Multi-photon microscopy (MPM) is an imaging technology that is used to obtain 3-D images from biological specimens with molecule specific contrast. The use of MPM for in vivo microscopy has multiple potential benefits over single photon microscopy, including applications such as non-invasive diagnostic imaging of the retina (the light sensitive tissue at the back of the eye). Compared to conventional microscopy with single photon excitation fluorescence, MPM uses light at longer wavelengths, in the near infrared (NIR), where tissue scattering and absorption is lower. The use of NIR light is particularly attractive for imaging the retina, which contains phototransduction pigments sensitive to the visible wavelengths. Unlike single photon processes, MPM techniques, such as two-photon excited fluorescence (TPEF), only occur at a narrow axial range around the focal point where the irradiance is the highest, providing an optical sectioning effect. However, a disadvantage of MPM imaging in ocular tissues is the high pulse energy required to elicit the non-linear effects. Minimizing the incident exposure energy is therefore important for non-invasive imaging, in particular for the delicate tissues of the retina.
Although MPM is relatively unaffected by low levels of out-of-focus scattering, wavefront aberrations from the sample and optical path cause blurring of the focal spot. Since the MPM signal is quadratically proportional to the focused spot size, significant improvements in the signal-to-noise ratio can be achieved through wavefront shaping to approach the diffraction-limited focus with a large numerical aperture.
Some conventional technologies apply adaptive optics (AO) to MPM to correct for refractive errors and promote diffraction-limited focusing in tissue. These conventional AO systems use a Hartmann-Shack Wavefront Sensor (HS-WFS) to detect the wavefront aberrations and, in a closed feedback loop control, guide the shape of an adaptive element, such as a deformable mirror, to correct the detected wavefront aberrations. Since the HS-WFS is sensitive to back-reflections, the conventional AO systems use curved mirrors instead of lenses, and long focal lengths to minimize the off-axis aberrations. Furthermore, the use of a wavefront sensor places significant design constraints on the system, requiring optical conjugation of the deformable element, WFS, and the pupil plane of the system. Additionally, the HS-WFS is generally only useful when there is a single scattering plane in the sample, because thick tissue samples or multi-layered samples negatively affect the ability to measure the wavefront.
Furthermore, conventional MPM techniques may require relatively long time, e.g., 6-7 minutes for image acquisition using high power laser excitation energy. Therefore, these conventional technologies subject the patient's eye to a relatively long period of high stress.
Accordingly, there remains a need for the eye imaging methods, systems and apparatuses that are relatively fast and do not cause high light-induced stress on the retina.