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In this chapter we present an approach to high spatial and temporal resolution three-dimensional (3-D) fluorescence imaging of living cells. This approach integrates sensitive CCD cameras, wide field epifluorescence microscopes and a computational method, image restoration, to obtain 3-D images with a lateral resolution of 100 nanometers, which is better than the resolution that can be achieved by either confocal fluorescence microscopy or conventional epifluorescence microscopy without image restoration. The optical and detector efficiency of the system allows us to obtain a time series of many 3-D images from the same cell with minimal photobleaching. When combined with the high speed camera and rapid focus drive described in figure 1, a 3-D image of a cell can be acquired in only 24 milliseconds.

The temporal carrier beat frequencies in an optical heterodyne interferometer can be generated by ramping the wavelength in a laser diode (LD) based on the frequency-modulated continuous-wave (FMCW) techniques [1,2]. The beat frequency is proportional to the optical path difference (OPD) of each pair of interfering beams in an interferometer [3]. It enables us to construct an optical heterodyne interferometer on an unbalanced OPD without auxiliary frequency modulators. Multiple-beam interferometry can produce the different beat frequencies by using a wavelength-tunable LD [4]. An LD interferometer has been applied to 3-D imaging by using the optical frequency domain reflectometry [5]. A technique of a holographic radar has been applied to measure the range information with a frequency-tunable laser [6]. 3-D object can be holographically reconstructed by the electronic tuning with beat signals by a current-modulated LD [7]. A phase-shifting common-path interferometer has been demonstrated with a biprism beam splitter [8].

Is there a way to see an object obscured by a strong diffuser such as a transmissive ground glass or a reflective opaque surface? Such a question has long been addressed in the context of inverse scattering problems [1], and a technique has been known that can detect a 2-D periodic grating structure hidden by a diffuser [2]. Recently a technique of ultrafast time-of-flight 3-D imaging that can look around a corner using diffusely reflected light was demonstrated [3]. Also a SLM-based technique that compensates the random phase and permits imaging a 3-D object through a diffuser has been reported [4].

Biomedical research truly needs new advances in imaging. Existing modalities of in vivo imaging, such as magnetic resonance imaging or ultrasound, lack the spatiotemporal resolution required to image the fundamental building block of living tissue. By contrast, existing high-resolution techniques for imaging cells and their sub-cellular features are technologies that are best suited for in vitro experiments in tissue slices. Yet, the ability to make direct connections between human pathological symptoms/behavior and the underlying cells and molecules responsible for such behavior requires in vivo techniques that can image cellular constituents. Our group research aim is to develop novel high-resolution optical endoscopes to satisfy unmet needs in the clinical environment. These differ from medical endoscopes, which are generally larger and designed to image macroscopic abnormalities. Most importantly, this novel optical endoscopic imaging might suggest new approaches to disease diagnosis and treatment. This talk will be focused on the development a novel confocal imaging modality integrated with microelectromechanical systems (MEMS) technology and their imaging applications. Confocal microscopy is an attractive tool for three-dimensional (3-D) imaging due to its optical sectioning property. Conventional single-axis confocal (SAC) microscopes have a tradeoff between resolution, field of view, and objective lens size, since a high numerical aperture (NA) lens is needed for sufficient resolution, and a long focal length is needed for a large FOV and working distance. A dual-axes confocal (DAC) microscope architecture has been proposed utilizing two overlapping low NA beams, which effectively decouples these tradeoffs. The other important advantage is the ability to achieve a much superior optical sectioning compared to the SAC design. The microscopes are miniaturized into two form factors (5 mm and 10 mm diameter). The imaging demonstrations of the probes were on both ex vivo and in vivo from mice and human for cancer oncology and genetic research.

Point source digital in-line holography with numerical reconstruction has been developed into a new microscopy, specifically for microfluidic and biological applications, that routinely achieves both lateral and depth resolution at the submi- cron level in 3-D imaging. This review will cover the history of this field and give details of the theoretical and experimental background. Numerous examples from microfluidics and biology will demonstrate the capabilities of this new microscopy. The motion of many objects such as living cells in water can be tracked in 3-D at subsecond rates. Microfluidic applications include sedimentation of suspensions, fluid motion around micron-sized objects in channels, motion of spheres, and forma- tion of bubbles. Immersion DIHM will be reviewed which effectively does holog- raphy in the UV. Lastly, a submersible version of the microscope will be introduced that allows the in situ study of marine life in real time in the ocean and shows images and films obtained in sea trials.

In this chapter we present three types of microscopy-related configurations while the first one is used for 3-D movement monitoring of the inspected samples, the second one is used for super-resolved 3-D imaging, and the last one presents an overview digital holographic microscopy applications. The first configuration is based on temporal tracking of secondary reflected speckles when imaged by properly defocused optics. We validate the proposed scheme by using it to monitor 3-D spontaneous contraction of rat’s cardiac muscle cells while allowing nanometric tracking accuracy without interferometric recording. The second configuration includes projection of temporally varying speckle patterns on top of the sample and by proper decoding exceeding the diffraction as well as the geometrical-related lateral resolution limitation. In the final part of the chapter, we overview applications of digital holographic microscopy (DHM) for real-time non-invasive 3-D sensing, tracking, and recognition of living microorganisms such as single- or multiple-cell organisms and bacteria.

Electron Tomography (ET) is uniquely suited to obtain three-dimensional (3-D) images of large pleiomorphic structures, such as supramolecular assemblies, organelles, or even whole cells. While the principles of ET have been known for decades, its use has gathered momentum only in recent years. Technological advances (namely computer controlled transmission electron microscopes and large area CCD cameras) have made it possible to develop automated data acquisition procedures. This, in turn, allowed to reduce the total electron dose to levels low enough for studying radiation sensitive biological materials embedded in vitreous ice. As a result, we are now poised to combine the power of high-resolution 3-D imaging with the best possible preservation of the specimen.

By making consistent use of the unique properties of synchrotron radiation it will be possible to push the resolution limit of X-ray tomography down to the sub-micron level. As exemplary steps of this development, the nondestructive 3-D imaging of alloy precipitates in Al, the imaging of the catalytic material in a supported catalyst, and the use of x-ray optical magnification are described.

Single Photon Emission Computed Tomography (SPECT) is a diagnostic 3-D imaging procedure of isotope distribution inside an object following internal administration of the isotopes that emit single photons such as Tc-99m (140 KeV) (1,2). The physical operation of SPECT stands on the basic 2-D computed tomography (CT) concept of projection data collection from an object slice over 2 π angle, and subsequent reconstruction process of filtered backprojection. A final 3-D volume image is formed by a stack of contiguous 2-D slice images reconstructed. Projection data of isotope distribution can be collected by either a stack of coplanar arrays of small discrete detectors or two-dimensional position-sensitive detectors such as Anger Scintillation gamma cameras.

Bright, spatially coherent soft x-ray sources are now becoming available and high-resolution imaging of biological specimens by x-ray holography has been discussed as an exciting application for the new sources (Robinson, 1985; Underwood and Attwood, 1984; Solem and Baldwin, 1982; Solem and Chapline, 1984; Matthews et al, 1985). However, even with fully coherent sources, there are some severe limitations for 3-D imaging which make it questionable if x-ray holography will ever be of any practical use for the imaging of microscopic objects. The purpose of this note is to point out these difficulties and speculate about possibilities to alleviate the problem.