Simultaneous optically sectioned fluorescence and optical coherence microscopy with full-field illumination

Abstract

Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis.

Full-Field Optical Coherence Tomography as a Diagnosis Tool: Recent Progress with Multimodal Imaging

“…Many such multimodal setups have been developed with both scanning OCT approaches [5,147] and full-field approaches, using either a flip mirror to switch from one modality to the other [18,148], or a simultaneous approach [41,42]. If all these multimodal approaches share a common interest, scanning OCT approaches are more easily combined with scanning fluorescence approaches, such as confocal or two photon microscopies, whereas FF-OCT systems have been developed in combination with widefield fluorescence techniques, such as structured illumination microscopy (SIM).…”

“…The SIM acquisition rate can go up to 100 frames per second in our measurements, even though the usual frame rate limit comes from the fluorophores' brightness, and from the minimal exposure time required to produce a decent image. Interestingly, simultaneous combination of FF-OCT and SIM [41,42] enable the parallel acquisition of both modalities, so images with both modalities can be acquired at more than 100 frames per second. However, SIM is not very robust to aberrations and multiple scattering, and can hardly penetrate inside scattering samples after 30 to 50 µm.…”

“…The second setup is a multimodal setup combining FF-OCT and structured illumination fluorescence microscopy (SIM) [41], in a relatively similar layout to the one developed by H.Makhlouf and colleagues [42]. To enhance the photon collection, we used higher NA objectives, either 40X, 0.8 NA in water, or 30X, 1.05 NA in silicone oil, achieving transverse resolution below 500 nm, and depth of focus between 1 and 2 µm.…”

En face coherence microscopy [Invited]

“…Fluorescence techniques can reveal or complement the OCT information that is obtained from a cell or a structure [77][78][79][80] using various approaches: single or 2 photon fluorescence, superficial observation or endoscopy [81][82][83][84]. Following similar developments in OCT [85][86][87][88], several multimodal setups associating full field microscopy and various fluorescence techniques have been developed [89][90][91]. While confocal or multi-photon microscopy can be naturally associated with point scanning OCT systems, it is less natural for full field coherence microscopy, where widefield fluorescence techniques are preferred.…”

Extended full-field optical coherence microscopy

“…Several extensions to the technique have also been proposed to provide additional information on the birefringence [26] or the spectroscopic properties [27] of the sample. Fluorescence microscopy was recently coupled to FF-OCM to yield complementary morphological and functional information [28].…”