Three-Dimensional Quantitative Phase Imaging for Characterizing Lymphocyte Subtypes

1. Flow Cytometry and Sorting of Lymphocyte Subtypes

NOTE: Sorting lymphocytes depending on cell type is essential for establishing the ground-truth (i.e., correct) cell type labels to train and test a cell type classifier in supervised learning. Flow cytometry, a gold standard method, is used to identify and separate lymphocytes.

1. Make a mixture of surface marker staining antibodies in 100 µL of fresh RPMI-1640 medium [with 10% FBS, 0.1 µg of CD3e (17A2), CD8a (53-6.7), CD19 (1D3), CD45R (B220, RA3-6B2), and NK1.1 (PK136)] and 0.25 µg of CD4 (GK1.5) antibodies to target B, CD4⁺ T, and CD8⁺ T lymphocytes.
2. Add 100 µL of the antibody mixture to the cell suspension.
3. Incubate for 25 min on ice.
4. Wash the cells by adding 5 mL of PBS and centrifuging at 400 x g for 5 min at 4 °C, twice.
5. Resuspend the cell pellet in 5 mL of fresh RPMI-1640 medium with 10% FBS and 2.5 µg of DAPI (4',6-diamidino-2-phenylindole).
6. Collect each lymphocyte type separately with flow cytometry using the fluorescence levels of the markers described above. Simultaneously exclude dead cells using the DAPI signals.

2. 3D Quantitative Phase Imaging

1. Keep the sorted lymphocytes on ice throughout the imaging procedures, which should be completed within 5 h (since lymphocyte isolation from the mouse) to avoid cell damage and biochemical alterations.
2. Select a sorted cell type (among B, CD4⁺ T, and CD8⁺ T lymphocytes) and dilute the sample to 180 cells/µL with RPMI medium for optimal imaging conditions (i.e., one cell per single field-of-view).
3. Load 120 μL of the diluted sample into an imaging chamber by slow injection. Thoroughly check the presence of bubbles in the imaging chamber with the sample. If there are bubbles, carefully remove them, as they will compromise the quality of the measurements.
4. Acquire 3D RI tomograms using a commercial 3D quantitative phase microscope, or holotomography, and its imaging software.
   NOTE: Detailed information about the experimental setup can be found in the original manuscript.
   1. Place a drop of distilled water on top of the objective lens of the microscope.
   2. Place the imaging chamber with the sample on the translation stage of the microscope and adjust its location so that the sample aligns with the objective lens.
   3. Adjust the axial positions of the objective and condenser lenses by clicking Focus and Surface, respectively, on the "Calibration" tab of the "Microscope" perspective of the imaging software.
   4. Align the objective and condenser lenses by clicking Auto Mode. Alternatively, use Scanning Mode and manually adjust the lenses so that the illumination patterns are localized at the central region of the field of view.
   5. Return to Normal Mode and adjust the translation stage to locate a cell in the field of view.
   6. Find the focal plane by adjusting the axial position of the objective lens. Perfect focusing makes the sample boundary visualized on the screen almost invisible.
   7. Adjust the translation stage to find a location without a cell.
   8. Click Calibrate to measure multiple 2D holograms with varying illumination angles.
   9. Adjust the translation stage to locate a cell at the center of the field of view.
   10. Move to the "Acquisition" tab and click 3D Snapshot to measure the holograms of the cell with identical illumination angles as done in step 2.4.8.
   11. When the acquired data is presented on the "Data Management" panel, right-click on the acquired data and click Process to reconstruct a 3D RI tomogram from the holograms measured in steps 2.4.8 and 2.4.10, using the diffraction tomography algorithm implemented in the imaging software.
   12. Repeat steps 2.4.5-2.4.11 to measure more than 100 cells to ensure statistical power for machine learning.
   13. All images that are processed through step 2.4.11. can be visualized. On the "Data Management" panel, right-click the data and click Open to visualize the data. Click RI Tomogram on the "Data Manager" panel. On the "Preset" tab, click Load and double click "lymphocyte.xml", which is a predefined transfer function provided by the imaging software to visualize the tomogram according to the 3D RI distributions.
5. Repeat steps 2.2-2.4 to measure 3D RI tomograms of all lymphocyte subtypes.