Live imaging using adaptive optics with fluorescent protein guide-stars

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.

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy

“…Electrically controllable spatial light modulators (SLMs), such as deformable mirror devices or liquid-crystalon-silicon devices, were shown to reduce not only the spherical aberration but also various other aberrations [15][16][17][18][19][20][21] while providing dynamic correction in three-dimensional (3-D) images. 21 For example, with a 40 × ∕NA1.3 oil immersion objective lens, the imaging depth in water was extended from approximately 3.4 to 46.2 μm by using an SLM in TPLSM. 15 Furthermore, the nonlinear guide-star concept was introduced to the adaptive optics technique using an SLM and succeeded in improving the image quality at deep regions within fixed and in vivo biological samples in TPLSM.…”

“…In these studies, researchers modified the conventional microscope and added a relay optical system in order to incorporate the refractive SLM in the system. [15][16][17][18][19][20][21] Such a modification would be difficult to implement for biologists who are not necessarily optics experts.…”

Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

“…[6,7] AO microscopy has successfully been used to cancel the disturbances caused by cellular structures in wide-field microscopy, [8] laser-scanning confocal microscopy, [9,10] multi-photon microscopy, [11][12][13] harmonic generation microscopy, [14,15] and superresolution stimulated emission depletion microscopy, [16,17] with or without the wavefront sensor. [18] So far, most AO microscopy has observed artificial samples and animal tissues; plant cells and tissues have not been examined by AO microscopy yet.…”

Snapshot coherence-gated direct wavefront sensing for multi-photon microscopy

“…One class of methods are sensorless methods [10][11][12][13][14] that iteratively optimise a metric such as the mean intensity or sharpness based on the fluorescence signal, without using a dedicated wavefront sensor. Alternatively, the fluorescence can be used to measure the wavefront directly, either using fluorescent beads [15,16], or fluorescent proteins [17][18][19][20] as the biological equivalent of astronomical guide stars [21,22].…”