A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed.

Figure 1. This Optoelectronic System is a custom-built system for two-photon fluorescence microscopy.

On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4× increase of penetration depth.

The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the longer-wavelength excitation light and passes the shorter-wavelength fluorescence light. Also, the confocal pinhole has been removed to increase the signal strength.

The laser beam is scanned by a two-perpendicular-axis pair of galvanometer mirrors through a pupil transfer lens into the side port of an inverted microscope. Finally, the beam is focused by a 63-magnification, 1.3-numerical-aperture oil-immersion objective lens onto a specimen. The pupil transfer lens serves to match the intermediate image planes of the scanning head and the microscope, and its location is critical.

In order to maximize the quality of the image, (that is, the point spread function of the objective lens for all scan positions), the entire system was modeled in optical-design software, and the various free design parameters (the parameters of the spatial-filter components as well as the separations of all of the system components) were determined through an iterative optimization process. A modular design was chosen to facilitate access to the optical train for future fluorescence correlation spectroscopy and fluorescence- lifetime experiments.

Figure 2. This Two-Photon Image of a human femur section shows several areas of remodeled bone. Thefield of view is 200 μm. The specimen in this image was provided by the Cleveland Clinic Foundation.

The spatial resolution of the microscope at an excitation wavelength of 780 nm was measured by scanning a 170-nm-diameter fluorescent bead throughout the focal region and found to be 320 nm in the transverse direction and 740 nm in the longitudinal direction. The sectioning capability of two-photon microscopy is demonstrated in Figure 2, which depicts a human bone specimen as imaged by use of two-channel spectral detection.

The two-photon microscope is now being employed to study osteoblast and osteoclast bone cells in cultures, including the effects of fluid flow and other environmental stimuli. Ultimately, these studies will be used to investigate and develop effective countermeasures to the bone loss experienced in a reduced- gravity environment. In addition, a variant of this instrument is being considered as an add-on module to the Light Microscopy Module, which will be deployed on the International Space Station.

This work was done by David G. Fischer and Gregory A. Zimmerli of Glenn Research Center and Marius Asipauskas of the National Center for Microgravity Research.

Inquiries concerning rights for the commercial use of this invention should be addressed to

NASA Glenn Research Center
Commercial Technology Office
Attn: Steve Fedor
Mail Stop 4–8
21000 Brookpark Road
Ohio 44135.

Refer to LEW-17573-1.