We present a multimodal optical microscope that incorporates six imaging modalities on one common platform. The imaging modalities include three staring modes, optical quadrature microscopy (OQM), differential interference contrast (DIC) microscopy, and epi-fluorescence microscopy, and three scanning modes, confocal reflectance microscopy (CRM), confocal fluorescence microscopy (CFM), and two-photon microscopy (2PM). OQM reconstructs the amplitude and phase of an optically transparent specimen within a modified Mach-Zehnder configuration. DIC microscopy images the phase gradient along a specified direction of an optically transparent specimen. CRM detects index of refraction changes that modulate backscatter. Epi-fluorescence microscopy, CFM, and 2PM detect endogenous and exogenous fluorophores within a specimen. The scanning modes are inherently capable of producing three-dimensional (3-D) images due to optical sectioning and localized probing. Illumination and imaging are performed coaxially with minimal changes of optical components between modes. Multimodal images of embryos are shown to demonstrate the microscope's imaging capabilities.
A mulitmodal microscope provides a researcher with the ability to capture images with multiple sources of contrast. Previous attempts at multimodal microscopy have been limited to capturing images at different instruments and digitally registering the images empirically using features common in the specimen. Other multimodal microscopes have combined different microscopy techniques, but have been limited in their sources of contrast. We present a unique microscope which containes 5 different imaging modalities, with four different sources of contrast. Quadrature microscopy measures the phase of the electric field of the light transmitted through an optically transparent specimen, by interference with a reference beam, using a polarimetric technique to resolve the ambiguities between phase and amplitude. Differential Interference Contrast (DIC) measures the phase gradient in an optically transparent specimen by combining two spatially separated images interferometrically. Confocal reflectance measures index of refraction changes in a plane or reflection from small scatterers. Confocal fluorescence and 2-photon laser scanning microscopy, measure fluorescent signatures of a specimen. The last three of these are inherently capable of producing three-dimensional images directly, through localized probing.
The Quadrature Tomographic Microscope measures the amplitude and phase of an image. This information allows the user to see contrast features not available in other microscopes, and is critical to any three-dimensional reconstruction. We report on development and use of test objects to measure the accuracy and repeatability of phase measurements. A simple binary phase grating, a series of glass beads, and preimplantation mouse embryos were used in these experiments. The gratings were fabricated on high-quality fused-silica substrates whose transmission phase error was determined to be less than one-tenth wave error across their 25 mm diameter before fabrication. The phase step of the binary phase grating was measured using both the optical quadrature technique and the usual fringe-counting techniques applied to the raw data. Phase unwrapping techniques were validated by measuring the diameter of glass beads of a known size. Results are presented showing that the phase measurements agree with each other, with the known data, and with the spatial resolution in preimplantation mouse embryos. More complicated objects will be fabricated in the future to validate 3-D imaging techniques.
The photon diffusion approximation provides an accurate description for the propagation of light in a highly scattering medium such as most tissue. However, complications arise at the boundaries between such a highly scattering medium and a clear medium, as at the locations where the light enters and exits the medium. Several approximations have been developed to address the input of light from a laser through an optical fiber, and several others have been developed to address the collection of light by an optical fiber and detector. The principle of reciprocity suggests that source and detector fibers could be treated identically, resulting in a simplification of the diffusive optical model. Such a formulation agrees quite well with Monte-Carlo and other models. In the present work, we apply the technique to some different situations, including the important practical problem of source and detector fibers which are not aligned to be normal to the surface. Simple analytical results based on this model will be compared to Monte-Carlo results.
Acousto-photonic imaging is a novel technique for non- invasive medical imaging that combines diffusive optical tomography with externally generated acoustic `virtual' sources to improve the resulting images. We lack, however, a detailed understanding of the nature of the interaction between the diffusive wave and the focused ultrasound. We present our recent theoretical and experimental work on determining the mechanism for the interaction between the acoustic and the optical fields.
The interaction of ultrasound with diffusive optical waves holds the promise of improved resolution and depth of penetration for non-ionizing, non-invasive medical imaging. Light offers the ability to make spectroscopic measurements, which can monitor metabolism but, while it can propagate deeply into the body at near infrared wavelengths, it is so strongly scattered in most tissues that imaging is achieved only with measures such as time-resolved or frequency-domain techniques. On the other hand ultrasound has the ability to penetrate deeply with considerably less scattering. We have proposed a mechanism for imaging through the body by mixing diffusive optical waves with focussed ultrasound to produce new, so-called virtual diffusive sources. In the present work, we consider various mechanisms of interaction and report some results suggesting that the interaction in a laboratory phantom is predominantly the result of particle motion rather than variations in the index of refraction of the background medium. This result may be important in development of techniques to maximize the strengths of the virtual sources.
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