Abstract
We present a new imaging method to record multicolor digital holograms from objects emitting fluorescent light. The fluorescent light specific to the emission wavelength of various fluorescent dyes after excitation of three dimensional (3D) objects is recorded on a digital monochrome camera after reflection from a diffractive optical element (DOE). For each wavelength of fluorescent emission, the camera sequentially records three holograms reflected from the DOE, each with a different phase factor of the DOE's function. The three holograms are superposed in a computer to create a complex valued Fresnel hologram of each fluorescent emission. The holograms for each fluorescent color are further combined in a computer to produce a multicolored fluorescence hologram and 3D color image.
Highlights
Fluorescence is one of the most widely used techniques in many areas of biology, material science and medical imaging because of its sensitivity, selectivity, low background and high contrast
Non-fluorescent holography is a well established technique in which holograms are recorded by interfering two mutually coherent beams [1]. This technique cannot be applied to the incoherent light which is characteristic of the emitted light in a fluorescent object
A Fresnel Zone Plate (FZP) pattern from a coherent fluorescence excitation source scans the fluorescent labeled object such that at each and every scanning position the emitted light intensity is integrated by a detector
Summary
Fluorescence is one of the most widely used techniques in many areas of biology, material science and medical imaging because of its sensitivity, selectivity, low background and high contrast. Non-fluorescent holography is a well established technique in which holograms are recorded by interfering two mutually coherent beams [1]. This technique cannot be applied to the incoherent light which is characteristic of the emitted light in a fluorescent object. A double channel interferometer with continuous temporal variation of the phase differences between the interfering beams to record non-fluorescent incoherent holograms has been described [7] This is in contrast to the much simpler motionless single channel FINCH system reported here which has been readily able to produce high quality 3D fluorescent images. To the best of our knowledge, the demonstrated holograms are the first fluorescence holograms recorded without scanning and the first fluorescence multiwavelength emission color holograms ever recorded
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