Nanoscale 3D co-Localization in Imaging Flow Cytometry via Point-Spread-Function Engineering
- Abstract number
- 29
- Corresponding Email
- [email protected]
- Session
- Stream 5: Seeing is Believing – Multiplexed Imaging Flow Cytometry
- Authors
- Dr. Lucien Weiss (1), Yael Shalev-Ezra (1), Dr. Sarah Goldberg (1), Boris Ferdman (1), Omer Adir (1), Dr. Avi Schroeder (1), Dr. Onit Alalouf (1), Dr. Yoav Shechtman (1)
- Affiliations
-
1. Technion - Israel Institute of Technology
- Keywords
Imaging flow cytometry, point-spread function engineering, 3D microscopy
- Abstract text
Attaining three-dimensional data at high throughput is a grand challenge in microscopy. Here we propose and demonstrate a novel solution to enable the collection of three-dimensional positions in thousands of cells each minute by merging two technologies: point-spread-function (PSF) engineering and imaging flow cytometry (IFC). We have applied our approach to monitor changes in DNA compaction-state in cells responding to stimuli and to characterize the uptake of synthesized nanoparticles in live cancer cells.
In PSF-engineering microscopy, the pattern of light emanating from a point source in a sample is modified to encode additional information such as the depth. This is achieved by adding an optical element into the imaging path. A theoretical model or experimental calibration is then used to decode the resulting image to attain the desired information, e.g. xyz positions. IFC operates by the same principles as traditional microscopy; however, the normally stationary sample is replaced with a flow cell. This design prohibits scanning the same object in z, thereby barring the canonical calibration method for 3D localization microscopy by PSF engineering, namely, translating the objective lens while imaging a static object to derive a calibration curve.
To overcome this technical limitation, we perform a “pseudo-scan,” by recording many images of different objects across a range of depths, then comparing that image library to the microfluidics-determined, depth-distribution probability. The result is a 3D calibration that can be applied to any images of localized fluorescent emitters, e.g. those within cells.
Using this approach, we have demonstrated (1) the co-localization of multicolor fluorescent beads in six spectral channels, (2) the measurement of length distributions of fluorescent DNA-origami nanorulers imaged in flow, (3) the 3D-distance measurements of DNA compaction in live yeast cells at orders of magnitude faster rates than previously realized methods, and (4) the application of our method to nanomedicine, namely, quantification of nanoparticle uptake in lymphocyte cancer cells.
In the context of nanomedicine, our technology enables an unprecedented combination of throughput and precision for intracellular localization of nanoparticles. A high-throughput system that measures the exact position of therapeutic nanoparticles upon cellular uptake would be critical for realizing the promise of directing nanomedicines to specific intracellular targets.
- References
L.E. Weiss, Y. S. Ezra, S. E. Goldberg, B. Ferdman, O. Adir, A. Schroeder, O. Alalouf, Y. Shechtman, “Three-dimensional localization microscopy in live flowing cells” Nature Nanotechnology 15, 500–506 (2020).