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 four-hours 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.

Antibody Engineering for Pursuing a Healthier Future

“…Labeled antibodies are used for the detection of target protein molecules or antigens on the surface of cells (Álvarez-Barrientos et al, 2000). FC has been used for the visualization of pulmonary clearance (Zhou et al, 2015), fluorescence decay lifetimes, control sorting (Houston et al, 2010), quantification of DNA-end resection in mammalian cells (Forment et al, 2012), microsphere-based protease assays, highthroughput screening (Saunders et al, 2010a), and botulinum neurotoxin type A light chain protease inhibitors (Saunders et al, 2010b).…”

Fluorescence calibration standards made from broadband emitters encapsulated in polymer beads for fluorescence microscopy and flow cytometry

“…Our results provide an important step towards an improved comparability of fluorescence data, across different instruments, laboratories, and even methods with special emphasis on spectroscopic methods studying micrometer-sized fluorescent beads or objects or relying on their use such as bead-based assays. Moreover, as shown for example by H. Zhou et al [42], NIR-bead standards are relevant not only for fluorescence microscopy but also for flow cytometry where applications of NIR flow cytometry have been emerging in the last years. Further experiments are currently focused on the screening of other dyes to identify fluorophores that even better meet the stringent requirements imposed by us on sets of spectral standards with overlapping emission spectra.…”

Imaging drug delivery to the lungs: Methods and applications in oncology

“…This can be problematic for lung imaging because of the physical proximity of the lungs and the GI tract, as shown by Zhou et al . [234] To avoid this issue, animals can be fed a chlorophyll-free diet for a week prior to imaging, but surprisingly this is not frequently reported in studies. Whilst ex vivo fluorescence imaging is useful to confirm drug delivery a posteriori , a different selection of fluorescent dyes more amenable to in vivo imaging might have allowed to visualise drug distribution longitudinally after lung delivery, potentially demonstrating the different fates of drugs and carriers over time.…”