2 METHODS
2.1 Sample Preparation
Thrombi were retrieved from the middle cerebral artery by stent-based
thrombectomy using a Solitaire stent (Covidien, Irvine, California) from
a patient with AIS in Soonchunhyang University, Bucheon Hospital. The
thrombi were sliced into sections (4-μm thickness) using a microtome.
Two consecutive sections of a thrombus were visualized using two
different imaging modalities: ODT and bright-field microscopy. Although
the two consecutive sections may differ in microstructure, we assumed
that they had nearly identical compositions of red blood cells (RBCs)
and fibrin. One thrombus section was prepared with H&E staining, while
the other was prepared without staining. Both thrombus sections were
mounted on standard glass slides and sealed with coverslips
(Figure 2 ).
2.2 Optical Measurement
The paired H&E stained and unstained slides of the thrombus were imaged
with a BF slide scanner (Axio Scan.Z1; Zeiss; Jena, Germany) and ODT,
respectively (Figure 2b). An ODT system was based on Mach–Zehnder
interferometry and equipped with a digital micromirror device (DMD) for
high-speed angle scanning (Figure S1 ). A blue continuous-wave
laser (λ0 = 457 nm, Cobolt Twist, Cobolt) was split into a sample and
reference beam using a beam splitter. The sample beam was modulated by
the DMD for illumination angle control by impinging on a sample slide
through a condenser objective (LUCPLFLN40X, NA = 0.6, Olympus). Then,
the scattered fields from the sample at each illumination angle were
collected by an objective lens (UPlanSAPO20X, NA = 0.75, Olympus) and
interfered with the reference beam at the camera plane, generating a
spatially modulated interferogram. Using field retrieval based on
spectral filtering, complex field information consisting of amplitudes
and phases was retrieved (Figure S1). The 2D optical field information
at various illumination angles was used to reconstruct a 3D refractive
index (RI) tomogram based on Fourier diffraction
theorem.[29,30] Our setup had a lateral resolution
of 0.17 μm and an axial resolution of 1.4 μm based on the Lauer
criteria.[31,32]
To obtain whole-slide RI tomograms, multiple tomograms were stitched
following the measurements in separate
locations.[33] A phase-correlation algorithm was
used to determine the relative position between overlapping tomogram
tiles, and stitch them into tomograms.[33,34] By
automatically stitching the scanned tomograms, the ODT setup achieved a
millimeter-scale FOV (10 mm2). The stitched tomogram
had a size of approximately 104 voxels in both lateral
directions and 102 voxels in the vertical direction.
The BF images acquired using a slide scanner were 2D images. Hence, the
3D RI tomograms were converted to 2D by identifying the optimal focal
plane. For training, RI values between 1.48 and 1.5 were normalized to a
grayscale from 0 to 255.
We registered the RI and BF images to annotate each RI patch based on
the corresponding BF patch. Subsequently, we adjusted the stained
bright-field image to match the size and FOV of the unstained QPI.
Rescale, rotation, skew, and distortions were applied to the
bright-field images using Adobe Photoshop (Version 2019, Adobe Systems,
San Jose, CA, USA).