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.     

结合线性回归的离轴数字全息去载波相位恢复算法

为实现仅用一幅离轴数字全息图便能直接恢复相位,提出一种利用空间载波相移技术(spatial carrier phase shift, SCPS)和线性回归相结合的离轴数字全息去载波相位恢复算法.首先,利用SCPS将一幅离轴数字全息图分为四幅含有载波相移的全息图,其中载波相移由沿行、列两个方向的正交载波所引入;然后,将四幅载波相移全息图作为输入,将所求物体相位和两个正交的载波作为未知量,结合最小二乘法和线性回归同时求出载波和相位信息.相较于已有的去载波技术,本算法无需背景全息图作为参考,便可准确地去除载波,实现高质量的相位重建.本文结合数值仿真和具体实验结果验证本算法的有效性和优越性.     

Fluorescence of bean leaves grown under low-light conditions

The patterns of changes of the spectral shapes of the fluorescence of bean leaves have been identified under low-light conditions of plant cultivation and decrease in chlorophyll concentration in the leaves. Under these experimental conditions, the ratio of peak intensities of fluorescence in the red spectral region, ω = F ₇₄₀/F ₇₆₀, which was recorded after the end of the induction period, was proportional to the chlorophyll content per 1 g of the wet weight of a leaf.     

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.     

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.     

Off-axis collimation of diode laser beams by means of single-element holographic diffractive optics

Principles of single-element holographic diffractive optics for collimation of diode laser beams with a large divergence, an elliptic cross-section, and astigmatism are presented. Holographic off-axis transformation enables collimation of the beam in two variants: one with a perpendicular input beam and an oblique output beam, and the other with the beams arranged vice-versa. Diffraction due to an elliptic aperture is analyzed. Inspection of experimental samples demonstrates an agreement with theory in the case of diffraction limited focal pattern and shows increase of astigmatism with the departure from diffraction only limitation.     

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.     

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.     

Heterodyne holographic microscopy of gold particles

We report experimental results on heterodyne holographic microscopy of subwavelength-size gold particles. The apparatus uses continuous green-laser illumination of the metal beads in a total internal reflection configuration for dark-field operation. Detection of the scattered light at the illumination wavelength on a charge-coupled-device array detector enables 3D localization of brownian particles in water.     

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.     

Phase reconstruction in lensless digital in-line holographic microscopy

The phase reconstruction in a digital in-line holographic microscopy is compared using two numerical reconstruction methods. The first method uses one Fourier transform and second one uses three Fourier transforms. It is shown that the latter method gives improved object phase reconstruction as compared to the former.     

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.     

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.     

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.     

Resolution enhancement in digital holographic microscopy with structured illumination

We present a digital holographic microscope wherein the sample is illuminated by structured light to enable the capture of additional object spatial frequencies. Reconstructed images with increased spatial resolution are obtained by separating and synthesizing bandwidths of different frequency regions in the Fourier domain. The theoretical analysis and experimental results are presented.     

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.     

Physical spherical phase compensation in reflection digital holographic microscopy

Reflection configured digital holographic microscopy (DHM) can perform accurate optical topography measurements of reflecting objects, such as MEMs, MOEMs, and semiconductor wafer. It can provide non-destructive quantitative measurements of surface roughness and geometric pattern characterization with nanometric axial resolution in real-time. However, the measurement results may be affected by an additional phase curvature introduced by the microscope objective (MO) used in DHM. It needs to be removed either by numerical compensation or by physical compensation.We present a method of physical spherical phase compensation for reflection DHM in the Michelson configuration. In the object arm, collimated light is used for illumination. Due to the use of the MO, the object wavefront may have a spherical phase curvature. In the reference arm, a lens and mirror combination is used to generate a spherical recording reference wave in order to physically compensate the spherical phase curvature of the object wavefront. By controlling the position of the mirror and the sample stage, the compensation process has been demonstrated. The relative positions of the test specimen and the reference mirror must be fixed for the physical spherical phase to be totally compensated. A numerical plane reference wave is preferred for the numerical reconstruction of the phase introduced by the test specimen. Experimental results on wafer pattern recognition are also given.