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Multiphoton laser scanning microscopy with focus on deep tissue imaging

Authors: Johan Borglin, Marica B. Ericson

Multiphoton laser scanning microscopy (MPM) has become a powerful complementary tool in biomedical research, enabling non-invasive three-dimensional imaging of tissue with high resolution [1]. The major advantage is that visualization of distribution of fluorophores can be performed without mechanical destruction of the sample through tissue sectioning. MPM makes it possible to visualize cellular morphology and the cutaneous distribution of topically applied compounds applied to intact biological tissue [2, 3]. In our group, the technique provides information about bioavailability of drugs by, for example, visualizing drug penetration pathways into skin [4], as demonstrated in Figure 1. MPM has also been implemented as a tool for obtaining non-invasive tissue biopsy based on skin autofluorescence in connection to diagnostics of skin cancer [5], and to understand mechanisms involved in contact allergy [6, 7].



Figure 1: Two two-photon images showing the distribution of a flourophore in human skin samples. (A) and (B) illustrate the same florophore in similar samples, but using different methods of florophore delivery. Reproduced from our work [4] with permission.




The most common non-linear optical process applied for MPM is two-photon excitation (2PE), predicted by Maria Göppert Mayer [8]. In this process, two photons are acting simultaneously to excite the fluorophore, illustrated by Figure 2. Since this process has very low probability, it requires a large amount of photons to be present at the same place at the same time. This can be achieved with a pulsed and tightly focused laser, as illustrated in the figure. By using 2PE, the signal is exclusively generated in the very focus of the laser where the photon flux is the highest.

 Figure 2. Illustration of the processes of one-photon versus two-photon excitation performed in our lab. The photos demonstrate the fluorescence signal arising from focusing a laser into a solution containing Rhodamine B. When a continuous visible laser (488 nm) is applied, one-photon excitation is obtained throughout the beam-path in the sample. When a near-infrared (740 nm) femtosecond pulsed laser is used, the two-photon excitation only occurs in the focus.


Multiphoton microscopy setups
In our research, we are presently using two different MPM systems:

• Commercial Zeiss LSM 710 NLO with MaiTai Ti:Saphhire laser, available at Centre for Cellular Imaging.

• Experimental MPM set up with Tsunami Ti:Sapphire laser in our lab

The aim with the experimental set up is to account for a fully flexible MPM system, optimized for deep tissue imaging and is presently under development.



1. Zipfel, W.R., R.M. Williams, and W.W. Webb, Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol, 2003. 21(11): p. 1368-1376.
2. Yu, B., et al., In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy. J Invest Dermatol, 2001. 117(1): p. 16-25.
3. König, K. and I. Riemann, High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt, 2003. 8(3): p. 432-9.
4. Bender, J., et al., Lipid cubic phases in topical drug delivery: Visualization of skin distribution using two-photon microscopy. J Control Release, 2008. 129(3): p. 163-169.
5. Paoli, J., M. Smedh, and M.B. Ericson, Multiphoton laser scanning microscopy--a novel diagnostic method for superficial skin cancers. Semin Cutan Med Surg, 2009. 28(3): p. 190-5.
6. Simonsson, C., et al., Caged fluorescent haptens reveal the generation of cryptic epitopes in allergic contact dermatitis. J Invest Dermatol, 2011. 131(7): p. 1486-93.
7. Simonsson, C., et al., The pilosebaceous unit--a phthalate-induced pathway to skin sensitization. Toxicol Appl Pharmacol, 2012. 264(1): p. 114-20.
8. Göppert-Mayer, M., Über Elementarakte mit zwei Quantensprüngen. Annalen der Physik, 1931. 9: p. 22.






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