Gabor domain optical coherence microscopy combined with laser scanning confocal fluorescence microscopy

Abstract

We report on the development of fluorescence Gabor domain optical coherence microscopy (Fluo GD-OCM), a combination of GD-OCM with laser scanning confocal fluorescence microscopy (LSCFM) for synchronous micro-structural and fluorescence imaging. The dynamic focusing capability of GD-OCM provided the adaptive illumination environment for both modalities without any mechanical movement. Using Fluo GD-OCM, we imaged ex vivo DsRed-expressing cells in the brain of a transgenic mouse, as well as Cy3-labeled ganglion cells and Cy3-labeled astrocytes from a mouse retina. The self-registration of images taken by the two different imaging modalities showed the potential for a correlative study of subjects and double identification of the target.

Fig. 1.

System layout of Fluo GD-OCM, a combination of GD-OCM and LSCFM.

Fig. 2.

Efficiencies of the dichroic mirrors and emission filter used in Fluo GD-OCM. (a) The dichroic mirror #1 for combining/splitting the optical paths of the tdTomato-fluorescence (red) and excitation (green), (b) the emission filter for blocking the excitation beam and passing the fluorescence, and (c) the dichroic mirror #2 for combining/splitting the optical paths of the GD-OCM and LSCFM. The light source of the GD-OCM (blue) was D-840-HP-I, Superlum.

Fig. 3.

A schematic of the imaging protocol: (a) when a 100-µm-thick brain slice is mounted on a glass slide and (b) when a <100-µm-thick retina is mounted on a glass slide and covered by a cover-slip.

Fig. 4.

Transverse imaging resolutions of the Fluo GD-OCM system. The USAF resolution target was imaged with (a) common-path modified GD-OCM and (b) LSCFM. The smallest resolvable group is marked in a red box. (c) The overlay of (a) and (b) demonstrate synchronization and transverse registration.

Fig. 5.

Longitudinal imaging resolution of Fluo GD-OCM. (a) The FWHMs of the axial PSFs over depth for the modified GD-OCM in common-path configuration (solid blue curve), the modified GD-OCM with the reference arm (solid black curve), and the original GD-OCM (solid red curve). The error bar is the standard deviation of the FWHM over ten measurements. (b) The optical sectioning capability of LSCFM was characterized by the FWHM of the through-focus intensity profile when a mirror was axially translated around the focus of the excitation beam (λ_EX=555-557 nm).

Fig. 6.

The implementation of Fluo GD-OCM showed two different working distances for GD-OCM and LSCFM over the range of electrical voltages applied to refocus the liquid lens of the probe. The operating wavelength, λ [nm], was 790-890 for GD-OCM (red) and 555-557 for LSCFM (blue). The bar is the standard deviation of the working distance, and ΔD is the averaged difference in the working distances over ten measurements.

Fig. 7.

Fluo GD-OCM images of a transgenic NG2-DsRed mouse brain slice. (a) An LSCFM image of the tissue showed several NG2-positive cells, confirmed by the DsRed fluorescence emission. NG2 was expressed by pericytes and vascular smooth muscle cells in the brain, and their cellular morphology allowed to differentiate segments of the vascular network: arterioles (yellow arrows), pre-capillary arterioles (green arrow), and capillaries (blue arrow) [56]. (b-c) Two GD-OCM en face images, taken at different depths separated by 30 µm, showing hyper-reflective strands of white matter surrounded by hypo-reflective gray matter. The hyper-reflective white matter corresponded to myelinated axon tracts that projected from the thalamus to cortex. (d-e) Overlays of the GD-OCM images in (b) and (c) with the LSCFM image in (a) showed that hypo-reflective spots corresponded to blood vessels that were drained during the perfusion-fixation process. The artifacts (e.g., the dark spots contained within the white oval dotted line in Fig. 7(b) and 7(c)) were caused by the difference in reflectivity of the reference beam deriving from non-uniform contact between the sample and the glass slide.

Fig. 8.

Fluo GD-OCM images of a mouse RBPMS-Cy3 labeled retina. (a) An LSCFM image of the tissue showing several RBPM-positive RGCs, confirmed by the Cy3-fluorescence emission. (b-c) Two GD-OCM en face images taken at different depths, separated by 78 µm. The LSCFM and the two GD-OCM images displayed co-localization of the optic disc (green arrow) and alignment between the hyper-reflective strands shown as yellow arrows in (c) and the hypo-reflective trajectories in (a), which corresponded to retinal vasculature. The granular feature visible in (b) mostly corresponded to the rods.

Fig. 9.

Fluo GD-OCM images of the mouse GFAP-Cy3 labeled retina. (a) An LSCFM image of the tissue showing several star-shaped GFAP-positive astrocytes whose end-feet wrap around the vasculature, confirmed by the Cy3-fluorescence emission. (b-c) Two GD-OCM en face images taken at different depths, separated by 84 µm. All images displayed co-localization of the optic disc (green arrow), and alignment between the hyper-reflective strands, shown as yellow arrows in (c), and the retinal vasculature in (a). The granular features shown in (b) mostly corresponded to the rods.

Fig. 10.

Optimization of the optical focus in GD-OCM for imaging either covered- or uncovered-sample fixed on a glass slide with two imaging configurations: (a) without or (d) with a coverslip. The corresponding theoretical GD-OCM B-scans are illustrated in (b) and (e), respectively, which contain images of the glass interfaces (red line) and the sample (thickened white line). Varying the focal plane locations, two 100-µm- and 25-µm-thick mouse brain slices were imaged in the setup of (a) and (d), respectively, where t_W=1 mm, t_G=1 mm, and t_c=160-190 µm. For the setups as shown in (a) and (d), the corresponding experimental A- and B-scans are shown in (c) and (f), respectively, within each region of interest (ROI).