Hyperspectral Confocal Fluorescence Microscope
abundant
immunola-
Figure 5: Tridimensional-hyperspectral micrograph of a fresh, unstained cross section of maize leaf. The color of the fluorescence in this RGB image corre- sponds to the relative intensity of the colors in the spectral pure-component graph (Figure 4A). Field of view = 100 µm.
beled proteins, in which signal was undetectable using traditional confocal microscopes, was also possible. Very weak fluorescence signals can now be detected and measured in the presence of a very strong leaf fluorescence background. (2) Detection of
fixation artifacts and false positives. Spectral changes were detected aſter fixation with typical plant fixatives. However, for 3.7% formaldehyde,
emission spectra from membrane-bound autofluorescent components were only significantly affected aſter the first eleven days in fixative, contrary to glutaraldehyde, acetone, or ethanol, which should be avoided. Tis new instrument also enabled the detection of false positives thus significantly improving the accuracy of diagnostic tests. For example, experiments with model systems transiently expressing green fluorescent protein (GFP) revealed that not all cells showing green fluorescence were expressing GFP. A spectral component with an emission peak close to that of GFP can be present on transfected cells and can be mistakenly quantified as GFP on filter-based systems. (3) Detection, identification, and quantification of
autofluorescent cellular components (such as chlorophyll a, b, carotenoids, and cell-wall components) as described above. Te upgrade of the current instrument to include a two-photon laser with a wide tuning range will strengthen the power of this technology for the study of cellular components with optimal excitations below 488 nm. (4) Separation of overlapping fluorescence signals, such
as green autofluorescence and green fluorescent protein (GFP) or Fluorescein Diacetate (FD). Tis capability enabled us to use combinations of dyes and probes impossible to use effectively with traditional microscopes and to discriminate and quantify autofluorescent components co-localized with the molecule(s) of interest.
Conclusion Te hyperspectral confocal fluorescence microscope
and MCR soſtware package developed by Sandia National Laboratories overcomes the limitations of traditional filter- based confocal systems, providing the ability to follow many spectrally and spatially overlapping tags simultaneously and to discriminate them against autofluorescence or impurity emissions. Apart from the conventional confocal microscopy applications, this instrument allows the detection, identifi- cation, and accurate measurement of autofluorescent cellular components, weak fluorescence signals, fixation artifacts, and false positives, among others. Tis diffraction-limited imaging microscope has proven to offer a new look into the living cell,
18
Figure 6: Spectra and distribution of chlorophylls a (red component) and b (green) in a maize chloroplast. Field of view = 25 µm.
enabling the observation and quantification of pure emission fluorescent components never before achieved.
Acknowledgments Te support of Monsanto and Sandia management is
gratefully acknowledged. Portions of this work were supported by the Laboratory Directed Research and Development program at Sandia National Laboratories. Sandia National Laboratories is a multi-program laboratory operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Company, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
References [1] M B Sinclair et al., Appl Optics 45 (2006) 6283–91. [2] JAT Ohlhausen et al., Appl Surf Sci 231/232 (2004) 230–34. [3] H D T Jones et al., J Chemometr 22 (2008) 491–99. [4] JA Timlin et al., BMC Genomics 6 (2005) 72. [5] W F J Vermaas et al., PNAS 105(10) (2008) 4050–55. [6] D M Haaland et al., Next-Generation Spectroscopic Technologies 6765 (2007) 67650.
[7] D M Haaland et al., J Applied Spectroscopy 63(3) (2009) 271–79.
www.microscopy-today.com • 2010 September
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76