title: [Two-photon laser scanning fluorescence microscopy for functional cellular imaging: Advantages and challenges or One photon is good... but two is better!].
abstract: One of the main challenges of modern biochemistry and cell biology is to be able to observe molecular dynamics in their functional context, i.e. in live cells in situ. Thus, being able to track ongoing molecular events with maximal spatial and temporal resolution (within subcellular compartments), while minimizing interference with tissue biology, is key to future developments for in situ imaging. The recent use of non-linear optics approaches in tissue microscopy, made possible in large part by the availability of femtosecond pulse lasers, has allowed major advances on this front that would not have been possible with conventional linear microscopy techniques. Of these approaches, the one that has generated most advances to date is two-photon laser scanning fluorescence microscopy. While this approach does not really provide improved resolution over linear microscopy in non absorbing media, it allows us to exploit a window of low absorbance in live tissue in the near infrared range. The end result is much improved tissue penetration, minimizing unwanted excitation outside the focal area, which yields an effective improvement in resolution and sensitivity. The optical system is also simplified and, more importantly, phototoxicity is reduced. These advantages are at the source of the success of two-photon microscopy for functional cellular imaging in situ. Yet, we still face further challenges, reaching the limits of resolution that conventional optics can offer. Here we review some recent advances in optics/photonics approaches that hold promises to improve our ability to probe the tissue in finer areas, at faster speed, and deeper into the tissue. These include super-resolution techniques, introduction of non paraxial optics in microscopy and use of amplified femtosecond lasers, yielding enhanced spatial and temporal resolution as well as tissue penetration.
authors.affiliation: Centre d'optique, photonique et laser, Département de Physique, de génie physique et d'optique, Université Laval, Québec, Canada.
authors.forename: Pascal
authors.initials: P
authors.lastname: Dufour
authors.forename: Suzie
authors.initials: S
authors.lastname: Dufour
authors.forename: Annie
authors.initials: A
authors.lastname: Castonguay
authors.forename: Nathalie
authors.initials: N
authors.lastname: McCarthy
authors.forename: Yves
authors.initials: Y
authors.lastname: De Koninck
mesh_terms: D002477:Cells / Q000648:ultrastructure*; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton