Fig 1.
(a) Photograph of the OPT plate detailing the stepper motor, water-tight chamber and position of the sample tube (shown in red). (b) Photograph of an inverted microscope adapted for OPT, with the plate inserted in the microscope stage aperture. (c) The custom chamber is suspended from an upper plate, with the lower plate seated in microscope stage. For alignment purposes, the axis of rotation (red line) is adjusted until it is orthogonal to the optical axis, setting the tilt angle φ = 0°. (d) In addition the axis of rotation should be approximately centred on the camera sensor and rotated to align with the sensor pixels, setting the shift δ ≈ 0 and rotation angle ζ ≈ 0°.
Fig 2.
(a) Schematic showing a microscope adapted for OPT. The only additional component inside the microscope (indicated by the dashed box) was the aperture placed directly behind the objective lens to reduce the NA. Simulated shift invariant axial PSF for (b) full-depth OPT, and (c) half-depth OPT. The sample cross section is indicated by red circle. Note that the PSFs are scaled for illustrative purposes.
Fig 3.
In vivo half-depth of field OPT reconstruction of a 5 days post fertilization transgenic mpx:GFP zebrafish, combining sequential fluorescence (for neutrophil GFP expression, shown in green) and transmission (for zebrafish morphology, shown in grey) acquisitions.
(a) Single slice through the reconstruction, accompanying (b) YZ cross-section (CS) along vertical yellow line in (a). (c) Maximum intensity projection (MIP) through entire reconstructed volume. (d) Magnified view of reconstruction within red box indicated in (b), and (e) line profile through neutrophils cells.
Fig 4.
(a) External relay added to the camera port of the microscope for higher magnification standard OPT and remote focal scanning OPT (RFS). A continuation of the components within the microscope frame (Fig 1(C)), CM removable cube mirror. External RFS-OPT relay (red box): L3 achromatic doublet, M2/M3 mirror cubes, ETL electrically tunable lens. Conventional OPT relay (blue box): M4 mirror, L4/L5 achromatic doublets, AS variable iris acting as aperture stop. (b) The variable iris or ETL act as the aperture stop in the conventional or RFS OPT systems respectively. The size of the iris or scan range of the tunable lens determine the respective DoF, but axial displacement away from the conjugate pupil plane can lead to non-telecentric performance. The 3D axial PSF is no longer shift invariant, resulting in a depth dependant magnification. Note the point spread functions are scaled for illustrative purposes. (c,d) Simulation showing the static axial PSF (exterior panels) and effective axial PSF(central panels) for RFS-OPT and region of interest (RoI) OPT. (c) RFS-OPT with a scan range covering the full axial extent (d) RoI-OPT uses a smaller SR to increase the contrast to noise ratio over a desired region of interest. The RoI is tracked in depth, by adjusting the focal offset required to track the RoI during an acquisition (i.e as the sample rotates to each projections angle).
Fig 5.
Fluorescence (green/cyan) and transmitted light (grey) reconstructions from an in vivo acquisition of a 5 dpf Tg(mpx:GFP) zebrafish using (a-e) an image relay for standard full-depth of field OPT at NA~0.035 and (f-j) RoI-OPT at full NA with an axial scan range (SR) of 130 μm.
(a,f) MIP of full reconstruction, (b,g) single YZ slice, (c,h) single XZ slice with depth of field and SR respectively indicated by dotted lines, (d,i) fluorescence reconstruction from region indicated by red box and (e,j) corresponding intensity line profile.