Spectral imaging fluorescence microscopy

Abstract

The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).Since the discovery of the jellyfish green fluorescent protein (GFP) and its colour variants, multiple wavelength fluorescence imaging has become a useful tool in studies of cell biology. For imaging multiple wavelength fluorescence images, computer-controlled microscope systems that automatically switch optical filters during observation have been developed (Hiraoka et al . 1991;Haraguchi et al . 1999). While such microscope systems can collect fluorescence images at multiple wavelengths, fluorescence emissions with a spectral overlap can cause cross-talk between the emitted signals, thus limiting the choice of fluorescent dyes.Here we introduce a microscope technique that is capable of resolving the spectra of fluorescence images without switching optical filters; the fluorescence spectra are obtained as a series of images as a function of fluorescence wavelength. This capability of spectral resolution makes it possible to unmix the cross-talk between overlapping fluoresence emissions. It also provides a unique capability for detecting spectral changes in fluorescent indicators; for example, an indicator for fluorescence resonance energy transfer (FRET). Such techniques have been devised by the use of an acoustooptic tunable filter (AOTF) or a grating (Wachman et al . 1997;Hanley et al . 2000;Tsurui et al . 2000;Ford et al . 2001;Lansford et al . 2001).The hardware design we used is an implementation of the previously reported technology of two-photon imaging spectroscopy (Lansford et al . 2001) to a commercial single-photon confocal scanning microscope; the technology is applicable to a single-photon or multiphoton fluorescence microscope. In this spectral imaging microscope system (LSM510 META; Carl Zeiss, Jena, Germany), fluorescence spectra are resolved using grating optics; resolved fluorescence spectra at each pixel are detected by an array of 32 photomultiplier tubes (PMT) placed at the confocal plane, and recorded on a pixel-by-pixel basis during scanning to generate a set of images, each corresponding to the fluorescence wavelength resolved at 10 nm intervals. The maximum range of wavelength is 380 -720 nm, resolved to 32 divisions. Spectral resolution of fluorescence imagesWe first demonstrate that the use of grating optics allows us to obtain multiple-wavelength images without switching optical filters. An example of spectral imaging, or imaging spectroscopy, is shown in Fig. 1A. ...

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