Wavefront optimized nonlinear microscopy of ex vivo human retinas.
A multiphoton microscope incorporating a Hartmann-Shack (HS) wavefront sensor to control the ultrafast laser beam's wavefront aberrations has been developed. This instrument allowed us to investigate the impact of the laser beam aberrations on two-photon autofluorescence imaging of human retinal tissues. We demonstrated that nonlinear microscopy images are improved when laser beam aberrations are minimized by realigning the laser system cavity while wavefront controlling. Nonlinear signals from several human retinal anatomical features have been detected for the first time, without the need of fixation or staining procedures. Beyond the improved image quality, this approach reduces the required excitation power levels, minimizing the side effects of phototoxicity within the imaged sample. In particular, this may be important to study the physiology and function of the healthy and diseased retina.
Gualda
Emilio J
EJ
Universidad de Murcia, Laboratorio de Optica, Campus de Espinardo (CiOyN), Murcia, 30100 Spain.
Bueno
Juan M
JM
Artal
Pablo
P
D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D066298:In Vitro Techniques; D007834:Lasers; D018613:Microscopy, Confocal / Q000295:instrumentation*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D017711:Nonlinear Dynamics; D015203:Reproducibility of Results; D012160:Retina / Q000473:pathology*; D042261:Retinoscopes; D012680:Sensitivity and Specificity; D014159:Transducers