Modulation image intensity

Bentzen SM (2005) Radiation therapy intensity modulated, image guided, biologically optimized and evidence based. Radiother Oncol 77 227-230... 

The image is formed by the signal of emitted secondary electrons, backscattered electrons. Auger electrons, absorbed specimen current (SC), or X-ray quanta, which modulate the intensity of a cathode-ray tube rastered in synchronism (Fig. 75). Conventional SEMs work with electron acceleration voltages of 5 - 30 kV, whereas a low-voltage scanning electron microscope (LVSEM) [222], [223] uses 0.5-5 kV. 

To use this type of microscope for FLIM, the camera has to be able to measure time resolved. Typically, a gain-modulated image intensifier is used for this, although recently the use of a modulated CCD camera for this has been reported [22,23]. The light source has to be intensity-modulated. This can be pulsed, for time-domain FLIM, or continuously, for frequency-domain FLIM. If a laser is used for excitation, the beam will have to be expanded to illuminate not a single point in the specimen but the entire field of view in order to obtain wide-field images. For references to wide-field FLIM instrumentation see Table 1, A1 and Bl. 

If the analyzer is set to accept electrons of an energy characteristic of a particular element, and if the incident X-ray beam is rastered over the surface to be analyzed, a visual display the intensity of which is modulated by the peak intensity will correspond to the distribution of that element over the surface. The result is also an image and this technique is realized with the Quantum 2000. 

Carlsson, K. and Liljeborg, A. (1998). Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique. J. Microsc. 191, 119-27. 

Fig. 7.9. Further FRET efficiency analysis. (A) Unbiased display of FRET efficiency. The ED image (upper left panel) is modulated with an intensity picture (in this case, s.e., upper right panel) to yield the lower left image. See text for further details. Lower right panel, example with several cells... 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

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