Development of a high-speed imaging MP imaging system. Several research projects currently being pursued at LOCI require fast, multiphoton imaging. On the basis of the rationale outlined below we are currently developing a high-speed, single beam laser-scanning MP system. The scanning system will be used in conjunction with the spectral/lifetime detector and will be tightly integrated with this device  

Considerations for high speed multiphoton imaging.

With the increasing use of Confocal and MP systems for dynamic imaging of living specimens, there is often a need for faster imaging speeds than these systems can provide. For example, imaging of calcium sparks usually has to be done in one-dimension in order to attain sufficient imaging speeds (Tanaka et al., 1998 J. Physiol.) 508: 145). In single-beam confocal imaging systems, fluorophore saturation imposes practical limits on the amount of fluorescent emission that can be obtained from the focal volume and effectively limits the scanning speed (higher scanning speeds just produce noisier images). We and others have developed fast confocal systems that circumvent the limits imposed by fluorophore saturation by using parallel beams either in the form of a slit (White & Amos, 1996 . In "Handbook of Biological Confocal Microscopy" (J. Pawley, Ed.Plenum Press p. 403) or an array of individual beams (Egger & Petran, 1967 . Science 157: 305).

Multiphoton parallel-beam scanning systems have been demonstrated (Brakenhof et al., 1996 J. Microsc. 181: 253; Guild & Webb, personal communication). However, they suffer from the systems used being too parallel. For example, for a slit equivalent to about 300 individual beams, this means that the signal from each of these equivalent beams is down by a factor of (300)2 for two-photon imaging compared to a single beam. This is because of the quadratic dependence of emission on excitation. This in turn means that the total image emission is reduced by a factor of three hundred compared to a single beam system run at levels below fluorophore saturation. Some of this loss can be retrieved by using more intense but less frequent pulses (Brakenhof et al., 1996 J. Microsc. 181: 253), thereby increasing the peak power in individual pulses. In the limit, with the pulse rates set to give the same signal from multiple beams as a single beam, fluorophore saturation sets in at the same mean power as a single beam system and there is no advantage. However a more limited degree of parallelism may be effective. Ideally, for an effective speed increase, there should be just enough parallelism so that all the beams are close to saturation at the highest powers that are available. For fluorophores excited at their optimum wavelength, saturation within the focal volume may occur at around 25mw mean power. This would suggest that ten beams may be optimal given a typical laser power of 500mw for 50% efficient optics. The scanning microlens array system recently described (Bewersdorf et al., 1998 Optics Letters 23(9), 655) comes closest to this although it is still rather high at 25 beams. They chose a lower mean power at the sample since their system utilized lower wavelength excitation where toxicity thresholds are a concern before typical exogenous fluorophores saturate.

Single beam MP systems can have very light efficient detection systems as it is possible to utilize light emitted from both sides of the sample (Wokosin et al., 1998. IEEE EMBS, 20:1707). Also, emitted light that is scattered within a specimen can contribute to an image if it is detected. This property is partly responsible for the considerably improved deep sectioning performance of MP compared to confocal systems (Centonze & White, 1998 J. Biophysics 75: 2015). Unfortunately, all these advantages are lost with multiple beam MP systems. When more than one beam is used there is no way of determining from which beam an emission signal originated except by some spatially-resolved imaging method. The detector that is generally used is a CCD camera. These devices are favored for low-light level microscopy because of their high quantum efficiency. However, read-out noise is a problem for high-speed imaging requiring the use of an additional image intensifier. These combined devices have similar quantum efficiencies to photomultipliers and are rather expensive.

Taken overall the prospects for high speed MP systems do not seem too promising. An optimally configured device could give 10 times the signal of a single beam device. However, scattered light and light emitted from the back of the specimen could not be used for imaging. This would reduce the speed advantage considerably. Also, images within scattering tissues would have considerably poorer signal-to-background than could be obtained with a single beam system.

With the considerations outlined above in mind, we have re-visited the possibilities offered by high-speed, single beam scanning MP imaging systems. In a confocal microscope, working at excitation levels at or beyond fluorophore saturation drastically reduces the signal-to-background ratio of an image (White et al., 1988). This is because, as power levels increase beyond saturation, no more signal is generated at the focal volume. However, background emission, generated away from the focal volume where the excitation power densities are below saturation levels, continues to increase. This is not the case for MP imaging systems. Firstly, there is essentially no out-of-focus background generated in an MP system. Secondly, when excitation levels are increased beyond the level where saturation occurs at the focal volume, more emission signal is produced as regions adjacent to the focal volume receive sufficient power density to enable significant levels of multiphoton excitation to occur. Therefore more signal is produced at the expense of resolution. For example, a x8 gain in signal could be obtained at the expense of a 50% loss in resolution. This is probably a better trade-off than the loss of signal-to-background ratio and lower photon detection efficiencies that result from the use of multiple beam MP systems.

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