ABSTRACT
Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy.
Subject(s)
Imaging, Three-Dimensional/methods , Optics and Photonics/methods , Animals , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/embryology , Embryo, Nonmammalian/anatomy & histology , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence , Time Factors , Wavelet AnalysisABSTRACT
We introduce a direct wavefront sensing method using structures labeled with fluorescent proteins in tissues as guide stars. An adaptive optics confocal microscope using this method is demonstrated for imaging of mouse brain tissue. A dendrite and a cell body of a neuron labeled with yellow fluorescent protein are tested as guide stars without injection of other fluorescent labels. Photobleaching effects are also analyzed. The results shows increased image contrast and 3× improvement in the signal intensity for fixed mouse tissues at depths of 70 µm.
Subject(s)
Green Fluorescent Proteins/metabolism , Microscopy, Confocal/instrumentation , Optical Devices , Animals , Brain/cytology , Brain/metabolism , Mice , PhotobleachingABSTRACT
Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images in deep-tissue imaging. We introduce a confocal fluorescence microscope with adaptive optics, which can correct aberrations based on direct wavefront measurements using a Shack-Hartmann wavefront sensor with a fluorescent bead used as a point source reference beacon. The results show a 4.3× improvement in the Strehl ratio and a 240% improvement in the signal intensity for fixed mouse tissues at depths of up to 100 µm.
Subject(s)
Microscopy, Confocal/methods , Optical Phenomena , Animals , Brain/cytology , Mice , Microscopy, Confocal/instrumentationABSTRACT
Adaptive optics scanning laser ophthalmoscopy allows for noninvasive, in vivo visualization of retinal abnormalities at a cellular level. We herein describe for the first time, to our knowledge, the utility of high-resolution retinal imaging in studying the photoreceptor mosaic in an otherwise unexplained visual disturbance. Imaging of the cone mosaic was performed in a 64-year-old man with a unilateral ringlike paracentral distortion that could not be explained using common clinical imaging instruments. Adaptive optics scanning laser ophthalmoscopy findings revealed a parafoveal circular abnormality of the cone mosaic approximately 3 degrees in diameter that corresponded to the ring of visual disturbance. Visualization of the cone mosaic with adaptive optics scanning laser ophthalmoscopy can reveal photoreceptor damage that may not be detectable with standard imaging devices. Optical axial sectioning of the retina may help in identifying and localizing abnormalities within the retinal layers.
Subject(s)
Ophthalmoscopy/methods , Retinal Cone Photoreceptor Cells/pathology , Retinal Diseases/diagnosis , Scotoma/diagnosis , Adult , Humans , Image Enhancement/methods , Lasers , Male , Middle Aged , Visual Field Tests , Visual FieldsABSTRACT
Adaptive optics scanning laser ophthalmoscopes have been used to produce noninvasive views of the human retina. However, the range of aberration compensation has been limited by the choice of deformable mirror technology. We demonstrate that the use of dual deformable mirrors can effectively compensate large aberrations in the human eye while maintaining the quality of the retinal imagery. We verified experimentally that the use of dual deformable mirrors improved the dynamic range for correction of the wavefront aberrations compared with the use of the micro-electro-mechanical-system mirror alone and improved the quality of the wavefront correction compared with the use of the bimorph mirror alone. We also demonstrated that the large-stroke bimorph deformable mirror improved the capability for axial sectioning with the confocal imaging system by providing an easier way to move the focus axially through different layers of the retina.
