N. B. E. Sawyer

Wide field amplitude and phase confocal microscope with parallel phase stepping

We describe a wide field phase sensitive confocal microscope based on correlation between speckles. In this system, parallel optical phase stepping provides for the simultaneous capture of four interferograms using a single camera, giving the system the potential to operate at high speed. The interferograms are mapped to a common frame of reference, removing any relative distortion in the process, so that phase, amplitude, and interference efficiency images can be obtained. We use a new phase stepping algorithm that accounts for the unequal phase steps and unbalanced beam amplitudes that are a consequence of the optical arrangement. Experimental results to show that the system operates as a wide field amplitude and phase confocal system are presented.

Tracking large solid constructs suspended in a rotating bioreactor: A combined experimental and theoretical study

We present a combined experimental and theoretical study of the trajectory of a large solid cylindrical disc suspended within a fluid‐filled rotating cylindrical vessel. The experimental set‐up is relevant to tissue‐engineering applications where a disc‐shaped porous scaffold is seeded with cells to be cultured, placed within a bioreactor filled with nutrient‐rich culture medium, which is then rotated in a vertical plane to keep the growing tissue construct suspended in a state of “free fall.” The experimental results are compared with theoretical predictions based on the model of Cummings and Waters (2007), who showed that the suspended disc executes a periodic motion. For anticlockwise vessel rotation three regimes were identified: (i) disc remains suspended at a fixed position on the right‐hand side of the bioreactor; (ii) disc executes a periodic oscillatory motion on the right‐hand side of the bioreactor; and (iii) disc orbits the bioreactor. All three regimes are captured experimentally, and good agreement between theory and experiment is obtained. For the tissue engineering application, computation of the fluid dynamics allows the nutrient concentration field surrounding a tissue construct (a property that cannot be measured experimentally) to be determined (Cummings and Waters, 2007). The implications for experimental cell‐culture protocols are discussed. Biotechnol. Bioeng. 2009; 104: 1224–1234.

Ultrastable absolute-phase common-path optical profiler based on computer-generated holography

A new scanning common-path interferometric profiler capable of absolute-phase measurement is described. The key element is a computer-generated hologram, which acts as the beam-splitting element. Unlike most absolute phase systems, it can be made entirely common path with respect to piston microphonics and is thus exceptionally stable. In addition to operating in scanning mode, the optical configuration permits simultaneous operation as a single-shot phase measuring interferometer and is thus capable of simultaneous form and texture measurements. The operation and stability of the scanning profiler are demonstrated experimentally.

Amplitude and phase microscopy for sizing of spherical particles

We describe a numerical vector diffraction model based on Mie theory that describes the imaging of spherical particles by bright-field, confocal, and interferometric microscopes. The model correctly scales the amplitude-scattered field relative to the incident field so that the forward-scattered and incident light can be interfered to correctly model imaging with copolarization transmission microscopes for the first time to our knowledge. The model is used to demonstrate that amplitude and phase imaging with an interferometric microscope allows subwavelength particle sizing. Furthermore, we show that the phase channel allows much smaller particles to be sized than amplitude-only measurements. The model is validated by experimental measurements.

Imaging of small particles using wide-field confocal microscopy

Particle measurement is important in many applications such as the manufacture of drugs and paints, and aerosols. In bioimaging there is interest understanding the imaging of nanoparticles and subcellular scatterers. We present in this paper a wide field, phase measuring confocal microscope that can be used for such measurements. The wide field confocal response is obtained by illuminating both sample and reference arms of an interferometric microscope with nominally identical speckle patterns. When the speckle patterns are highly correlated the interference is significant. Contributions from out of focus planes result in uncorrelated speckle patterns and no interference. This provides a wide field confocal response. High speed measurements are enabled by parallel phase stepping using polarization optics. We have also developed a vector diffraction microscope model, using Mie theory as a scattering function, to validate the images of small particles. Correctly scaling the amplitudes of the unscattered and scattered electric fields enables co-polar transmission imaging to be modeled. Finally it is demonstrated that the phase is a more sensitive measurement of particle size than the amplitude.

Wide field, phase measuring confocal microscopy of small particles

Amplitude and phase measurements of small particles using a wide field, phase measuring confocal microscope are demonstrated. The wide field confocal capability of the microscope is achieved by illuminating both sample and reference arms of a Linnik interferometer with a moving speckle pattern. In addition a rigorous vector diffraction microscope model based upon Mie scattering theory has been developed. The model is particularly useful as by careful consideration of the scattered and unscattered light, quantitative transmission images can be achieved.

Differential optical profilometer using a single probe beam

This paper describes a heterodyne common path differential phase interferometer which is resistant to the thermally and microphonically induced measurement errors of non-common path systems. The system utilizes a single probe beam which is focused onto the sample and then imaged onto the detector plane. Differentiation in any direction can be performed by altering the positions of the detectors. A theoretical derivation of the system transfer function is presented, the results from which show excellent agreement with experimental measurements. Initial theoretical results of the recovery of a sample profile from its differential profile are presented. Finally, a simplified and more stable differential system is introduced.