Mulitiphoton imaging techniques such as Two-Photon Excitation Fluorescence (TPEF) and Second-Harmonic Generation (SHG) microscopy provide high-resolution images of sub-cellular biological structures in a noninvasive manner. Compared to confocal microscopy, TPEF eliminates fluorescence outside the focal plane and increases axial resolution. By using IR radiation instead of visible or UV light to illuminate the sample during the two-photon process, sample phototoxicity is reduced and deeper penetration is possible.
Comparison of cross-sectional TPEF images (upper panels) of a histological section of a human retina and SHG images (lower panels) of a human sclera section obtained with (frames b and d) and without (frames a and c) AO. The intensity scale was kept constant for each pair of images.
During the experiment, Bueno et al. used a Shack-Hartmann Wavefront Sensor and Deformable Mirror (DM) to monitor and correct wavefront aberrations (WAs), respectively. These aberrations primarily resulted from misalignments in the laser cavity of their illumination source (110 fs, 76 MHz Ti:Sapphire laser with 760 nm output). This led to an improvement in the beamquality.The average power at the sample plane ranged from 2 to 200 mW depending on the sample studied. Using the AO-equipped multiphoton setup, Bueno et al. obtained TPEF and SHG images of 5 μm thick non-stained transversal (XZ) sections of human ocular tissues (retina and sclera) embedded in paraffin. As shown in Fig. 1, the improvement in signal intensity using AO was threefold for TPEF images and twofold for SHG images. The increased image contrast achieved with AO enables structures such as the retinal fiber layer (RNFL), photoreceptor layers (PRL), and different intermediate layers to be better visualized. Each retinal layer was imaged without noticeable photodamage.
Thorlabs' AO kit is a cost-effective solution for improving multiphoton image contrast and axial resolution since it allows low-order aberrations, attributed to the laser source, to be monitored and corrected with relatively little effort and computational expense. Its use for retinal studies could have potential clinical applications for early diagnosis of ocular pathologies such as glaucoma, which is linked to a loss of ganglion cells (refer to Figure 2).
TPEF images of a retinal area containing ganglion cells (GC) and blood vessels (BV) obtained without (a) and with (b) AO correction. The total intensity in (b) is three times higher than that in (a).