title: Hyperspectral confocal fluorescence imaging: exploring alternative multivariate curve resolution approaches.
abstract: Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution (MCR), provides a powerful new tool for improved quantitative imaging of multi-fluorophore samples. Generally, fully non-negatively constrained models are used in the constrained alternating least squares MCR analyses of hyperspectral images since real emission components are expected to have non-negative pure emission spectra and concentrations. However, in this paper, we demonstrate four separate cases in which partially constrained models are preferred over the fully constrained MCR models. These partially constrained MCR models can sometimes be preferred when system artifacts are present in the data or where small perturbations of the major emission components are present due to environmental effects or small geometric changes in the fluorescing species. Here we demonstrate that in the cases of hyperspectral images obtained from multicomponent spherical beads, autofluorescence from fixed lung epithelial cells, fluorescence of quantum dots in aqueous solutions, and images of mercurochrome-stained endosperm portions of a wild-type corn seed, these alternative, partially constrained MCR analyses provide improved interpretability of the MCR solutions. Often the system artifacts or environmental effects are more readily described as first and/or second derivatives of the main emission components in these alternative MCR solutions since they indicate spectral shifts and/or spectral broadening or narrowing of the emission bands, respectively. Thus, this paper serves to demonstrate the need to test alternative partially constrained models when analyzing hyperspectral images with MCR methods.
authors.affiliation: Sandia National Laboratories, Albuquerque, New Mexico 87185-0895, USA. dmhaala@sandia.gov
authors.forename: David M
authors.initials: DM
authors.lastname: Haaland
authors.forename: Howland D T
authors.initials: HD
authors.lastname: Jones
authors.forename: Mark H
authors.initials: MH
authors.lastname: Van Benthem
authors.forename: Michael B
authors.initials: MB
authors.lastname: Sinclair
authors.forename: David K
authors.initials: DK
authors.lastname: Melgaard
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Stork
authors.forename: Maria C
authors.initials: MC
authors.lastname: Pedroso
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.forename: Allan R
authors.initials: AR
authors.lastname: Brasier
authors.forename: Nicholas L
authors.initials: NL
authors.lastname: Andrews
authors.forename: Diane S
authors.initials: DS
authors.lastname: Lidke
mesh_terms: D000465:Algorithms; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015999:Multivariate Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity