Digital In-line Holographic Microscopy Research Articles

Hologram reconstruction corrected for measurements through layers with different refractive indices in digital in-line holographic microscopy

Digital in-line holographic microscopy (DIHM) using point sources has been shown to be a versatile technique, especially for three-dimensional tracking of particles or microorganisms. However, the spherical source wave is altered when measurements are performed through layers with different refractive indices, such as water cuvettes. The situations where a layer of medium with a refractive index different than that of the predominant surrounding propagation medium (usually air) is situated behind or in front of the plane to be reconstructed are analyzed in detail, and a general approach for reconstruction under such circumstances is developed. The proposed refractive index correction is tested experimentally and compared to conventional reconstruction algorithms. Using 3D traces of swimming algal spores, the influence on the velocity calculation is also shown.

A Quantitative 3D Motility Analysis of Trypanosoma brucei by Use of Digital In-line Holographic Microscopy

We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming.

Partially coherent light-emitting diode illumination for video-rate in-line holographic microscopy

The light of a light-emitting diode or a common thermal source, such as a tungsten filament lamp, is known to be quasi-incoherent. We generated partially coherent light of these sources with a volume of coherence in the micrometer range of 5-100 μm3 by spatial and spectral filtering. The corresponding degree of partial coherence was adapted for microscopic interference setups, such as a digital in-line holographic microscope. The practicability of the sources was determined by the spectral emittance and the resulting signal-to-noise ratio (SNR) of the detector. The microscale coherence in correlation with the SNR and its resolution for microscopy were analyzed. We demonstrate how low-light-level, non-laser sources enable holographic imaging with a video frame rate (25 frames/s), an intermediate SNR of 8 dB, and a volume of coherence of 3.4×10(4) μm3. Holograms of objects with a lateral resolution of 1 μm were achieved using a microscope lens (50×/NA=0.7) and a CCD camera featuring a 4-12 bit dynamic range.

Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy

Point-source digital in-line holographic microscopy with numerical reconstruction is ideally suited for quantitative phase measurements to determine optical path lengths and to extract changes in refractive index within accuracy close to 0.001 on the submicrometer length scale. This is demonstrated with simulated holograms and with detailed measurements on a number of different micrometer-sized samples such as suspended drops, optical fibers, as well as organisms of biological interest such as E. coli bacteria, HeLa cells, and fibroblast cells.

Quantitative phase microscopy using dual-plane in-line digital holography

We present detailed theoretical evaluation and thorough experimental investigation of quantitative phase imaging using our previously demonstrated dual-plane in-line digital holographic microscopy technique [Opt. Lett. 35, 3426 (2010)]. This evaluation is based on the recording of two interferograms at slightly different planes and numerically reconstructing the object information. The zero-order diffracted wave is eliminated by using the method of subtraction of average intensity of the entire hologram, and the twin-image diffracted wave is removed by Fourier domain processing of the two recorded holograms. Experiments are performed using controlled amplitude and phase objects and human muscle cells to demonstrate the potential of this technique.

A Lensless, Automated Microscope for Disease Diagnostics

Optical microscopy is widely accepted as one of the gold standards in disease diagnosis. However, factors such as cost and the need for a trained eye limit the prevalence of such equipment, particularly in resource-limited areas such as rural clinics. Lensless microscopy, which is based on principles of digital holography, has illustrated the possibility of using simple and cheap optical components combined with software algorithms to implement microscope platforms. We present a digital in-line holographic microscope (DIHM) platform to be used with image processing and classification algorithms to provide a low cost, portable and automated microscope. Initial results show that the images obtained using the DIHM platform are similar to those obtained using a conventional bright field microscope. Applications of this work are targeted towards the implementation of a full blood count, which could provide resource-limited areas with improved healthcare facilities and diagnosis times.

Compact system design based on digital in-line holographic microscopy configuration

We present our study regarding a compact system design for cell counting and simultaneous 3D imaging, based on digital in-line holographic microscopy configuration. The system is built around the known experimental configuration which includes a pinhole but we also investigate the configuration with a monomode fiber as a light source. Considered samples consist of a very low concentration of cells in flow in a microchannel. The main challenge in our design is to obtain the digital hologram of one cell on a regular video camera sensor in proper resolution conditions, as opposed to the usual configurations where the aim is to visualize a large area. This fact is possible with shorter distances between pinhole and sample and with pinholes with diameters slightly larger than 1micron. These can now be realized by considering the microtechnological processes for microchannel and pinhole fabrication on the same substrate with high refractive index - to increase the numerical aperture of the system The geometrical parameters are established after the numerical analysis of the diffracted field from a single cell and of the entire system numerical aperture values.

Three-dimensional positioning of optically trapped nanoparticles

We firstly demonstrate the three-dimensional (3D) measurement of a nanometer-sized sphere held in optical tweezers in water using an in-line digital holographic microscope with a green light emitting diode. Suppressing the movement with optical tweezers enabled us to detect the three-dimensional position of a polystyrene sphere with a diameter of 200 nm. The positioning resolutions of the microscope were 3.2 nm in the transverse direction and 3.4 nm in the axial direction, from the standard deviation of measurements of the 200 nm sphere fixed on glass. Changes in the Brownian motion in response to a change in the trapping laser power were measured. We also demonstrated that this holographic measurement is an effective method for determining the threshold power of the optical trapping.

High-accuracy three-dimensional position measurement of tens of micrometers size transparent microspheres using digital in-line holographic microscopy

Digital in-line holographic microscopy has a strong potential in measuring various three-dimensional (3D) microscale flow phenomena. However, the axial elongation problem in reconstructing particles severely degrades the measurement accuracy along the light propagation direction. In this Letter, we utilize the lenslike characteristic of tens of micrometers size transparent spherical particles to extract their 3D position. A sharp intensity peak is observed in the reconstructed wave field, resulting from the light-focusing effect of the particle. As a result, the depth-of-focus constraint caused by the particle size is eliminated and the measurement accuracy is drastically improved up to submicrometer resolution.

High resolution (NA=08) in lensless in-line holographic microscopy with glass sample carriers

For lensless digital in-line holographic microscopy a new state-of-the-art spatial resolution corresponding to an NA of 0.8 is shown based on the tile superposition propagation. The result is proved using a common glass sample carrier with a refraction index of 1.52. Single-shot high-resolution imaging is possible by suppression of coherent reflections in an optimized arrangement using partially coherent laser light illumination.

High-Resolution Soft X-ray Digital In-Line Holographic Microscopy

High-spatial-resolution soft X-ray digital in-line holographic microscopy was developed. The scheme uses highly coherent and divergent lights generated by a combination of a Fresnel zone plate (FZP) and a pinhole of 200 nm radius with incidence incoherent lights tightly focused on the pinhole. At a wavelength of 2.38 nm, objects such as a carbon powder particle and a gold pattern were imaged with a spatial resolution of ∼200 nm. It approached the theoretically achievable resolution (i.e., the pinhole's radius) of typical pinhole-based in-line holographic microscopy. It is better than the previously reported best result by a factor of 2.

High-Resolution Soft X-ray Digital In-Line Holographic Microscopy

High-spatial-resolution soft X-ray digital in-line holographic microscopy was developed. The scheme uses highly coherent and divergent lights generated by a combination of a Fresnel zone plate (FZP) and a pinhole of 200 nm radius with incidence incoherent lights tightly focused on the pinhole. At a wavelength of 2.38 nm, objects such as a carbon powder particle and a gold pattern were imaged with a spatial resolution of ∼200 nm. It approached the theoretically achievable resolution (i.e., the pinhole's radius) of typical pinhole-based in-line holographic microscopy. It is better than the previously reported best result by a factor of 2.

A new methodology for pixel size retention in lensless digital holographic microscopyapplied to micro-particle analysis

A new method to control the pixel size corresponding to appropriate magnification ispresented for a lensless digital holographic microscopy system. Maintaining the correctpixel size corresponding to the desired magnification at different depths is accomplishedby suitably zero padding the digital hologram prior to reconstruction. This isparticularly significant for three-dimensional (3D) micro-particle analysis underconverging wave reconstruction geometry. Experimental results are presentedto demonstrate the improvement in the measurement of the size and depth ofmicro-particles in three dimensions using lensless in-line digital holographic microscopy.

