Tracking particles in four dimensions with in-line holographic microscopy

We describe a simple holographic method that has enabled us to capture as a single data set the trajectories of micrometer-sized objects suspended in water. By subtracting consecutive holograms of a particle suspension and then adding these difference holograms, we constructed a final data set that contains the time evolution of the particle trajectories free from spurious background interference effects. The method is illustrated by a recording of the motion of 5-10-microm diameter algae in water.     

Automatic three-dimensional tracking of particles with high-numerical-aperture digital lensless holographic microscopy

We present an automatic procedure for 3D tracking of micrometer-sized particles with high-NA digital lensless holographic microscopy. The method uses a two-feature approach to search for the best focal planes and to distinguish particles from artifacts or other elements on the reconstructed stream of the holograms. A set of reconstructed images is axially projected onto a single image. From the projected image, the centers of mass of all the reconstructed elements are identified. Starting from the centers of mass, the morphology of the profile of the maximum intensity along the reconstruction direction allows for the distinguishing of particles from others elements. The method is tested with modeled holograms and applied to automatically track micrometer-sized bubbles in a sample of 4 mm3 of soda.     

Three-dimensional holographic fluorescence microscopy

Most commonly used methods for three-dimensional (3D) fluorescence microscopy make use of sectioning techniques that require that the object be physically scanned in a series of two-dimensional (2D) sections along the z axis. The main drawback in these approaches is the need for these sequential 2D scans. An alternative approach to fluorescence imaging in three dimensions has been developed that is based on optical scanning holography. This novel approach requires only a 2D scan to record 3D information. Holograms of 15-microm fluorescent latex beads with longitinal separation of ~2 mm have been recorded and reconstructed. To our knowledge, this is the first time holograms of fluorescent specimens have been recorded by an optical holographic technique.     

Common-path phase-shifting lensless holographic microscopy

We present an approach capable of high-NA imaging in a lensless digital in-line holographic microscopy layout even outside the Gabor's regime. The method is based on spatial multiplexing at the sample plane, allowing a common-path interferometric architecture, where two interferometric beams are generated by a spatial light modulator (SLM) prior to illuminating the sample. The SLM allows phase-shifting interferometry by phase modulation of the SLM diffracted beam. After proper digital processing, the complex amplitude distribution of the diffracted object wavefront is recovered and numerically propagated to image the sample. Experimental results are reported that validate the proposed method.     

Immersion digital in-line holographic microscopy

Digital in-line holographic microscopy is a promising new tool for high resolution imaging. We demonstrate, by using latex beads, that a considerable increase in numerical aperture, and, therefore, resolution can be achieved if the space between a source and a CCD camera chip is filled with a high refractive index medium. The high refractive index medium implies a shorter effective wavelength so that submicrometer resolution can be obtained with laser light in the visible range.     

Multiframe full-field heterodyne digital holographic microscopy

Digital holographic microscopy using multiframe full-field heterodyne technology is discussed in which two acousto-optic modulators are applied to generate low-frequency heterodyne interference and a high-speed camera is applied to acquire multiframe full-field holograms. We use a temporal frequency spectrum analysis algorithm to extract the object's information. The twin-image problem can be solved and the random noise can be significantly suppressed. The relationship between the frame number and the reconstruction accuracy is discussed. The typical objects of microlenses and biology cells are reconstructed well with 100-frame holograms for illustration.     

Dual plane in-line digital holographic microscopy

We report a dual plane in-line digital holographic microscopy technique that exploits the method of subtraction of average intensity of the entire hologram to suppress the zero-order diffracted wave. Two interferograms are recorded at different planes to eliminate the conjugate image. The experimental results demonstrate successful reconstruction of phase objects as well as of amplitude objects. The two interferograms can be recorded simultaneously, using two CCD or CMOS sensors, in order to increase the acquisition rate. This enhanced acquisition rate, together with the improved reconstruction capability of the proposed method, may find applications in biomedical research for visualization of rapid dynamic processes at the cellular level.     

Synthetic aperture fourier holographic optical microscopy

We report a new synthetic aperture optical microscopy in which high-resolution, wide-field amplitude and phase images are synthesized from a set of Fourier holograms. Each hologram records a region of the complex two-dimensional spatial frequency spectrum of an object, determined by the illumination field's spatial and spectral properties and the collection angle and solid angle. We demonstrate synthetic microscopic imaging in which spatial frequencies that are well outside the modulation transfer function of the collection optical system are recorded while maintaining the long working distance and wide field of view.     

Reflected light holographic microscopy of moving objects

A holographic system that images front-illuminated, fast-moving microscopic objects is described. Focused micrographs can be generated under circumstances when ordinary microscopy (due to object movements) and transilluminated holographic microscopy (due to object opaqueness) cannot be used. Details of the experimental arrangements, easy-to-use working formulae for obtaining optimum image reproduction, and results from the application of the system to studies of droplet and solid particle suspensions in liquids are presented.     

Off-axis compressed holographic microscopy in low-light conditions

This Letter reports a demonstration of off-axis compressed holography in low-light level imaging conditions. An acquisition protocol relying on a single exposure of a randomly undersampled diffraction map of the optical field, recorded in the high heterodyne gain regime, is proposed. The image acquisition scheme is based on compressed sensing, a theory establishing that near-exact recovery of an unknown sparse signal is possible from a small number of nonstructured measurements. Image reconstruction is further enhanced by introducing an off-axis spatial support constraint to the image estimation algorithm. We report accurate experimental recovering of holographic images of a resolution target in low-light conditions with a frame exposure of 5 μs, scaling down measurements to 9% of random pixels within the array detector.     

