Abstract
Optical microscopy provides noninvasive imaging of biological tissues at subcellular level. The optical aberrations induced by the inhomogeneous refractive index of biological samples limits the resolution and can decrease the penetration depth. To compensate refractive aberrations, adaptive optics with Shack-Hartmann wavefront sensing has been used in microscopes. Wavefront measurement requires light from a guide-star inside of the sample. The scattering effect limits the intensity of the guide-star, hence reducing the signal to noise ratio of the wavefront measurement. In this paper, we demonstrate the use of interferometric focusing of excitation light onto a guide-star embedded deeply in tissue to increase its fluorescent intensity, thus overcoming the excitation signal loss caused by scattering. With interferometric focusing, we more than doubled the signal to noise ratio of the laser guide-star through scattering tissue as well as potentially extend the imaging depth through using AO microscopy.
Highlights
With the advantages of high-resolution and viewing of live organisms, optical microscopy has become an important tool for biological research and continues to open new avenues in its capabilities
This paper demonstrates the use of the interferometric focusing (IF) method, rather than conventional geometric focusing to concentrate excitation light onto a guide-star in tissue for direct wavefront measurement using a Shack-Hartmann wavefront sensor (SHWS)
We have demonstrated that IF, rather than conventional geometric focusing, of excitation light onto a guide-star that is embedded deeply in tissue, increases its fluorescence intensity
Summary
With the advantages of high-resolution and viewing of live organisms, optical microscopy has become an important tool for biological research and continues to open new avenues in its capabilities. In a recent study, ultrasonically encoded focusing has been utilized to generate a guide-star as a coherent point source for phase measurement [12,13,14] Another method, called interferometric focusing (IF), is used to estimate the optimal phase of the scattering light field by modulating the phase of illumination light while analyzing the variation of emission light from the sample [15, 16]. Scattering will exponentially reduce the intensity of ballistic light with the imaging increasing depth, correction of refractive aberration still benefits the imaging resolution and contrast. The wavefront can be measured by a SHWS with fluorescence from the illuminated laser guide-star These measurements will subsequently be used in our AO microscope to overcome refractive image aberrations using adaptive geometric optics. The correction of the refractive aberration when using AO provides a larger correction field, compared to the scattering compensation while using IF
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