These authors contributed equally to this work.
Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)
Article first published online: 21 JUN 2010
Published 2010. This article is a US Government work and is in the public domain in the USA
Journal of Microscopy
Volume 241, Issue 2, pages 153–161, February 2011
How to Cite
COMBS, C.A., SMIRNOV, A., CHESS, D., MCGAVERN, D.B., SCHROEDER, J.L., RILEY, J., KANG, S.S., LUGAR-HAMMER, M., GANDJBAKHCHE, A., KNUTSON, J.R. and BALABAN, R.S. (2011), Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED). Journal of Microscopy, 241: 153–161. doi: 10.1111/j.1365-2818.2010.03411.x
- Issue published online: 11 JAN 2011
- Article first published online: 21 JUN 2010
- Received 9 February 2010; accepted 22 April 2010
Vol. 243, Issue 2, 220, Article first published online: 18 JUL 2011
- light collection improvement;
- photon diffusion simulations;
- two-photon microscopy
A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330–337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ∼8-fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ∼4-fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2-fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal-to-noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo-damage).