The Role of Amplitude and Phase in Fluorescence Coherence Imaging: From Wide Field to Nanometer Depth Profiling A. Bilenca, C. Joo, A. Ozcan, J. F. de Boer, B. Bouma and G. Tearney Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom Street, BAR 7, Boston, Massachusetts, 02114 abilenca.partners.org Abstract: We investigate the amplitude and phase of fluorescence self-interference fields and their implication for new imaging strategies. Wide-field (y > 1mm, z > 100μm) high resolution (micron-scale) imaging and phase-contrast profiling with nanometer sensitivity are demonstrated. ©2007 Optical Society of America OCIS codes: (170.1650) Coherence imaging; (110.6960) Tomography; (120.5050) Phase measurement The manipulation of low coherence optical fields for sensing deep inside biological media has had a significant impact on the field of biomedical optical imaging. In general, both amplitude and phase of low coherence fields carry a wealth of information. Using the amplitude of low coherence fields has proven to be useful for biological and biomedical imaging of tissue reflectance [1-3]. For example, coherence gating techniques, such as optical coherence tomography (OCT) [1-3], simultaneously provide micron-scale optical sectioning while maintaining a large depth of field. Recently, the use of phase information of low coherence fields for imaging and microscopy has been demonstrated, including Fourier phase microscopy [4] and spectral-domain coherence phase microscopy [5, 6]. These techniques show great potential for quantitative studies of biological systems. Here we employ concepts of spectral-domain coherence gated imaging and coherence phase microscopy to manipulate fluorescence fields, instead of reflected light. Use of low coherence interferometric techniques for fluorescent light imaging provides new capabilities, such as wide field optical sectioning with low NA lenses and determination of fluorophore depth positions to within tens of nanometers. Wide field fluorescence imaging with high axial resolution and large depth range (referred to as ‘Spectral-domain Fluorescence Coherence Tomography’ or SD-FCT [7]) is achieved by exciting the entire sample depth with a wide field-of-focus line beam and then employing self-interference of fluorescence light [8] and imaging spectrometry to detect spectral fringe patterns of excited fluorescent probes along the illumination beam. Since the micron-scale axial (depth) location of excited fluorophores is encoded by the spectral fringe frequency, it may be retrieved by extracting the amplitude information (that is, the modulus) of the inverse Fourier transform of the recorded spectral interferograms. The depth range and transversal field of SD-FCT depend on the excitation optics. The former can be on the order of a few hundreds of microns for low NA objectives and the later on the order of millimeters. Furthermore, unlike the transversal resolution, the axial resolution of SD-FCT is decoupled from the optics and is determined by the coherence length of the fluorescent light (typically a few microns). Nanometer profiling is obtained by evaluating the differential phase information from the spectral interferograms of the self-interference fluorescence signals using numerical algorithms similar to those reported in [5, 6]. In this context, it is worth mentioning spectral self-interference fluorescence microscopy which also extracts phase information for detecting fluorophores on reflecting surfaces [8]. Hereon, we present the SD-FCT experimental setup and the corresponding amplitude/phase measurements. An object consisting of a fluorescently labeled specimen is located near the zero differential path length point (z 0 ) of a two opposing matched objectives interferometer and is illuminated with an excitation line focus (dashed line) as shown in Fig. 1. Similar to 4Pi microscopy [9], fluorescence emission (solid line) is collected by the two opposing objectives and combined at the beam splitter. The resulting interference as a function of emission wavelength is detected by an imaging spectrometer. We note that this experimental arrangement provides one-shot ‘y-z’ cross-sectional imaging capabilities due to the parallel detection of multiple spectra corresponding to different points along the transversal (‘y’) direction of the sample. a2668_1.pdf CTuV1.pdf