Visualization of pulmonary clearance mechanisms via noninvasive optical imaging validated by near-infrared flow cytometry

Abstract

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as 4-h post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies.

Keywords: alveolar macrophage; flow cytometry; in vivo imaging; lung imaging; near-infrared.

Figure 1

Research plan to investigate the uptake of the NIR contrast agents by cells (i.e. macrophages) using NIR flow cytometry

Figure 2

Optical path diagram of the modified NIR flow cytometer that allowed observation of two emissions above 820 nm from the sample and a potential secondary emissions below 770 nm. H957-15 – a detector module with an R928 PMT, HQ820/60 - a NIR band filter. HQ740SP - a short pass filter, T770lpxr -a beam-splitter

Figure 3

NIR flow cytometry of unstained and NIR stained beads. The histograms show the number of cells vs. fluorescence signal in the NIR channel.

Figure 4

Flow cytometry of fluorecently labeled cell lines. A: Structure of a NIR dye cypate, B: eGFP expressing U87 cells (green) treated with Lysotracker Red (blue); no fluorescence in NIR channel, C: U87 eGFP cells (green) + Lysotracker Red (blue) + cypate (red); D: No eGFP expressing cells A427-7 cells + Lysotracker Red (blue) + cypate (red); no fluorescence in eGFP channel.

Figure 5

A: Fluorescent dye cypate delivered IT shows retention in the lung area within 24 hours. Red - NIR fluorescence channel (785/810 nm), green –fluorescence channel (685/710 nm). B: Region of interest (ROI) C: change of total fluorescent signal with time (ROI, dorsal position). Fluorescence in the 700 nm region (green) originates from digestion of chlorophyll-containing food in the stomach and intestines. Fluorescence from chlorophyll-containing chow is much more intense at about 700 nm than 800 nm and identified the location of the stomach and intestine relative to the expected signal from the nanoparticles in the lungs.

Figure 6

Flow cytometry of bronchoalveolar lavage cells: not treated with cypate (A-B) and 4 hours after cypate treatment (C-D). Y-axis – NIR channel, X-axis – APC channel