Quantitative phase microscopy: automated background leveling techniques and smart temporal phase unwrapping.

In order for time-dynamic quantitative phase microscopy to yield meaningful data to scientists, raw phase measurements must be converted to sequential time series that are consistently phase unwrapped with minimal residual background shape. Beyond the initial phase unwrapping, additional steps must be taken to convert the phase to time-meaningful data sequences. This consists of two major operations both outlined in this paper and shown to operate robustly on biological datasets.

Quantitative Phase Microscopy: how to make phase data meaningful.

The continued development of hardware and associated image processing techniques for quantitative phase microscopy has allowed superior phase data to be acquired that readily shows dynamic optical volume changes and enables particle tracking. Recent efforts have focused on tying phase data and associated metrics to cell morphology. One challenge in measuring biological objects using interferometrically obtained phase information is achieving consistent phase unwrapping and -dimensions and correct for temporal discrepanices using a temporal unwrapping procedure.

Performance enhancement and background removal to improve dynamic phase imaging of biological organisms.

This paper describes recent advances in enhancing optical imaging performance and removal of background shape for a new, novel interference dynamic microscope system. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for labels or contrast agents. This instrument utilizes a pixelated phase mask enabling simultaneous measurement of multiple interference patterns.

Dynamic quantitative phase imaging for biological objects using a pixelated phase mask.

This paper describes research in developing a dynamic quantitative phase imaging microscope providing instantaneous measurements of dynamic motions within and among live cells without labels or contrast agents. It utilizes a pixelated phase mask enabling simultaneous measurement of multiple interference patterns derived using the polarization properties of light to track dynamic motions and morphological changes. Optical path difference (OPD) and optical thickness (OT) data are obtained from phase images.

Dynamic 4-dimensional microscope system with automated background leveling.

This paper describes recent advances in developing an automatic background leveling algorithm for a new, novel interference microscope system and presents images and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. "Label-free" measurements of biological objects in reflection using harmless light levels are possible without the need for scanning and vibration isolation.

Dynamic phase imaging for in vitro process monitoring and cell tracking.

This paper describes a new, novel quantitative interference microscope system and presents images, videos and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with a variety of magnifications and wavelengths with fields of view from several hundred microns up to a millimeter.

Dynamic phase imaging utilizing a 4-dimensional microscope system.

This paper describes a new, novel interference Linnik microscope system and presents images and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with a variety of magnifications and wavelengths with fields of view from several hundred microns up to a millimeter.

Dynamic quantitative phase images of pond life, insect wings, and in vitro cell cultures.

This paper presents images and data of live biological samples taken with a novel Linnik interference microscope. The specially designed optical system enables instantaneous and 3D video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with current magnifications of 10X (NA 0.

Measuring effects of music, noise, and healing energy using a seed germination bioassay.

Objective: To measure biologic effects of music, noise, and healing energy without human preferences or placebo effects using seed germination as an objective biomarker.

Methods: A series of five experiments were performed utilizing okra and zucchini seeds germinated in acoustically shielded, thermally insulated, dark, humid growth chambers. Conditions compared were an untreated control, musical sound, pink noise, and healing energy.

Window function influence on phase error in phase-shifting algorithms.

We present five different eight-point phase-shifting algorithms, each with a different window function. The window function plays a crucial role in determining the phase (wavefront) because it significantly influences phase error. We begin with a simple eight-point algorithm that uses a rectangular window function.

Comparison of phase-unwrapping algorithms by using gradient of first failure.

The comparison of phase-unwrapping algorithms has been an enigma because there has been no quantitative means of comparison. Noting that unwrapping routines are sensitive to noise and the local gradient of the phase array as well as fringe modulation, we have linked unwrapping performance to the gradient of first failure of the algorithm. When the gradient of first failure is plotted versus the signal-to-noise ratio, this can be used as an indicator of which algorithm to use in a given situation without the need for user intervention during the measurement and calculation.

Liquid-crystal point-diffraction interferometer for wave-front measurements.

