Confocal single-photon

Protein motions in single FlAsH-labeled CaM molecules tethered to glass slides have been measured by anisotropy using time-correlated single-photon counting in a confocal microscope [46]. Average anisotropy values were similar to bulk measurements but showed wide variability from molecule to molecule. Decay rates indicated that rapid-scale protein motions occur in the N-terminal domain on a nanosecond timescale but limited signal-to-noise levels precluded detailed analysis. Comparable experiments with CaM labeled with Texas Red failed to detect such motions because of faster dye rotation, independent of the protein motions. 

Perhaps the most significant contrihution that two-photon excited fluorescence has made in biological research to date is in the area of fluorescent imaging. Prior to the invention of two-photon microscopy in 1990, depth resolution in fluorescence microscopy was achieved using the confocal microscope, the principles of which are illustrated in Figure 11.9. Fluorescence is excited via single-photon excitation... 

Figure 11.21 StimuLated emission depletion (STED) microscopy. The sample is excited using single-photon excitation (PUMP pulse) in a confocal microscope arrangement. A time-delayed DUMP pulse selectively depletes close to 100% of the exdted state population in a region around the focus of the PUMP pulse. Using this approach. Hell and co-workers were able to obtain a 5-fold reduction in the fluorescent spot size in the vertical (Z-direction) and a greater than a 2-fold reduction in the horizontal Y/X) direction, leading to a final image size of 97 by 104 nm...

Two-Photon Confocal Microscopy. Confocal microscopy provides the possibilities of optical sectioning of a sample by the use of a spatial filter to improve axial resolution of the optical microscope (20). These optical sections can be used to reconstruct the 3D structure of a polymeric specimen. Confocal microscopy using fluorescence is usually performed by a single-photon excitation (linear absorption) of a fluorophore which may be chemically an integral part of the system (polymer) being investigated or just... 

Confocal/multi-photon decay necessary Single photon sensitivity Slow each photrai has to be... 

The PSF in single-photon confocal and two-photon epi-fluorescence illumination systems... 

Fig. 15 Zinc salen complexes and colocalisation in endoplasmic reticulum via single-photon confocal microscopy before and after irradiation for 315 s. ...

In fact, confocal single element mapping aehieves higher spatial resolution than apertureless imaging systems sueh as FPA, in particular when dealing with extremely brilliant SR sources. However, not all experiments need the ultimate resolution and, as addressed by reeent experimental data, SR emission is characterized by a sufficient photon flux density to illuminate the array detector in order to operate with a high S/N. ... 

For example, the Rayleigh scattering of UV wavelength photons is a particular problem in single-photon confocal microscopy [17]. Furthermore, another point to note is that the emission wavelength for MPM is substantially shorter than the excitation wavelength, which makes for easier differentiation and hence detection. 

---