Versatile high-speed confocal microscopy using a single laser beam.

We present a new flexible high speed laser scanning confocal microscope and its extension by an astigmatism particle tracking velocimetry (APTV) device. Many standard confocal microscopes use either a single laser beam to scan the sample at a relatively low overall frame rate or many laser beams to simultaneously scan the sample and achieve a high overall frame rate. The single-laser-beam confocal microscope often uses a point detector to acquire the image. To achieve high overall frame rates, we use, next to the standard 2D probe scanning unit, a second 2D scan unit projecting the image directly onto a 2D CCD-sensor (re-scan configuration). Using only a single laser beam eliminates crosstalk and leads to an imaging quality that is independent of the frame rate with a lateral resolution of 0.235 µm. The design described here is suitable for a high frame rate, i.e., for frame rates well above the video rate (full frame) up to a line rate of 32 kHz. The dwell time of the laser focus on any spot in the sample (122 ns) is significantly shorter than those in standard confocal microscopes (in the order of milli- or microseconds). This short dwell time reduces phototoxicity and bleaching of fluorescent molecules. The new design opens up further flexibility and facilitates coupling to other optical methods. The setup can easily be extended by an APTV device to measure three dimensional dynamics while being able to show high resolution confocal structures. Thus, one can use the high resolution confocal information synchronized with an APTV dataset.