Our findings demonstrate that epiTED can be used to increase the efficiency of fluorescence emission collection in multi-photon experiments performed in vivo. This was shown in three different tissue types in living animals using a relatively high NA (0.95) water immersion lens with a long working distance (2 mm), demonstrating that the TED imaging concept can be applied not only to thin slices and cellular imaging but also to intravital imaging. The main advantage of epiTED is that it can be attached directly to the objective and can enhance collection efficiency by a factor of two or more.
As expected, the highest gains were observed in brain imaging (). We assume that this is due to less light absorption than in the other tissues and because brain is a high light scattering tissue that allows many forward-directed photons to scatter back into a detectable zone for the optics in the epiTED system. The thinned skull preparation provided much less increase in gain than did the extracted brain. This is likely due to the bone absorption of emitted light outside of a narrow cone delimited by the boundaries of the thinned region in the skull. Although exposed brain imaging showed the best results, other more absorptive tissues like kidney and skeletal muscle showed a twofold improvement in light collection as well ().
The observed gains in light collection could lead to decreased laser excitation powers (by a factor of the square root of the gain for the TPFM excitation mode) and/or an increase in temporal resolution (lower pixel dwell times necessary for integration of the signal). The gains observed in this work ranged from 2.9 to 1.2 fold increase in light collection (average ~ 2 fold). This would enable a decrease in laser power by 42% or could allow for ~ 2 fold increase in imaging speed (in photon counting mode) depending on the characteristics of the fluorophores. Both of these factors are crucial for many in vivo
imaging applications. Lower laser powers would decrease photo-toxicity and background signal. Faster imaging can decrease the chance of motion artifacts during imaging and/or allow for monitoring faster physiological events in vivo
by limiting the need for signal averaging. When no averaging and minimum dwell times are used, for example in resonance scanners (Leybaert et al., 2005
), the benefit is limited to the improvement in signal to noise alone.
The epiTED design has advantages over the hybrid objective and fiber optic external detection schemes in their published forms. First, the potential gain is inversely related to the NA of the objectives (Combs et al., 2007
). Here we have shown that emission collection gains of two or more are possible even when using a relatively high NA (0.95) objective. This is similar to the gains found in the aforementioned studies (Engelbrecht et al., 2009
; Vucinic et al., 2006
) where 0.8 NA objectives were used. This may be of further importance when considering co-localization studies or multi-photon STED (Stimulated Emission Depletion Microscopy) where two color excitation is necessary and the use of highly corrected, wider acceptance angle lenses is important. Although similar gains are predicted for hybrid objective systems (Vucinic et al., 2006
), the epiTED device is much simpler in design and could be retrofitted to commercially available objectives. Further, in theory the epiTED device should be superior to the ancillary fiber optic ring collection due to the light losses inherent in the solid angle interrupted by the fiber optic ring system (non-collecting regions due to fiber NA and cladding-limited packing). This can be seen from a comparison of the numerical simulations (). Engelbrecht et al. 2009
predict a maximum gain of < 4 fold for a 0.8 NA water immersion lens, which is less than the gain calculated here for a higher NA water immersion lens. Our simulations suggest a gain of greater than 7 fold is possible using a comparable NA objective (). As already mentioned, another benefit of the epiTED system is that it does not contact the tissue or sample being imaged. This gives added room for tissue access and manipulation and does not cause potential physiological perturbation due to the pressure of contact by the fiber optic ring.
The gains observed in the prototype epiTED system are considerably less than predicted by the numerical simulations (). This is most likely due to light loss at the face of the objective or in the conjugate cylindrical body of the system leading to the detector. It is likely that the greatest loss mechanism could be related to either absorption of photons in the lens outside the collectible NA (the front element of the objective) or reflection of some of the emitted light back into the tissue from the objective face. The current epiTED design has the objective face and chamfer body simply wrapped in reflective aluminum foil. Instead, a small oblique conical mirror on the face of the objective would be more efficient in redirecting such light sideways to the parabola, esp. for longer working distance objectives.
In summary, we demonstrate that the newly constructed epiTED device significantly increases TPFM fluorescence light collection, which has the potential to markedly improve in vivo imaging. The advantages of this device are that it does not touch the tissue, has a similar or higher light gain potential when compared to other systems, and is easy to implement using the standard optics of an inverted TPFM microscope. It is likely that future developments will include a compact version of this device for TPFM microscopes in both the upright and inverted configurations.