HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence

Abstract

The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.

Assessment of unmixing approaches for the quantitation of SERS nanoparticles in highly multiplexed spectral images

“…Our results with the HLP algorithm show much promise thanks to its augmentation with characteristic variance information and use of regularization which helps regression with collinearities 19 . Sophisticated regression techniques designed to combat crosstalk in fluorescence applications could also be applied to Raman imaging 44–46 . Alternative variance analyses, normalization, preconditioning, or optical models may be useful in some cases 16,35,47–52 …”

“…19 Sophisticated regression techniques designed to combat crosstalk in fluorescence applications could also be applied to Raman imaging. [44][45][46] Alternative variance analyses, normalization, preconditioning, or optical models may be useful in some cases. 16,35,[47][48][49][50][51][52] Spatial Raman spectroscopy with nanoparticle contrast agents is a high-resolution, sensitive, and extensively multiplexable technique that can be leveraged for proteomic imaging.…”

New approaches for low phototoxicity imaging of living cells and tissues

“…[68] A combination of MFM with hyperspectral phasors and linear unmixing has recently been presented. [69] Data were collected from hyperspectral and two-dimensional histograms based on natural and imaginary Fourier components. Spectral denoising was also performed.…”

Achieving two-photon fluorescence bioimaging and photodynamic therapy for D–A conjugated polymers through manipulating twisted intramolecular charge transfer

“…Fluorescence imaging is an indispensable tool for augmenting our comprehension of biological systems because of its noninvasiveness, sensitivity, and real-time spatial imaging. 1–5 Specifically, two-photon (TP) fluorescence microscopy enables noninvasive and real-time imaging with intrinsic 3D optical sections of living organisms. 6,7 In addition, the used excitation light is in the NIR region (700–1700 nm), which allows for better light penetration and deeper imaging on account of diminished light absorption and scattering in biological tissues.…”