Hyperspectral confocal fluorescence imaging

Exploring alternative multivariate curve resolution approaches

David M. Haaland, Howland D T Jones, Mark H. Van Benthem, Michael B. Sinclair, David K. Melgaard, Christopher L. Stork, Maria C. Pedroso, Ping Liu, Allan R. Brasier, Nicholas L. Andrews, Diane S. Lidke

Research output: Contribution to journalArticle

42 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)271-279
Number of pages9
JournalApplied Spectroscopy
Volume63
Issue number3
DOIs
StatePublished - Mar 2009

Fingerprint

Fluorescence
Imaging techniques
fluorescence
curves
Environmental impact
Merbromin
Fluorophores
Confocal microscopy
Fluorescence microscopy
artifacts
Semiconductor quantum dots
Seed
corn
Derivatives
beads
lungs
seeds
emission spectra
quantum dots
aqueous solutions

Keywords

  • ALS
  • Autofluorescence
  • Corn endosperm
  • Fluorescence imaging
  • Hyperspectral confocal microscope
  • MCR
  • Multivariate curve resolution
  • Partially constrained alternating least squares
  • Quantum dots

ASJC Scopus subject areas

  • Spectroscopy
  • Instrumentation

Cite this

Haaland, D. M., Jones, H. D. T., Van Benthem, M. H., Sinclair, M. B., Melgaard, D. K., Stork, C. L., ... Lidke, D. S. (2009). Hyperspectral confocal fluorescence imaging: Exploring alternative multivariate curve resolution approaches. Applied Spectroscopy, 63(3), 271-279. https://doi.org/10.1366/000370209787598843

Hyperspectral confocal fluorescence imaging : Exploring alternative multivariate curve resolution approaches. / Haaland, David M.; Jones, Howland D T; Van Benthem, Mark H.; Sinclair, Michael B.; Melgaard, David K.; Stork, Christopher L.; Pedroso, Maria C.; Liu, Ping; Brasier, Allan R.; Andrews, Nicholas L.; Lidke, Diane S.

In: Applied Spectroscopy, Vol. 63, No. 3, 03.2009, p. 271-279.

Research output: Contribution to journalArticle

Haaland, DM, Jones, HDT, Van Benthem, MH, Sinclair, MB, Melgaard, DK, Stork, CL, Pedroso, MC, Liu, P, Brasier, AR, Andrews, NL & Lidke, DS 2009, 'Hyperspectral confocal fluorescence imaging: Exploring alternative multivariate curve resolution approaches', Applied Spectroscopy, vol. 63, no. 3, pp. 271-279. https://doi.org/10.1366/000370209787598843
Haaland, David M. ; Jones, Howland D T ; Van Benthem, Mark H. ; Sinclair, Michael B. ; Melgaard, David K. ; Stork, Christopher L. ; Pedroso, Maria C. ; Liu, Ping ; Brasier, Allan R. ; Andrews, Nicholas L. ; Lidke, Diane S. / Hyperspectral confocal fluorescence imaging : Exploring alternative multivariate curve resolution approaches. In: Applied Spectroscopy. 2009 ; Vol. 63, No. 3. pp. 271-279.
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