Coherent Light Microscopy

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 submicron 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 formation of bubbles. Immersion DIHM will be reviewed which effectively does holography 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.

We investigate the use of partially spatial coherent illuminations for digital holographic microscopes (DHMs) working in transmission. Depending on the application requirements, the sources are made from a spatially filtered LED or from a decorrelated laser beam. The benefits gained with those sources are indicated. A major advantage is the drastic reduction of the speckle noise making possible high image quality and the proper emulation of phase contrast modes such as differential interference contrast (DIC). For biomedical applications, the DHMs are coupled with fluorescence sources to achieve multimodal diagnostics. Several implementations of biomedical applications where digital holography is a significant improvement are described. With a fast DHM permitting the analysis of dynamical phenomena, several applications in fluid physics and biomedical applications are also provided.

In this chapter we show how digital holography (DH) can be efficiently used as a free and noninvasive investigation tool capable of performing quantitative and qualitative mapping of biological samples. A detailed description of the recent methods based on the possibility offered by DH to numerically manage the reconstructed wavefronts is reported. Depending on the sample and on the investigation to be performed it is possible, by a single acquired image, to recover information on the optical path length changes and, choosing the more suitable numerical method, to obtain quantitative phase map distributions. The progress achieved in the reconstruction methods will certainly find useful applications in the field of the biological analysis.

Holography is an attractive imaging technique as it offers the ability to view a complete three-dimensional volume from one image. However, holography is not widely applied to the regime of fluorescence microscopy, because fluorescent light is incoherent and creating holograms requires a coherent interferometer system. We review two methods of generating digital Fresnel holograms of three-dimensional microscopic specimens illuminated by incoherent light. In the first method, a scanning hologram is generated by a unique scanning system in which Fresnel zone plates (FZP) are created by a coherently illuminated interferometer. In each scanning period, the system produces an on-axis Fresnel hologram. The twin image problem is solved by a linear combination of at least three holograms taken with three FZPs with different phase values. The second hologram reviewed here is the Fresnel incoherent correlation hologram. In this motionless holographic technique, light is reflected from the 3-D specimen, propagates through a spatial light modulator (SLM), and is recorded by a digital camera. Three holograms are recorded sequentially, each for a different phase factor of the SLM function. The three holograms are superposed in the computer, such that the result is a complex-valued Fresnel hologram that does not contain a twin image. When these two types of hologram are reconstructed in the computer, the 3-D properties of the specimen are revealed.

Microlens arrays are of fundamental importance in a wide variety of applications in optics and photonics. This chapter deals with an accurate digital holography-based characterization of both liquid and polymeric microlenses fabricated by an innovative pyro-electrowetting process. The actuation of liquid and polymeric films is obtained through the use of pyroelectric charges generated into polar dielectric lithium niobate crystals.

In this chapter, we present a new method capable of recovery of the quantitative phase information of microscopic samples. Essentially, a spatial light modulator (SLM) and digital image processing are the basics to extract the sample’s phase distribution. The SLM produces a set of misfocused images of the input sample at the CCD plane by displaying a set of lenses with different power at the SLM device. The recorded images are then numerically processed to retrieve phase information. Computations are based on the wave propagation equation and lead to a complex amplitude image containing information of both amplitude and phase distributions of the input sample diffracted wave front. The proposed configuration becomes a non-interferometric architecture (conventional transmission imaging mode) where no moving elements are included. Experimental results are provided in comparison with conventional digital holographic microscopy.

Interferometric phase measurements of wide-field images of biological cells provide a quantitative tool for cell biology, as well as for medical diagnosis and monitoring. Visualizing rapid dynamic cell phenomena by interferometric phase microscopy can be performed at very fast rates of up to several thousands of full frames per second, while retaining high resolution and contrast to enable measurements of fine cellular features. With this approach, no special sample preparation, staining, or fluorescent labeling is required, and the resulting phase profiles yield the optical path delay profile of the cell with sub-nanometer accuracy. In spite of these unique advantages, interferometric phase microscopy has not been widely applied for recording the dynamic behavior of live cells compared to other traditional phase microscopy methods such as phase contrast and differential interference contrast (DIC) microscopy, which are label free but inherently qualitative. Recent developments in the field of interferometric phase microscopy are likely to result in a change in this situation in the near future. Through careful consideration of the capabilities and limitations of interferometric phase microscopy, important new contributions in the fields of cell biology and biomedicine will be realized. This chapter presents the current state of the art of interferometric phase microscopy of biological cell dynamics, the open questions in this area, and specific solutions developed in our laboratory.

