Three-Dimensional Fluorescence Microscopy by Optical Scanning Holography

Abstract

As three-dimensional (3D) imaging and fluorescence techniques become standard in optical microscopy, novel approaches to 3D fluorescence microscopy are emerging. One such approach is based on the incoherent holography technique called optical scanning holography (OSH). The main advantage of OSH-based microscopy is that only a single two-dimensional (2D) scan is required to record 3D information, whereas most current 3D microscopes rely on sectioning techniques. To acquire a 3D representation of an object, current microscopes must physically scan the specimen in a series of 2D sections along the z-axis.

In order to record holograms by OSH, the fluorescent specimen is scanned with an optically heterodyned laser field consisting of a Fresnel zone pattern. A unique acousto-optic modulator configuration is employed to generate a suitable heterodyne frequency for excitation of the fluorescent object. The optical response of a solution containing a high concentration of 15 um fluorescent latex beads to this type of excitation field has been recorded. In addition, holograms of the same beads have been recorded and reconstructed. To demonstrate the 3D imaging capability of the technique, the hologram includes beads with longitudinal separation of about 2 mm.

A detailed comparison of 3D fluorescence microscopy by OSH and the confocal approach was conducted. Areas for comparison were 3D image acquisition time, resolution limits and photobleaching. The analysis shows that an optimized OSH-based fluorescence microscope can offer improved image acquisition time with equal lateral resolution, but with degraded longitudinal resolution when compared to confocal scanning optical microscopy (CSOM). For the photobleaching investigation, the parameter of concern is the fluence received by the specimen during excitation, which takes into account both the irradiance level and the time of illumination. Both peak and average fluence levels are addressed in the comparison. The analysis shows that during a 3D image acquisition, the OSH system will deliver lower peak fluence but higher average fluence levels to the specimen when compared to CSOM.