James P. Ryle

Generalized in-line digital holographic technique based on intensity measurements at two different planes

In-line digital holography based on two-intensity measurements [Zhang Opt. Lett. 29, 1787 (2004)], is modified by introducing a pi shifting in the reference phase. Such an improvement avoids the assumption that the object beam must be much weaker than the reference beam in strength and results in a simplified experimental implementation. Computer simulations and optical experiments are carried out to validate the method, which we refer to as position-phase-shifting digital holography.

Lensless multispectral digital in-line holographic microscope

An compact multispectral digital in-line holographic microscope (DIHM) is developed that emulates Gabors original holographic principle. Using sources of varying spatial coherence (laser, LED), holographic images of objects, including optical fiber, latex microspheres, and cancer cells, are successfully captured and numerically processed. Quantitative measurement of cell locations and percentage confluence are estimated, and pseudocolor images are also presented. Phase profiles of weakly scattering cells are obtained from the DIHM and are compared to those produced by a commercially available off-axis digital holographic microscope.

Dual wavelength digital holographic Laplacian reconstruction

Access to the spatial derivatives of an optical wave field can be used to enhance edge detection, focusing, and holographic imaging. It was recently shown that, by using digital holographic techniques, the Laplacian of an object field can be extracted. Here it is demonstrated that equivalent results can be found using two holograms captured at either two distances or with two appropriately related wavelengths. Experimental and numerical results confirming the theoretical analyses are presented. The proposed two-wavelength-based system requires no mechanical repositioning of the object and is shown to provide superior performance.

Digital inline holography of biological specimens

Digital Holography is the technique of numerically reconstructing a three-dimensional (3D) image containing both amplitude and phase information from a two dimensional (2D) interference pattern recorded by the CCD. In this paper, we study the effects of varying the coherence length by using light from two types of sources (1) coherent laser light and (2) spatially filtered incoherent light from a Light Emitting Diode (LED). We present results using both calibrated test objects and biological samples with view to developing a 3D object recognition and classification system.

Self-written waveguides in a dry acrylamide/polyvinyl alcohol photopolymer material

For the first time it is demonstrated that permanent optical waveguides can be self-written in a solid acrylamide/polyvinyl alcohol photopolymer material. The novel (to our knowledge) technique used to prepare the polymeric medium used is described. It is demonstrated that the resulting waveguides formed can be used to guide different wavelengths. A standard theoretical model is used to predict both the evolution of the light intensity distribution and the channel formation inside the material during the exposure. The experimental results and the numerical simulations are compared, and good agreement is obtained.

Calibration of a digital in-line holographic microscopy system: depth of focus and bioprocess analysis

Digital in-line holographic microscopy (DIHM) allows access to both intensity and phase information with conventional microscopic lateral resolutions. Such imaging techniques can, however, be used to increase the depth of focus compared to conventional compound microscopes. We present a simple DIHM capable of imaging weakly scattering 10 μm diameter microspheres as well as Hs578T cells over a depth of 1 mm; i.e., we demonstrate an increase by a factor of 100 over the depth of focus of a conventional microscope.

Compact portable ocular microtremor sensor: design, development and calibration

Ocular microtremor (OMT) is a physiological high-frequency (up to 150 Hz) low-amplitude (25-2500 nm peak-to-peak) involuntary motion of the human eye. Recent studies suggest a number of clinical applications for OMT that include monitoring the depth of anesthesia of a patient in surgery, prediction of outcome in coma, and diagnosis of brain stem death. Clinical OMT investigations to date have used mechanical piezoelectric probes or piezoelectric strain gauges that have many drawbacks which arise from the fact that the probe is in contact with the eye. We describe the design of a compact noncontact sensing device to measure OMT that addresses some of the above drawbacks. We evaluate the system performance using a calibrated piezoelectric vibrator that simulates OMT signals under conditions that can occur in practice, i.e., wet eye conditions. We also test the device at low light levels well within the eye safety range.

Multispectral lensless digital holographic microscope: imaging MCF-7 and MDA-MB-231 cancer cell cultures

Digital holography is the process where an objects phase and amplitude information is retrieved from intensity images obtained using a digital camera (e.g. CCD or CMOS sensor). In-line digital holographic techniques offer full use of the recording devices sampling bandwidth, unlike off-axis holography where object information is not modulated onto carrier fringes. Reconstructed images are obscured by the linear superposition of the unwanted, out of focus, twin images. In addition to this, speckle noise degrades overall quality of the reconstructed images. The speckle effect is a phenomenon of laser sources used in digital holographic systems. Minimizing the effects due to speckle noise, removal of the twin image and using the full sampling bandwidth of the capture device aids overall reconstructed image quality. Such improvements applied to digital holography can benefit applications such as holographic microscopy where the reconstructed images are obscured with twin image information. Overcoming such problems allows greater flexibility in current image processing techniques, which can be applied to segmenting biological cells (e.g. MCF-7 and MDA-MB- 231) to determine their overall cell density and viability. This could potentially be used to distinguish between apoptotic and necrotic cells in large scale mammalian cell processes, currently the system of choice, within the biopharmaceutical industry.

Experimental evaluation of inline free-space holography systems

It is important to be able to quantify, theoretically and experimentally, the performance of coherent digital systems, so that their suitability for a given metrology application can be assessed. Here, a free-space inline digital holographic system is investigated. To isolate the scattered object field, phase-shifting interferometry (PSI) techniques are used. Several sequential holographic measurements are made, where the phase of the reference field is stepped by a known amount relative to the scattered object field between captures. Under ideal conditions such as noise-free electronics, vibration-free environments, and perfect reference and illuminating object waves, this system will be diffraction limited. However, real systems suffer from experimental error and noise effects. In this paper, we examine a PSI digital holographic imaging system considering all prominent error sources. An experimental metric is defined that quantifies how far from the theoretical ideal a real system is performing. By carefully optimizing our system, following our recommended guidelines, we approach diffraction limited imaging, surpassing the Nyquist sampling rate of the CCD/CMOS device.

Multispectral lensless digital in-line holographic microscope: LED illumination

Holography is the process where two coherent wavefields interfere resulting in an interference pattern from which whole field information can be retrieved. Digital holography is the process where the intensity of the superposition of the two waves is recorded using a light sensitive opto-electronic detector array such as a CCD or CMOS camera. From this recorded hologram it is possible to numerically reconstruct the object wavefield. When an optical beam is focused on a pinhole whose diameter is of the order of a few times the wavelength of the illumination beam, a spherically divergent wavefield is emitted. We use the emitted optical beam to illuminate weakly scattering objects resulting in a geometrically magnified diffraction pattern at the camera face. Scattered light from the object is the called the object wavefield, while unscattered light acts as the reference wavefield. The hologram is captured digitally before numerical reconstruction is applied to yield whole field information about the object. It is possible to reconstruct the objects wavefield using this method from coherent laser or incoherent LED illumination. The emitted light from the pinhole acts a pointsource of spatially coherent light enabling holography. This, in combination with the use of multiple wavelength LEDs multispectral amplitude images can be reconstructed. The multispectral lensless DIHM described here can be used to holographically image biological specimens such as cells grown for use in the biopharmaceutical industry or for research purposes. In analysing cell viability based on the trypan blue assay, the outer membrane of non-viable cells is penetrated by violet blue dye. Using such a Digital In-line Holographic Microscope as described here, automatic classification of viable and non-viable cells could be performed.