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Abstract
We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.
Highlights
Widefield single-photon microscopy is still extensively used for live cell Ca2+ imaging using fluorescent reporter molecules
With an image acquisition rate of 1 Hz, the plots were similar for single-photon and two-photon excitation, with the normalised average fluorescence intensity having decreased by 5% for two-photon excitation and by 6% for singlephoton excitation
The error bars show the standard deviation from an average of four images. It is clear from the intensity profile that the illuminated field was not perfectly homogeneous, but the fluorescence intensity level varied by less than 10% across the field of view. In evaluating this widefield two-photon microscopy method, it is natural to ask what advantages it confers in relation to single-photon widefield microscopy
Summary
Widefield single-photon microscopy is still extensively used for live cell Ca2+ imaging using fluorescent reporter molecules. As well as problems with loading, compartmentalisation and leaking of fluorescent Ca2+ indicators, one of the main limitations of their use in single-photon microscopy is photo-bleaching, which can severely limit the number of experiments possible with a given specimen and restrict the useful duration of imaging experiments [1]. We demonstrate a new method of live cell Ca2+ imaging using widefield twophoton excitation without scanning, which has the advantage of greatly reduced photo-bleaching in comparison with single-photon excitation. Its chief advantages are its ability to penetrate more deeply into tissues than single-photon excitation, the creation of optical sections (by the combination of an excitation proportional to the square of the intensity with the conical beam geometry) and the more efficient utilization of scattered emission than is possible in a confocal microscope. The chief drawbacks were the slow scanning speed, which restricted the original instruments to a rate of approximately one image per second, often
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