Spectral domain phase microscopy (SDPM) is a functional extension of optical coherence tomography (OCT) using common-path interferometry to produce phase-referenced images of dynamic samples. Like OCT, axial resolution in SDPM is determined by the source coherence length, while lateral resolution is limited by diffraction in the microscope optics. However, the quantitative phase information SDPM generates is sensitive to nanometer-scale displacements of scattering structures. The use of a common-path optical geometry yields an imaging system with high phase stability. Due to coherence gating, SDPM can achieve full depth discrimination, allowing for independent motion resolution of subcellular structures throughout the sample volume. Here we review the basic theory of OCT and SDPM along with applications of SDPM in cellular imaging to measure topology, Doppler flow in single-celled organisms, time-resolved motions, rheological information of the cytoskeleton, and optical signaling of neural activation. Phase imaging limitations, artifacts, and sensitivity considerations are discussed.

Quantitative phase imaging (QPI) of live cells has received significant scientific interest over the past decade or so, mainly because it offers structure and dynamics information at the nanometer scale in a completely noninvasive manner. Fourier transform light scattering (FTLS) relies on quantifying the optical phase and amplitude associated with a coherent image field and propagating it numerically to the scattering plane. It combines optical microscopy, holography, and light scattering for studying inhomogeneous and dynamic media. We present recent developments of QPI technology and FTLS for biological system structure and dynamics study. Their applications are classified into static and dynamic according to their temporal selectivity. Several promising prospects are discussed in the summary section.

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.

Applications of phase microscopy based on either coherent or partially coherent sources are widely distributed in today's biological and biomedical research laboratories. But the quantitative phase information derivable from these techniques is often not fully understood, because in general, no universal theoretical model can be set up, and each of the techniques has to be treated specifically. This chapter is dedicated to the fundamental understanding of the methodologies that derive optical phase information using imaging techniques and microscopic instrumentation. Several of the latest and most significant techniques are thoroughly studied through the theoretical formalism of the optical transfer function. In particular, we classify these systems into two main categories: those based on coherent illumination, such as digital holographic microscopy (DHM) and its extension into tomography, and those based on partially coherent illumination, such as differential interference contrast (DIC) and differential phase contrast (DPC). Our intention is that the models described in this chapter give an insight into the behaviour of these phase imaging techniques, so that better instrumentation can be designed and improved phase retrieval algorithms can be devised.

In this chapter an approach using a 2D phase grating to enhance the resolution in digital holographic microscopy exploiting the electro-optic effect is proposed. We show that, by means of a tunable lithium niobate phase grating, it is possible to increase the numerical aperture of the imaging system, thus improving the spatial resolution of the images in two dimensions. The enhancement of the numerical aperture of the optical system is obtained by recording spatially multiplexed digital holograms. Furthermore, thanks to the flexibility of the numerical reconstruction process, it is possible to selectively use the diffraction orders carrying useful information for optimizing the diffraction efficiency and to increase the final spatial resolution.

In this chapter, we present two novel approaches for 3-D object shape measurement and range estimation based on digital image processing of speckle patterns. In the first one, 3-D mapping and range measurement are retrieved by projecting, through a ground glass diffuser, random speckle patterns on the object or on the camera for a transmissive and reflective configuration, respectively. Thus, the camera sensor records in time sequence different speckle patterns at different distances, and by using correlation operation between them, it is possible to achieve 3-D mapping and range finding. In the second one, the 3-D mapping and ranging are performed by sensing the visibility associated with the coherence function of a laser source used to illuminate the object. In this case, the object depth is encoded into the amplitude of the interference pattern when assembling a typical electronic speckle pattern interferometric (ESPI) layout. Thus, the 3-D object shape is reconstructed by means of a range image from the visibility of the image set of interferograms without the need for depth scanning. In both cases, we present experimental implementation validating the proposed methods.