A new instrument, the liquid-crystal point-diffraction interferometer (LCPDI), is developed for the measurement of phase objects. This instrument maintains the compact, robust design of Linnik's point-diffraction interferometer and adds to it a phase-stepping capability for quantitative interferogram analysis. The result is a compact, simple to align, environmentally insensitive interferometer capable of accurately measuring optical wave fronts with very high data density and with automated data reduction.

Extended averaging technique for derivation of error-compensating algorithms in phase-shifting interferometry.

Phase-shifting interferometry suffers from two main sources of error: phase-shift miscalibration and detector nonlinearity. Algorithms that calculate the phase of a measured wave front require a high degree of tolerance for these error sources. An extended method for deriving such error-compensating algorithms patterned on the sequential application of the averaging technique is proposed here.

Liquid-crystal point-diffraction interferometer.

A new point-diffraction interferometer has been developed with a liquid-crystal filter that permits arbitrary phase shifts to be introduced between the object and reference beams. A microsphere embedded within the liquid-crystal layer provides a locally generated reference beam. We phase shift the object beam by varying a voltage across the liquid crystals, thereby altering the refractive index of the birefringent nematic liquid crystals.

Interferometric testing of high-numerical-aperture convex surfaces.

Interferometric tests of convex surfaces for large-N.A. lens systems often present problems.

Phase-shifting errors in interferometric tests with high-numerical-aperture reference surfaces.

The errors introduced by moving a spherical reference surface to generate a phase shift between the beams in a Fizeau interferometer are discussed.

Testing spherical surfaces: a fast, quasi-absolute technique.

A technique for measuring the quality of spherical surfaces that provides a quasi-absolute result is presented. It requires only two measurement positions rather than the traditional method of absolute sphere measurement that requires three measurement positions. A measurement is taken with a mirror at the focus of the interferometer diverger lens and is subtracted from a measurement of the sphere tested at its center of curvature.

Absolute measurement of surface roughness.

In an interferometer which uses a reference surface, the measured surface heights correspond to the difference between the test and reference surfaces. To accurately determine the rms roughness of supersmooth surfaces, the effects of the reference surface roughness need to be removed. One technique for doing this involves averaging a number of uncorrelated measurements of a mirror to generate a reference surface profile which can then be subtracted from subsequent measurements so that they do not contain errors due to the reference surface.

Calibration of numerical aperture effects in interferometric microscope objectives.

The numerical aperture (N.A.) of a microscope objective can affect the measurement of surface profiles.

Holographic contour and deformation measurement using a 1.4 million element detector array.

Holographic interferometry enables the measurement of object deformations due to stress induced by pressure, heat, or applied force and the measurement of surface shape. Real-time double-exposure holographic interferometry and phase-measurement interferometry have been combined to measure both static changes in objects and their shape. However, the number of detector points available has limited the number of fringes which can be measured.

Combining multiple-subaperture and two-wavelength techniques to extend the measurement limits of an optical surface profiler.

A method has been developed for extending the measurement range of an optical surface profiler using the techniques for combining multiple subapertures and two-wavelength phase-shifting interferometry. The effective trace length of the optical profiler is augmented by making a series of partially overlapping collinear measurements and then piecing the subapertures into a composite profile, thus extending the field of view of the instrument. The vertical dynamic range of the optical profiler is extended by taking phase measurements at two wavelengths and then subtracting these measurements to obtain the same result as if the object had been tested at a longer equivalent wavelength.

Step height measurement using two-wavelength phase-shifting interferometry.

Two-wavelength phase-shifting interferometry is applied to an interference phase-measuring microscope enabling the measurement of step features. The surface is effectively tested at a synthesized equivalent wavelength lambda(eq) = lambda(a)lambda(b)/| lambda(a) - lambda(b)| by subtracting phase measurements made at visible wavelengths lambda(a) and lambda(b). The rms repeatability of the technique is lambda/1000 at the equivalent wavelength.