Lensless multispectral digital in-line holographic microscope

An compact multispectral digital in-line holographic microscope (DIHM) is developed that emulates Gabor's original holographic principle. Using sources of varying spatial coherence (laser, LED), holographic images of objects, including optical fiber, latex microspheres, and cancer cells, are successfully captured and numerically processed. Quantitative measurement of cell locations and percentage confluence are estimated, and pseudocolor images are also presented. Phase profiles of weakly scattering cells are obtained from the DIHM and are compared to those produced by a commercially available off-axis digital holographic microscope.

Imaging mechanisms analysis of compact digital holographic microscope for microparticles measurement

Conventional optical microscopy suffers from small depth of focus due to its high numerical aperture and magnification of the microscope objective. In comparison, digital in-line holographic microscopy (DIHM) provides information about the entire 3D volume through numerical reconstruction of the single hologram at several depths. This advantage makes DIHM an effective tool for the measurement of microparticles in suspension. Recently, our group has demonstrated the potential of DIHM for accurate measurement of particles with sizes ranging from 40 microns to a few millimetres. In this paper, the applicability of DIHM is extended for measurement of near-micron sized particles. A compact digital holographic microscope with a single microscope objective is presented. The system imaging mechanisms of the microscope is analyzed first and the recording distance of digital hologram is calculated using spatial frequency analysis. Then the system magnification, lateral resolution and depth resolution are analyzed in terms of the hologram recording distance. Finally, the characterization of microparticles with a diameter of 1 micron and 10 microns is demonstrated with the compact setup. The experimental results show the efficiency and accuracy of this method with a measured error less than 1.55% in the diameter of certified particles.

Flow conditions in the vicinity of microstructured interfaces studied by holography and implications for the assembly of artificial actin networks

Microstructured fluidic devices have successfully been used for the assembly of free standing actin networks as mechanical model systems on the top of micropillars. The assembly occurs spontaneously at the pillar heads when preformed filaments are injected into the channel. In order to reveal the driving mechanism of this localization, we studied the properties of the flow profile by holographic tracking. Despite the strong optical disturbances originating from the pillar field, 2 μm particles were traced with digital in-line holographic microscopy (DIHM). Trajectories in the pillar free region and local alterations of the flow profile induced by the channel structure in the pillar decorated region can be distinguished. Velocity histograms at different z-positions reveal that the laminar flow profile across the channel shows a difference between the minimum in the z-component of the velocity field and the maximum of the overall velocity. This minimum drag in vertical direction is present at the top of the pillars and explains why biopolymer networks readily assemble in this region instead of forming a homogeneous three-dimensional network in between the pillars. On the basis of the observations we propose a new mechanism for actin network formation on top of the microstructures.

Real time numerical reconstruction of digitally recorded holograms in digital in-line holographic microscopy by using a graphics processing unit

Digital in-line holographic microscopy (DIHM) is perhaps the simplest methodology to obtain three-dimensional information from the micrometre world. The recovery of complex amplitude scattered by the specimens, relies on quite robust algorithms that consume large amounts of time. In this letter is presented the numerical reconstruction in real-time of holograms acquired in DIHM. The use of a graphics processing unit combine with a reduced number of operations allows for reconstructing DIHM holograms of 1024x1024 pixels up to 32 frames per second. Full text: PDF

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.

Magnified reconstruction of digitally recorded holograms by Fresnel–Bluestein transform

A method for numerical reconstruction of digitally recorded holograms with variable magnification is presented. The proposed strategy allows for smaller, equal, or larger magnification than that achieved with Fresnel transform by introducing the Bluestein substitution into the Fresnel kernel. The magnification is obtained independent of distance, wavelength, and number of pixels, which enables the method to be applied in color digital holography and metrological applications. The approach is supported by experimental and simulation results in digital holography of objects of comparable dimensions with the recording device and in the reconstruction of holograms from digital in-line holographic microscopy.

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.