FACED imaging flow cytometer setup

A multimodal FACED IFC platform was used to obtain the quantitative phase and FL images of single cells in microfluidic flow at an imaging throughput of ~70,000 cells/s. The light source consisted of an Nd:YVO picosecond laser (center wavelength = 1064 nm, Time-Bandwidth) and a periodically-poled lithium niobate (PPLN) crystal (Covesion) for second harmonic generation of a green pulsed beam (center wavelength = 532 nm) with a repetition rate of 20 MHz. The beam was then directed to the FACED module, which mainly consists of a pair of almost-parallel plane mirrors. This module generated a linear array of 50 beamlets (foci) which were projected by an objective lens (40X, 0.6NA, MRH08430, Nikon) on the flowing cells in the microfluidic channel for imaging. Each beamlet was designed to have a time delay of 1 ns with the neighboring beamlet in order to minimize the FL crosstalk due to the FL decay. Detailed configuration of the FACED module can be referred to Wu et al.¹³. The epi-fluorescence image signal was collected by the same objective lens and directed through a band-pass dichroic beamsplitter (center: 575 nm, bandwidth: 15 nm). The filtered orange FL signal was collected by the photomultiplier tube (PMT) (rise time: 0.57 ns, Hamamatsu). On the other hand, the transmitted light through the cell was collected by another objective lens (40X, 0.8NA, MRD07420, Nikon). The light was then split equally by the 50:50 beamsplitter into two paths, each of which encodes different phase-gradient image contrasts of the same cell (a concept similar to Scherlien photography⁶⁶). The two beams are combined, time-interleaved, and directed to the photodetector (PD) (bandwidth: >10 GHz, Alphalas) for detection. The signals obtained from both PMT and PD were then passed to a real-time high-bandwidth digitizer (20 GHz, 80 GS/s, Lecroy) for data recording.