Experimental setup

Our design derived from our previous work on oblique scanning laser ophthalmoscopy (oSLO) that essentially implements scanning OPM using natural ocular optics (27). We previously simulated the diffraction-limited 3D resolutions at varying NA of the objective lens from 0.1 to 0.9 (24), and demonstrated oSLO in rodent in vivo and in human in vitro (27-29). Here we implemented and remodel the optical design used in oSLO in a microscopic setting. The schematic layout of the experimental setup is shown in Figure 1A. The objective lens OL1 is a low NA and low magnification objective lens, which makes it possible for a wide FOV in OPM.

The experiment setup of mesoscopic OPM. (A) The system schematic. (B) The angle enlargement of the image plane. L, lens; OL, objective lens; F, filter; IP, intermediate image plane; M, mirror; GM, galvanometer mirror; LS, light source; OA, optical axis; TS, translation stage; OPM, oblique plane microscopy.

As shown in Figure 1A, excitation light is marked by blue, and emission light is marked by pale green, green, and orange. The excitation light was generated by a 488 nm laser (LS), first coupled into single-mode fiber and then collimated by lens L1 (f=10 mm). An additional focus-tunable lens L2 (Edmund: EL-3-10-VIS-26D) was used to make fine adjustments such that excitation and emission light could be confocal over a large FOV. The collimated light was first scanned by GM1 (Thorlabs: GVS201) and then redirected into a 1:1 relay lens group (L3–L4) by a right-angle prism mirror M1 (MRAK25-P01). The GM1 was placed at the focal plane of L3. The intersection point of the scanning beams was generated at the pupil plane of objective lens OL1 after two relay lens groups (L3: f=100 mm, L4: f=100 mm; L5: f=100 mm, L6: f=50 mm), and then a scanned light-sheet was created within the specimen. To maximize the angle of the oblique light-sheet, the excitation light was offset from the optical axis OA1-OA3 and incident on the edge of OL1. Another galvanometer GM2 (Nutfield: QS-12 OPD, 20 mm aperture), which was conjugated to the pupil plane of the OL1, was used to sweep the oblique light-sheet through the sample along the Y direction. As GM2 steered the excitation light-sheet and created a moving light-sheet within the sample, fluorescence emission mapped back on the same GM2 could be descanned. As a result, an intermediate stationary image plane (IP) could be created between OL2 (UPLSAPO 20×/0.75) and OL3 (UplanFL20×/0.5). The angle between the optical axis OA2 and OA3 was minimized to facilitate a more effective descanning over a large FOV. M1 was used to direct the fluorescence into OL2. An emission filter F1 (500–550 nm) was placed in the back of OL2. As the relay lens groups (L3–L6) were in the arrangement of a typical 4f-imaging system, the magnification can be calculated directly by the ratio of the focal lengths. The magnification from L4 to L3 can be calculated as follows:

Similarly, the magnification from L6 to L5 can be calculated as follows:

So, the magnification from L6-L3 can be obtained by:

The magnification of these two relay lens groups (L3–L6) was chosen to maximize the use of the numerical aperture of OL2. The lateral (perpendicular to OA1) magnification MXY_OL1−OL2 from the sample to the conjugated IP can be calculated in the same way as:

The lateral magnification MXY_OL1−OL2 was designed to be less than 1. As the angle of the IP was significantly increased by the demagnification design, the remote imaging system could achieve sufficient light collecting efficiency. A translation stage TS of 4 degrees of freedom (X, Y, Z, and rotation around Z) was used to adjust the position and the angle of the remote imaging system.

OL1 can be switched between different low NA objective lenses, such as UplanFL10×/0.3 and UplanFL20×/0.5 from Olympus, to change FOVs and resolutions. The focal length of the collimator lens L1, as well as the two groups of relay lenses (L3–L4 and L5–L6), were carefully designed so that the Gaussian beam width and the Rayleigh range of the scanned light-sheet were ~9 µm and ~490 µm for 10× configuration. As for 20× configuration, these beam parameters were ~4.5 µm and ~122 µm, respectively. The axial magnification MZ_OL1−OL2 (along the OA1) from the sample to intermediate image (IP) could be calculated as follows (30):

It’s true that the refractive indices should be taken into consideration when calculating the magnification. However, the thickness of the mounting medium involved in imaging was <1 mm, which is small in comparison with the focal length (18 mm for 10×; 9 mm for 20×) and working distance (10 mm for 10×; 2.1 mm for 20×) of the objective. So, Eq. [5] would still hold reasonably, which is evident by our imaging results. As shown in Figure 1A, the XZ’ plane was imaged by the remote focusing system (from OL3 to L9) imaged after the optical refocusing of the sample to the intermediate image (IP) plane. As the angle of the sample plane with respect to the OA1 is small, we made the following approximation.

To ensure equal magnification on the image plane in the camera (Andor: Zyla 4.2, 2,448×2,048 pixels, 6.5 µm pixel pitch), we inserted anamorphic telescope composed of cylindrical lens L7 (Edmund: #35024, f=50 mm) and L8 (Edmund: 2 × #68-046, f=12.5 mm) between OL3 and L9 (Navitar: MVL75M1, f=75 mm), as described in (27). The magnifications of the remote focusing system in two directions are denoted as and and can be calculated as:

Then the ratio of MZ'_Remote and MX_Remote is calculated to be 4. The anamorphic telescope has optical power only in one dimension such that it can create a difference of 4× in the two directions. The overall magnification in the axial and lateral direction can be calculated as:

The magnification difference between axial and lateral magnification was corrected. Thus, the magnification of the whole optical system along all three directions (X, Y, and Z) is 2. As for 20× configuration, the later and axial magnification were 4 and 8, respectively.