Three-dimensional holographic fluorescence microscopy

Abstract

Most commonly used methods for three-dimensional (3D) fluorescence microscopy make use of sectioning techniques that require that the object be physically scanned in a series of two-dimensional (2D) sections along the z axis. The main drawback in these approaches is the need for these sequential 2D scans. An alternative approach to fluorescence imaging in three dimensions has been developed that is based on optical scanning holography. This novel approach requires only a 2D scan to record 3D information. Holograms of 15-microm fluorescent latex beads with longitinal separation of ~2 mm have been recorded and reconstructed. To our knowledge, this is the first time holograms of fluorescent specimens have been recorded by an optical holographic technique.

Principles and techniques of digital holographic microscopy

“…The detected light may in fact be a secondary emission, such as fluorescence, and does not need to be coherent. For this reason, OSH has a unique potential for 3-D fluorescence microscopy [216]. In fact, the detection mode can be varied so that the coherence property of the imaging process ranges from linear in amplitude to linear in intensity [217].…”

Optical Scanning Holography - A Review of Recent Progress

“…The reason is that more than 90% of biomedical specimens are fluorescent and with OSH we can capture holograms of fluorescent samples. Poon et al has issued a patent on three-dimensional holographic fluorescence microscopy [20] and the captures of fluorescent specimens holographically, for the first time, have been reported by Schilling et al with resolution about 15 μm [21]. Most recently, holograms of Oscillatoria strands illuminated at 532 nm and fluoresced at 600 nm have been demonstrated with resolution better than 1 μm [18].…”

Space–time digital holography: A three-dimensional microscopic imaging scheme with an arbitrary degree of spatial coherence

“…The recording of holograms of fluorescent specimens using temporal, rather than spatial heterodyning has been demonstrated recently with a scanning method. 8 In this letter, we describe and demonstrate a digital holographic method capable of recording holograms of threedimensional objects with an arbitrary degree of spatial coherence, and in a single exposure, without any mechanical scan. The coherence of the illumination can be varied as in a conventional microscope by changing the size of the source, a versatility which is important in microscopy where incoherent illumination is preferred if scattering and speckle noise is a problem, but partially coherent illumination is needed for example to visualize phase variations and unstained specimens.…”