Changgeng Liu

Review of digital holographic microscopy for three-dimensional profiling and tracking

Abstract. Digital holographic microscopy (DHM) is a potent tool to perform three-dimensional imaging and tracking. We present a review of the state-of-the-art of DHM for three-dimensional profiling and tracking with emphasis on DHM techniques, reconstruction criteria for three-dimensional profiling and tracking, and their applications in various branches of science, including biomedical microscopy, particle imaging velocimetry, micrometrology, and holographic tomography, to name but a few. First, several representative DHM configurations are summarized and brief descriptions of DHM processes are given. Then we describe and compare the reconstruction criteria to obtain three-dimensional profiles and four-dimensional trajectories of objects. Details of the simulated and experimental evidences of DHM techniques and related reconstruction algorithms on particles, biological cells, fibers, etc., with different shapes, sizes, and conditions are also provided. The review concludes with a summary of techniques and applications of three-dimensional imaging and four-dimensional tracking by DHM.

Measurement of the traction force of biological cells by digital holography

The traction force produced by biological cells has been visualized as distortions in flexible substrata. We have utilized quantitative phase microscopy by digital holography (DH-QPM) to study the wrinkling of a silicone rubber film by motile fibroblasts. Surface deformation and the cellular traction force have been measured from phase profiles in a direct and straightforward manner. DH-QPM is shown to provide highly efficient and versatile means for quantitatively analyzing cellular motility.

Four-dimensional motility tracking of biological cells by digital holographic microscopy

Abstract. Three-dimensional profiling and tracking by digital holography microscopy (DHM) provide label-free and quantitative analysis of the characteristics and dynamic processes of objects, since DHM can record real-time data for microscale objects and produce a single hologram containing all the information about their three-dimensional structures. Here, we have utilized DHM to visualize suspended microspheres and microfibers in three dimensions, and record the four-dimensional trajectories of free-swimming cells in the absence of mechanical focus adjustment. The displacement of microfibers due to interactions with cells in three spatial dimensions has been measured as a function of time at subsecond and micrometer levels in a direct and straightforward manner. It has thus been shown that DHM is a highly efficient and versatile means for quantitative tracking and analysis of cell motility.

Quantitative phase-contrast confocal microscope

We present a quantitative phase-contrast confocal microscope (QPCCM) by combining a line-scanning confocal system with digital holography (DH). This combination can merge the merits of these two different imaging modalities. High-contrast intensity images with low coherent noise, and the optical sectioning capability are made available due to the confocality. Phase profiles of the samples become accessible thanks to DH. QPCCM is able to quantitatively measure the phase variations of optical sections of the opaque samples and has the potential to take high-quality intensity and phase images of non-opaque samples such as many biological samples. Because each line scan is recorded by a hologram that may contain the optical aberrations of the system, it opens avenues for a variety of numerical aberration compensation methods and development of full digital adaptive optics confocal system to emulate current hardware-based adaptive optics system for biomedical imaging, especially ophthalmic imaging. Preliminary experiments with a microscope objective of NA 0.65 and 40 × on opaque samples are presented to demonstrate this idea. The measured lateral and axial resolutions of the intensity images from the current system are ~0.64μm and ~2.70μm respectively. The noise level of the phase profile by QPCCM is ~2.4nm which is better than the result by DH.

Fourier transform digital holographic adaptive optics imaging system

A Fourier transform digital holographic adaptive optics imaging system and its basic principles are proposed. The CCD is put at the exact Fourier transform plane of the pupil of the eye lens. The spherical curvature introduced by the optics except the eye lens itself is eliminated. The CCD is also at image plane of the target. The point-spread function of the system is directly recorded, making it easier to determine the correct guide-star hologram. Also, the light signal will be stronger at the CCD, especially for phase-aberration sensing. Numerical propagation is avoided. The sensor aperture has nothing to do with the resolution and the possibility of using low coherence or incoherent illumination is opened. The system becomes more efficient and flexible. Although it is intended for ophthalmic use, it also shows potential application in microscopy. The robustness and feasibility of this compact system are demonstrated by simulations and experiments using scattering objects.

Quantitative imaging and measurement of cell–substrate surface deformation by digital holography

Quantitative phase microscopy by digital holography (DH-QPM) is introduced to study the cell–substrate interactions and migratory behavior of adhesive cells. A non-wrinkling elastic substrate, collagen-coated polyacrylamide (PAA) has been employed and its surface deformation due to cell adhesion and motility has been visualized as certain tangential and vertical displacement and distortion. The surface deformation on substrates of different elasticity and thickness has been quantitatively imaged and the corresponding cellular traction force of motile fibroblasts has been measured from phase profiles by DH-QPM. DH-QPM is able to yield quantitative measures directly and provide efficient and versatile means for quantitatively analyzing cellular motility.

Digital adaptive optics line-scanning confocal imaging system

Abstract. A digital adaptive optics line-scanning confocal imaging (DAOLCI) system is proposed by applying digital holographic adaptive optics to a digital form of line-scanning confocal imaging system. In DAOLCI, each line scan is recorded by a digital hologram, which allows access to the complex optical field from one slice of the sample through digital holography. This complex optical field contains both the information of one slice of the sample and the optical aberration of the system, thus allowing us to compensate for the effect of the optical aberration, which can be sensed by a complex guide star hologram. After numerical aberration compensation, the corrected optical fields of a sequence of line scans are stitched into the final corrected confocal image. In DAOLCI, a numerical slit is applied to realize the confocality at the sensor end. The width of this slit can be adjusted to control the image contrast and speckle noise for scattering samples. DAOLCI dispenses with the hardware pieces, such as Shack–Hartmann wavefront sensor and deformable mirror, and the closed-loop feedbacks adopted in the conventional adaptive optics confocal imaging system, thus reducing the optomechanical complexity and cost. Numerical simulations and proof-of-principle experiments are presented that demonstrate the feasibility of this idea.

Phase aberration correction by correlation in digital holographic adaptive optics

We present a phase aberration correction method based on the correlation between the complex full-field and guide-star holograms in the context of digital holographic adaptive optics (DHAO). Removal of a global quadratic phase term before the correlation operation plays an important role in the correction. Correlation operation can remove the phase aberration at the entrance pupil plane and automatically refocus the corrected optical field. Except for the assumption that most aberrations lie at or close to the entrance pupil, the presented method does not impose any other constraints on the optical systems. Thus, it greatly enhances the flexibility of the optical design for DHAO systems in vision science and microscopy. Theoretical studies show that the previously proposed Fourier transform DHAO (FTDHAO) is just a special case of this general correction method, where the global quadratic phase term and a defocus term disappear. Hence, this correction method realizes the generalization of FTDHAO into arbitrary DHAO systems. The effectiveness and robustness of this method are demonstrated by simulations and experiments.

Ophthalmic adaptive optics by digital holography

We are developing adaptive optics systems for aberration corrections in retinal imaging based on digital holography. Compared to existing technologies of adaptive optics, our systems do not have hardware components such as lenslet arrays or deformable mirrors. Instead, wavefront sensing and correction are done by acquisition and numerical manipulation of optical phase by digital holography, thereby substantially reducing hardware complexity and introducing novel imaging capabilities. Experimental results are presented to demonstrate capabilities of this novel imaging system.

Holographic Line-Scanning Confocal Microscope

A new imaging system is proposed by combining line-scanning confocal microscope with digital holography. By this combination, the proposed system will posses characteristics of these two imaging technologies, such as low coherent noise, optical sectioning and accessibility to complex optical field.