Compared to competing scanning spot OCM designs, the authors implemented an alternative approach, full-field optical coherence microscopy (FF-OCM). In this acquisition scheme, a thin en-face slice of the imaged structure is reconstructed rather than a depth scattering profile. This was done by processing two phase shifted interferometric images recorded by a OCM CCD camera. To limit the effect of speckle and to achieve uniform illumination of the sample, a halogen lamp setup in Kohler illumination was used. Additionally, a Linnik interferometer with identical water immersed microscope objectives at both arms was used to limit negative effects of aberrations in sample and reference arms.
To complement the wide field image acquisition offered by FF-OCM, the authors used the structured illumination microscopy (SIM) technique to acquire the fluorescent image. To achieve that, three images of the sample illuminated with a shifting grid pattern were acquired and processed to get rid of the modulated illumination and to reconstruct an optically sectioned fluorescent image. Here a 488nm solid state laser was used as the light source, with a spinning ground glass plate inserted into the path of the laser beam to suppress speckle.
The performance of presented system was demonstrated by imaging two samples of excised mouse colon and tongue tissues, topically stained with acridine orange (AO). The images of both samples clearly demonstrate different morphological information captured by fluorescence (nucleic structure of the sample) and FF-OCM (gross morphology of the sample).
In summary, this novel dual modality imaging system can provide simultaneously wide field high resolution FF-OCM and fluorescent images of biological tissues. Optical sectioning of acquired data was achieved by coherence gating properties of the light source used in OCM and by implementing structured illumination for fluorescent images. The combined fluorescent and tissue scattering information offered by this system should be useful for pathological diagnosis and for studying cellular function.
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