Christopher Mann

High-resolution quantitative phase-contrast microscopy by digital holography

Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating holographic optical field is seen to have significant advantages including tight control of spurious noise components. Holographic phase images are obtained with 0.5 mum diffraction-limited lateral resolution and largely immune from the coherent noise common in other holographic techniques. The phase profile is accurate to about 30 nm of optical thickness. Images of SKOV-3 ovarian cancer cells display intracellular and intranuclear organelles with clarity and quantitative accuracy.

Interference techniques in digital holography

Interference techniques in digital holography are discussed and experimental results from each technique are presented. Numerical reconstruction algorithms for digital holography are reviewed. The angular spectrum method is seen to be particularly advantageous, with the ability to remove noise and unwanted holographic terms. The dual wavelength optical unwrapping technique offers an unambiguous method of removing 2π phase discontinuity. Application of wavelength-scanning digital interference holography is used to obtain tomographic images with synthesized short coherence length.

Movies of cellular and sub-cellular motion by digital holographic microscopy

BackgroundMany biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy.Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope.MethodsA modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features.ResultsBoth cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration.ConclusionDigital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.

Digital Holography and Multi-Wavelength Interference Techniques

Recent developments in digital holography techniques are presented. The Fourier transform and convolution methods to calculate the diffraction fields are compared with the angular spectrum method, which allows short reconstruction distances down to zero with flexibility in the control of image noise. Examples of applications of digital holography in biological microscopy are presented with submicron lateral resolution. The phase imaging digital holography using two wavelengths is described that achieves unambiguous phase unwrapping, even for noisy images common in biological imaging. The wavelength scanning digital interference holography (WSDIH) is described that allows tomographic imaging by numerical superposition of many holograms using a series of different wavelengths to synthesize short coherence length. The WSDIH technique is applied to image sub-surface structures of an animal tissue down to 1.5 mm depth with 10 µm axial resolution.

Quantitative phase-contrast microscopy by angular spectrum digital holography

Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating the holographic optical field is seen to have several advantages over the more commonly used Fresnel transformation or Huygens convolution method. Spurious noise and interference components can be tightly controlled through the analysis and filtering of the angular spectrum. The reconstruction distance does not have a lower limit and the off-axis angle between the object and reference can be lower than the Fresnel requirement and still be able to cleanly separate out the zero-order background. Holographic phase images are largely immune from the coherent noise common in amplitude images. Together with the use of a miniature pulsed laser, the resulting images have 0.5μm diffraction-limited lateral resolution and the phase profile is accurate to about 30 nm of optical path length. SKOV-3 (ovarian cancer cells) and HUVEC (human umbilical vein endothelial cells) are imaged that display intra-cellular and intra-nuclear organelles with clarity and quantitative accuracy. The technique clearly exceeds currently available methods in phase-contrast optical microscopy in the level of resolution and detail, and provides a new modality for imaging morphology of cellular and intracellular structures that is not currently available.

Phase contrast movies of cell migration by multi-wavelength digital holography

Quantitative phase unwrapped movies of cell migration are generated by multi-wavelength phase imaging digital holography. Two or more wavelengths are used for simultaneous illumination of the cells in the holographic system.

Phase Contrast Movies of Cell Migration by Multi-Wavelength Digital Holography

Quantitative phase unwrapped movies of cell migration are generated by phase imaging digital holography. Two or more wavelengths are used for simultaneous illumination of the cells and real-time acquisition of holographic images.

Transmission digital holography microscopy applied to the study of coal palynofacies

For the first time, transmission digital holography microscopy is applied to observe coal palynofacies, which are organic fossil microcomponents contained in the coal grains. The recorded holograms were produced by using microscope lenses with 20x and 40x of lateral magnification respectively, and He-Ne laser of wavelength 594.5 nm. The results show that reflection digital holography microscopy is required for observing relative opaque particles, because the phase recovery is strong diminished by light transmission in those cases. On the other hand, the phase distribution is related to the relief of the particles and the variations of their refraction index. Therefore, a priori information should be necessary to properly relate the phase information to physical features of the particles. Numerical unwrapping procedures are also crucial. Procedures with special requirements can be needed for analysing fast varying phase distributions. However, digital holography microscopy becomes a high performance tool for 3D modelling of fossil particles if the above requirements are enough fulfilled.

Phase-imaging digital holographic movies of animal cells

Multi-wavelength phase imaging digital holography is applied in acquiring holographic movies of animal microbes. Two or three lasers of different wavelengths are used to generate movies of phase maps without 2 pi discontinuities.

Digital Gabor holography for particle field imaging

We describe the digital Gabor holography as an effective method for particle field and cellular imaging. It has a particularly simple optical set up and acquisition procedure.