We present a technique of visible spectrum quantitative phase imaging with ten millimeter range without phase ambiguity while achieving better than ten micrometer precision. A suitable synthetic wavelength for absolute phase measurement, without unwrapping, is implemented by two-wavelength digital holography. This results in amplified phase noise which is then reduced by an iterative optical and algorithm based process involving one or two additional source wavelengths. We demonstrate that these source wavelengths can be acquired from a single laser diode source by realizing the wavelength output response to fine temperature control.
We present a technique of three-dimensional (3-D) differential imaging by the way of incoherent digital holography. We demonstrate that the method is suitable for fluorescence microscopy without the need for unfavorable scanning. We acquire the complex optical field of an incoherent volumetric scene at various times and calculate a complex difference hologram representing only the information, which has changed throughout the volumetric space during the time intervals in-between. We first demonstrate the advanced capability of self-interference incoherent digital holography combined with difference holography to track 3-D changes in a broadband, unfiltered, sunlit scene containing macroscopic continuous objects. This case is particularly remarkable due to the exceptionally short temporal coherence length and excessive build-up of noninterfering source points. We then demonstrate the ease of adaptation to the versatile, functional imaging of fluorescence microscopy.
Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: a wavefront sensor, wavefront corrector, and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, for example, lenslet arrays for sensing or multiactuator deformable mirrors for correcting. We have previously introduced an alternate approach based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile are possible not only with conventional coherent digital holography, but also with a new type of digital holography using incoherent light: selfinterference incoherent digital holography (SIDH). The SIDH generates a complex—i.e., amplitude plus phase—hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. Adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.
Fluorescence microscopy is an indispensable imaging tool in modern biomedical research. Holography is well-known to have many interesting and useful imaging capabilities. But the requirement of coherent illumination has all but precluded holography as a means for fluorescence imaging, which is inherently incoherent. Recent developments in digital holography, however, including self-interference incoherent digital holography (SIDH), provide highly effective and versatile capabilities for 3D holographic imaging with incoherent light, that can remove the barrier between fluorescence and holography. Recent progress in holographic fluorescence microscopy is presented.
We present a new technique of three-dimensional tracking by way of incoherent digital holography suitable for non-scanning fluorescence microscopy. By acquiring complex holograms of a sample at different times, a difference hologram can be calculated. This result is another complex hologram representing only the information which has changed throughout the volumetric space during the time interval between one hologram and the next. We first demonstrate the advanced capability of self-interference incoherent digital holography combined with difference holography to track three-dimensional changes in a broadband, unfiltered, sunlit scene containing macroscopic continuous objects. This case is particularly remarkable due to the exceptionally short temporal coherence length and excessive build-up of noninterfering source points. We then demonstrate the ease of adaptation to microscopy, successfully converting a standard two-dimensional commercial microscope into a powerful three-dimensional tool. By overcoming these challenges, we show the feasibility and ease at which this technique may be adapted to the versatile, functional imaging of fluorescence microscopy.
Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: wavefront sensor, wavefront corrector and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, e.g., lenslet arrays for sensing or multi-acuator deformable mirrors for correcting. We have previously introduced an alternate approach to adaptive optics based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile is possible not only with the conventional coherent type of digital holography, but also with a new type of digital holography using incoherent light: self-interference incoherent digital holography (SIDH). The SIDH generates complex – i.e. amplitude plus phase – hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using a guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. The adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.
Digital Holographic Microscopy produces quantitative phase analysis of a specimen with excellent optical precision. In
the current study, this imaging method has been used to measure induced thermal lensing by optical excitation in the
time-resolved regime with excellent agreement to model predictions. We have found that the thermal effect should not
be dismissed when pursuing optical radiation pressure experiments, even when the media involved are transparent. We
have developed a unified model and simulated methods of decoupling the two effects. The results of this study and
simulations suggest that our near term goal of nanometric measurement of an optical pressure induced deformation will
prove successful. Precise measurement of this phenomenon can be useful in determining physical properties of
interfacial surfaces, such as surface tension, and characterizing physical properties of cellular structures.
A new form of near-field microscopy is presented using digital holography for quantitative phase imagery and
characterization of cell-substrate interfaces. This imaging technique, termed total internal reflection holographic
microscopy (TIRHM), utilizes an evanescent wave phase shift from the presence of cellular organisms, membranes,
adhesions, and tissue structures on a prism face in order to modulate an object beam wavefront in a digital holographic
microscope. Quantitative phase images of live cellular specimens are presented.
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