995); (2) the confocal aperture's size and its shape (i.e., pinhole vs. slit apertures) as well as its orientation relative to the sample in the case of slit-scanning CLSM (Gard, 1993;Wilson, 1995); (3) the depth of the optical plane within the sample ; and (4) refractive index mismatches within the imaging pathway (Hell and Steltzer, 1995).…”

Adenovirus Type-5 Entry and Disassembly Followed in Living Cells by FRET, Fluorescence Anisotropy, and FLIM

“…Fluorescence anisotropy decays were collected by measuring the components of the fluorescence decay parallel [I par (t)] and perpendicular [I per (t)] to the linearly polarized excitation light: r(t) ¼ (I par ÿ I per ) / (I par 1 2I per ) with a polarizing microscope as described elsewhere (Martin-Fernandez et al, 1998). Confocal reflection and fluorescence images were collected using a purpose-built confocal microscope equipped with a continuous wave Ar1 laser as a light source (Van der Oord et al, 1996). Pinholes (50 mm) were used in the excitation and emission paths.…”