Three-Dimensional Quantitative Phase Imaging for Characterizing Lymphocyte Subtypes

Load a lymphocyte subtype suspension into an imaging chamber.

Position the chamber on the 3D  quantitative phase imaging microscope stage.

Adjust microscope parameters and the focal plane for optimum imaging.

During lymphocyte imaging, the laser beam splits into reference and sample beams.

The Digital Micromirror Device, featuring a micromirror array in the sample beam's path, allows the beam to rotate 360 degrees around the optical axis.

Each beam passing through the lymphocyte undergoes a phase shift based on refractive index variations within the cell.

The resulting sample and reference beams recombine, forming a 2D hologram.

A sequence of holograms containing phase and amplitude information from different angles is captured through the beam rotation around the lymphocyte.

Acquire multiple background holograms with varying illumination angles, excluding lymphocytes.

Using the diffraction tomography algorithm, reconstruct the holograms into a 3D refractive index tomogram, providing morphological and biochemical information of lymphocytes without the need for labeling.

For optimal imaging, dilute each cell sample to a concentration of 180 cells per microliter of RPMI medium, and slowly inject 120 microliters of the first diluted sample into an imaging chamber. After confirming a lack of bubbles within the chamber, place a drop of distilled water onto the objective lens of a 3D quantitative phase microscope, and place the imaging chamber onto the translation stage of the microscope. Adjust the stage so that the sample aligns with the objective lens, and click Focus and Surface in the Calibration tab of the microscope perspective of the imaging software, to adjust the axial positions of the objective, and condenser lenses respectively.

Click Auto Mode to align the objective, and condenser lenses. To optimize the alignment, open Scanning Mode and manually adjust the lenses to align the digital micromirror device pattern to the center. Then, return to Normal Mode, and adjust the translation stage to locate a cell in the field of view. Adjust the axial position of the objective lens to find the focal plane until the sample boundary visualized on the screen is almost invisible.

It is important to perfectly adjust the focus of the cell to generate an optimal 3D RI tomogram. If the image is not taken properly, 3D reconstruction will be impaired, resulting in a noisy tomogram.

Adjust the translation stage to find a location without a cell, and click Calibrate to measure multiple 2D holograms with varying illumination angles. Adjust the translation stage to locate a cell at the center of the field of view, and under the Acquisition tab, name the sample being imaged.

Click 3D Snapshot to measure the holograms of the cell using the same illumination angles as for the 2D holograms just measured. When the acquired data appears in the data management panel, right-click the Data, and click Process to reconstruct a 3D refractive index tomogram from the 2D holograms using the diffraction tomography algorithm implemented in the imaging software. After imaging, in the Data Management panel, right-click on the Data and click Open to visualize the data.

Click the center of the cell to reposition it, and click RI Tomogram on the Data Manager panel. On the Preset tab, click Load and double-click lymphocytes.xml, which is a predefined transfer function provided by the imaging software to visualize the tomogram according to the 3D RI distributions. Scroll the mouse to zoom in, and drag the cell to rotate it in any direction.