Hyperspectral Confocal Fluorescence Microscope
previously, MCR analysis of hyperspectral images enables us to determine the relative spatial concentration of each pure spectral component per voxel and visualize its distribution in the sample. Te relative concentration maps for maize leaf cross sections revealed differences in the amounts and distri- butions of chloroplast pigments between mesophyll and bundle sheath chloroplasts (Figure 4B). Tose differences were clearly visualized in the corresponding RGB image (Figure 5) where the color assigned to each pure component in the spectral pure-component graph (Figure 4A) corresponds to the three RGB colors (pures 1, 2, and 3) in the image generated (Figure 5). High spectral resolution and minimization of sources of noise during image processing also enabled the visualization of regions of grana in the chloroplasts (Figure 3C versus Figure
3D), and this allowed the detection of the spatial distribution of chlorophylls a and b in each chloroplast (Figure 6). Tis proved to be a powerful application of this technology allowing the quantification and comparison of chloroplast pigment differ- ences between plants with different leaf phenotypes, as well the correlation of hyperspectral imaging results with chloroplast ultrastructure and photosynthetic efficiency.
Advantages and Potential Te evaluation studies performed using our hyperspectral
confocal fluorescence microscope revealed significant advantages over traditional confocal microscopes, namely the ability to detect and measure fluorescent components in the plant cell not detected by commercial microscopes. When compared to a high-end commercial confocal microscope, the hyperspectral microscope proved superior for the following: (1) Experiments using dyes and/or fluorophores, thus
allowing the use of more labels per cell, lower concentrations of dyes, and detection of weak signals. Multiple labeling experi- ments can be carried out in a cell with Alexa probes and/or quantum dots, and/or coupled with a fluorescent staining. Te upper limit of the number of fluorescent species that can be detected per sample has not been established yet. As fluorescent molecules have different photobleaching rates, photobleach- ing can be used to separate covarying spectral components. Double staining of organs, tissues, or cells can now be achieved for dyes with overlapping spectra (that is, staining of protein and lipid bodies using dyes that emit in the red). Also due to the sensitivity of the instrument, common cell viability or nuclear dyes can be diluted 10- to 100-fold. Detection of low
Figure 4: MCR analysis of hyperspectral images of maize leaf revealed the presence of five pure spectral components in those samples (A), and provided relative concentrations and the 3D spatial location of those pure components (B). Pure 2 is an autofluorescent component of cell wall; and pures 1, 3, 4, and 5 originate from chloroplast pigments.
2010 September •
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