We investigate tissue and instrument parameters affecting the penetration depth in two-photon microscopy. We show that the temporal redistribution of the same average power into fewer pulses of higher peak energy by means of a regenerative amplifier results in an increase in excitation depth by approximately 2-3 scattering mean free paths. We then measure the excitation scattering mean free path in vitro, using rat brain slices, as a function of the excitation wavelength and tissue age. We find that young-animal tissue (< P18) is two-fold less scattering than adult tissue (P90). We quantify the fall-off of the collected fraction of generated fluorescence in a backward detection geometry, in vivo. At large depths, we observe that the collected fraction scales as the angular acceptance squared (related to the effective field-of-view) of the detection optics. Matching the angular acceptance of the detection optics to that of the objective (63X NA-0.90) results in a factor 3-4 of the collected fluorescence. The collection efficiency can be further increased (10X) by using an objective with large field-of-view and high numerical aperture (20X NA-0.95). These gains translate into approximately 120 micrometers additional depth penetration when working in the rat brain in vivo with a standard Ti:sapphire source.
The evanescent wave (EW) elicited by total internal reflection of light provides a means to selectively excite fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epi-illumination microscopy, and can offer a 5-fold enhancement of axial resolution compared to confocal and two- photon microscopy. The decay length of the evanescent field is a function of the refractive indices at the interface, the wavelength of the light, and is modulated by the beam-angle. EW microscopy has been used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution on top of the interface. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. 3-D reconstruction of image stacks by an inverse Laplace transform results in topographical information with an axial resolution of 10's of nanometers. We have labeled subcelluar storage organelles ('vesicles') of approximately equal 300 nm diameter and visualized the trajectories of single vesicles in the 'footprint' region of living neuroendocrine cells, grown on the interface.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.