Hyperspectral confocal fluorescence imaging of cells

David M. Haaland, Howland D T Jones, Michael B. Sinclair, Bryan Carson, Catherine Branda, Jens F. Poschet, Roberto Rebeil, Bing Tian, Ping Liu, Allan R. Brasier

Research output: Chapter in Book/Report/Conference proceedingConference contribution

6 Citations (Scopus)

Abstract

Confocal fluorescence imaging of biological systems is an important method by which researchers can investigate molecular processes occurring in live cells. We have developed a new 3D hyperspectral confocal fluorescence microscope that can further enhance the usefulness of fluorescence microscopy in studying biological systems. The new microscope can increase the information content obtained from the image since, at each voxel, the microscope records 512 wavelengths from the emission spectrum (490 to 800 nm) while providing optical sectioning of samples with diffraction-limited spatial resolution. When coupled with multivariate curve resolution (MCR) analyses, the microscope can resolve multiple spatially and spectrally overlapped emission components, thereby greatly increasing the number of fluorescent labels, relative to most commercial microscopes, that can be monitored simultaneously. The MCR algorithm allows the "discovery" of all emitting sources and estimation of their relative concentrations without cross talk, including those emission sources that might not have been expected in the imaged cells. In this work, we have used the new microscope to obtain time-resolved hyperspectral images of cellular processes. We have quantitatively monitored the translocation of the GFP-labeled RelA protein (without interference from autofluorescence) into and out of the nucleus of live HeLa cells in response to continuous stimulation by the cytokine, TNFα. These studies have been extended to imaging live mouse macrophage cells with YFP-labeled RelA and GFP-labeled IRF3 protein. Hyperspectral imaging coupled with MCR analysis makes possible, for the first time, quantitative analysis of GFP, YFP, and autofluorescence without concern for cross-talk between emission sources. The significant power and quantitative capabilities of the new hyperspectral imaging system are further demonstrated with the imaging of a simple fluorescence dye (SYTO 13) traditionally used to stain the nucleus of live cells. We will demonstrate the microscope system's ability to actually discover and quantify the presence of two separate SYTO 13 fluorescent species shifted in wavelength by only a few nm. These two emission components exhibit very different spatial distributions in macrophage cells (i.e., nucleus vs. cytoplasm + nucleus). Two highly overlapped autofluorescence components in addition to the two SYTO 13 components were also observed, and the spatial distributions of the two autofluorescence components were quantitatively mapped throughout the cells in three dimensions.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
Volume6765
DOIs
StatePublished - 2007
EventNext-Generation Spectroscopic Technologies - Boston, MA, United States
Duration: Sep 10 2007Sep 11 2007

Other

OtherNext-Generation Spectroscopic Technologies
CountryUnited States
CityBoston, MA
Period9/10/079/11/07

Fingerprint

Microscopes
Fluorescence
microscopes
Imaging techniques
fluorescence
cells
nuclei
macrophages
Macrophages
Biological systems
Spatial distribution
spatial distribution
curves
proteins
Proteins
Wavelength
cytoplasm
Fluorescence microscopy
stimulation
wavelengths

Keywords

  • Confocal microscopy
  • Fluorescence imaging
  • HeLa cells
  • Hyperspectral
  • Macrophage cells
  • Multivariate curve resolution

ASJC Scopus subject areas

  • Electrical and Electronic Engineering
  • Condensed Matter Physics

Cite this

Haaland, D. M., Jones, H. D. T., Sinclair, M. B., Carson, B., Branda, C., Poschet, J. F., ... Brasier, A. R. (2007). Hyperspectral confocal fluorescence imaging of cells. In Proceedings of SPIE - The International Society for Optical Engineering (Vol. 6765). [676509] https://doi.org/10.1117/12.738152

Hyperspectral confocal fluorescence imaging of cells. / Haaland, David M.; Jones, Howland D T; Sinclair, Michael B.; Carson, Bryan; Branda, Catherine; Poschet, Jens F.; Rebeil, Roberto; Tian, Bing; Liu, Ping; Brasier, Allan R.

Proceedings of SPIE - The International Society for Optical Engineering. Vol. 6765 2007. 676509.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Haaland, DM, Jones, HDT, Sinclair, MB, Carson, B, Branda, C, Poschet, JF, Rebeil, R, Tian, B, Liu, P & Brasier, AR 2007, Hyperspectral confocal fluorescence imaging of cells. in Proceedings of SPIE - The International Society for Optical Engineering. vol. 6765, 676509, Next-Generation Spectroscopic Technologies, Boston, MA, United States, 9/10/07. https://doi.org/10.1117/12.738152
Haaland DM, Jones HDT, Sinclair MB, Carson B, Branda C, Poschet JF et al. Hyperspectral confocal fluorescence imaging of cells. In Proceedings of SPIE - The International Society for Optical Engineering. Vol. 6765. 2007. 676509 https://doi.org/10.1117/12.738152
Haaland, David M. ; Jones, Howland D T ; Sinclair, Michael B. ; Carson, Bryan ; Branda, Catherine ; Poschet, Jens F. ; Rebeil, Roberto ; Tian, Bing ; Liu, Ping ; Brasier, Allan R. / Hyperspectral confocal fluorescence imaging of cells. Proceedings of SPIE - The International Society for Optical Engineering. Vol. 6765 2007.
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