Fluorescence line-scanning

Figure 4. X-Ray fluorescence line scan mapping performed on Au/Ag nanoparticles using the Ag-L and Au-M atomic emission lines.

Another variation of the FRAP technique scans the laser in one dimension across the cell producing a line-scan profile of fluorescence intensity (14). For a round cell whose surface is evenly labeled with a fluorescently conjugated antibody, a line scan typically produces a profile with two peaks of fluorescence, since the laser beam is illuminating more fluorophores at the edges... 

Guild, J. B., and Webb, W. W. 1995. Line scanning microscopy with two-photon fluorescence excitation. Biophys. J. 68 290a. 

FIGURE 6.11. (a) A total internal reflection fluorescence image of the same conditions as shown in Figure 6.10a. (b) Line scan of fluorescence intensity (dotted line) across the microchannels. 

FIGURE 10.9 (a) Bulk-phase epifluorescence image of Alexa 594 dye-labeled anti-DNP inside 12 bilayer-coated microchannels. Starting from the left-hand side, the antibody concentrations in these channels are 13.2, 8.80, 5.87, 3.91, 2.61, 1.74, 1.16, 0.77, 0.52, 0.34, 0.23, and 0.15 mM. A line scan of fluorescence intensity across the 12 microchannels is plotted in (b) [1029]. Reprinted with permission from the American Chemical Society. 

Figure 3 shows the excitation spectrum of CaO doped with Eu2+ when fluorescence from all sites in the sample was monitored. The dye laser was scanned over the possible absorption lines and each time the wavelength matched a transition on any site, the fluorescence intensity increased and gave a line. Figure 4 shows the same procedure on the same crystal except now a high resolution monochromator was used to monitor the fluorescence that occurred at a wavelength characteristic of a specific site. Now, one sees increases in the fluorescence only when the dye laser matches an absorption line of the same site that has the fluorescence line being monitored. 

Fig. 3.5. Scanning confocal image of a glycerol thin film with a low concentration of fluorescent dye molecules at 204 K. The polarization of the fluorescence, recorded by two independent detectors, is rendered as the color of the pixels. The three circled single molecules demonstrate visible differences in their tumbhng patterns. The upper one tumbles at the scanning rate (about f s per line scan), while the lower molecule keeps its orientation during a few successive scans. The middle molecule reorients faster...

A line-scan of the illumination laser light has been used to enhance the scan rate, at least compared to the point-by-point scan [18, 21-25]. It remains compatible with a spectral acquisition of high resolution, since total fluorescence from the linear region can be projected onto the slit of an imaging polychromator. A CCD camera at the exit port records the fluorescence intensity as a function of the spatial coordinate and the wavelength in the two-dimensional sensitive area of the camera. This method... 

Figure 30.5 Fluorescence images of two neighboring chloroplasts in a mesophyll cell of Zea mays at different Z positions, (a) Integrated intensities between 665 and 681 nm (red image). The scale bar is 5.0 Xm. The direction of the line scan is alongtheXaxis. (b) Integrated intensities between 715 and 740nm (far-red image). In (a) and (b), the maximum intensity in each image is indicated in white and a threshold intensity...

Kumazaki, S., Hasegawa, M., Ghoneim, M., Shimizu, Y., Okamoto, K., Nishiyama, M., Oh-oka, H. and Terazima, M. (2007) A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120. J. Microsc., 228, 240-254. 

Fig. 7, Line scan image from a vascular smooth muscle cell. Image shows a spark originating at a site towards the edge of the cell. The line scan has had the background subtracted and it has been colorized in order to clearly visualise the spark. In this image time is in the horizontal direction and distance across the cell is in the vertical direction. Beneath the line scan is the averaged fluorescence signal, measured from the section of the line scan indicated by the bar. The figure also shows an example of a STOC to demonstrate the similarity in the time course of these events, (taken from figure 1, Nelson et 0.11995).

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