Optimized coherence parameters for high-resolution holographic microscopy

A holographic in-line microscope setup with a glass sample carrier commonly uses a coherent laser light source, with the disadvantage of an incomplete suppression of disturbing interferences and coherence-induced noise.     

Polarization phase shifting in digital holographic microscopy

In the present work we have made use of polarization phase shifting in digital holographic microscopy (DHM) for three dimensional phase profiling of transmissive and reflecting microscopic samples. The Mach–Zehnder arrangement with proper polarizing elements (polarizer-masked cube beam splitter, quarter wave plate and a linear polarizer) is used for recording the phase-shifted digital holograms. The suggested procedure is simple and accurate and obviates the need of piezo devices for phase shifting. The phase profile of the specimen is reconstructed from the holograms by using standard phase shifting algorithms.     

Confocal microscopy with a volume holographic filter

We describe a modified confocal microscope in which depth discrimination results from matched filtering by a volume hologram instead of a pinhole filter. The depth resolution depends on the numerical aperture of the objective lens and the thickness of the hologram, and the dynamic range is determined by the diffraction efficiency. We calculate the depth response of the volume holographic confocal microscope, verify it experimentally, and present the scanned image of a silicon wafer with microfabricated surface structures.     

Digital holographic shape measurement using Fizeau microscopy

We present a Fizeau interferometer using a microscopic objective as a tool for surface contouring without the need for a numerical lens for reconstruction. The interferometer is associated with a telescope system to feature the object with collimated light. The experiment is conducted on two objects possessing different step heights.The phase maps from the captured off-axis holograms are calculated numerically, which allows us to deduce the contours of the objects. The great advantages of the presented technique are that it can be done in real time and there is no need for numerical lenses for micro-objects reconstruction.     

预放大数字全息显微系统的特点

为了提高预放大数字全息显微系统的成像质量,采用理论分析与实验验证相结合的方法,分别对采用平面及球面参考光记录的预放大数字全息显微系统进行了研究和比较。结果表明：在通常的实验条件下,系统的横向分辨力主要取决于显微物镜的成像分辨力;记录距离较小时,两种系统的横向分辨力均随着记录距离的增大略有降低;但当记录距离较大时,球面参考光预放大数字全息系统的横向分辨力降低得更为明显,即平面参考光预放大数字全息显微系统较为优越;在记录距离为0的情况下,即像面数字全息成像情况下,两种系统的再现像均具有最高的分辨力,在利用普通工业用传感器条件下,横向分辨力远超过了2.19 m,且像质较好。因此,尽可能减小全息图的记录距离,或者采用像面数字全息系统,可以有效提高数字全息系统的成像分辨力。     

Time-resolved digital holographic microscopy of laser-induced forward transfer

We develop a method for time-resolved digital holographic microscopy to obtain time-resolved 3-D deformation measurements of laser-induced forward transfer (LIFT) processes. We demonstrate nanometer axial resolution and nanosecond temporal resolution of our method which is suitable for measuring dynamic morphological changes in LIFT target materials. Such measurements provide insight into the early dynamics of the LIFT process and a means to examine the effect of laser and material parameters on LIFT process dynamics.     

预放大数字全息显微系统的特点

为了提高预放大数字全息显微系统的成像质量,采用理论分析与实验验证相结合的方法,分别对采用平面及球面参考光记录的预放大数字全息显微系统进行了研究和比较。结果表明:在通常的实验条件下,系统的横向分辨力主要取决于显微物镜的成像分辨力;记录距离较小时,两种系统的横向分辨力均随着记录距离的增大略有降低;但当记录距离较大时,球面参考光预放大数字全息系统的横向分辨力降低得更为明显,即平面参考光预放大数字全息显微系统较为优越;在记录距离为0的情况下,即像面数字全息成像情况下,两种系统的再现像均具有最高的分辨力,在利用普通工业用传感器条件下,横向分辨力远超过了2.19 m,且像质较好。因此,尽可能减小全息图的记录距离,或者采用像面数字全息系统,可以有效提高数字全息系统的成像分辨力。     

Methods of Fourier optics in digital holographic microscopy

In this paper, processing methods of Fourier optics implemented in a digital holographic microscopy system are presented. The proposed methodology is based on the possibility of the digital holography in carrying out the whole reconstruction of the recorded wave front and consequently, the determination of the phase and intensity distribution in any arbitrary plane located between the object and the recording plane. In this way, in digital holographic microscopy the field produced by the objective lens can be reconstructed along its propagation, allowing the reconstruction of the back focal plane of the lens, so that the complex amplitudes of the Fraunhofer diffraction, or equivalently the Fourier transform, of the light distribution across the object can be known. The manipulation of Fourier transform plane makes possible the design of digital methods of optical processing and image analysis. The proposed method has a great practical utility and represents a powerful tool in image analysis and data processing. The theoretical aspects of the method are presented, and its validity has been demonstrated using computer generated holograms and images simulations of microscopic objects.     

Digital holographic imaging of aerosol particles in flight

This work describes the design and application of an apparatus to image aerosol particles using digital holography in a flow-through, contact-free manner. Particles in an aerosol stream are illuminated by a triggered, pulsed laser and the pattern produced by the interference of this light with that scattered by the particles is recorded by a digital camera. The recorded pattern constitutes a digital hologram from which an image of the particles is computationally reconstructed using a fast Fourier transform. This imaging is validated using a cluster of ragweed pollen particles. Examples involving mineral-dust aerosols demonstrate the technique's in situ imaging capability for complex-shaped particles over a size range of roughly 15-500 μm micrometers. The focusing-like character of the reconstruction process is demonstrated using a NaCl aerosol particle and is compared to a similar particle imaged with a conventional microscope.