Method to analyze effects of low-level laser therapy on biological cells with a digital holographic microscope.

Low-level laser therapy (LLLT) is a therapeutic tool that uses the photobiochemical interaction between light and tissue. Its effectiveness is controversial due to a strong dependence on dosimetric parameters. In this work, we demonstrate that digital holographic microscopy is an effective label-free imaging technique to analyze the effects of LLLT on biological cells, and we propose the full methodology to create correct synthetic aperture phase maps for further extensive, highly accurate statistical analysis. The proposed methodology has been designed to provide a basis for many other biological experiments using quantitative phase imaging. We use SHSY-5Y and HaCaT cells irradiated with different doses of red light for the experiment. The analysis shows quantitative changes in cell dry mass density and the projected cell surface in response to different radiation doses.

Real-time quantitative phase imaging by single-shot dual-wavelength off-axis digital holographic microscopy.

A single-shot dual-wavelength digital holographic microscopy with an adjustable off-axis configuration is presented, which helps realize real-time quantitative phase imaging for living cells. With this configuration, two sets of interference fringes corresponding to their wavelengths can be flexibly recorded onto one hologram in one shot. The universal expression on the dual-wavelength hologram recorded under any wave vector orientation angles of reference beams is given. To avoid as much as possible the effect of zero-order spectrum, we can flexibly select their carry frequencies for the two wavelengths using this adjustable off-axis configuration, according to the distribution feature of object's spatial-frequency spectrum. This merit is verified by a quantitative phase imaging experiment for the microchannel of a microfluidic chip. The reconstructed phase maps of living onion epidermal cells exhibit cellular internal life activities, for the first time to the best of our knowledge, vividly displaying the progress of the nucleus, cell wall, cytoskeleton, and the substance transport in microtubules inside living cells. These imaging results demonstrate the availability and reliability of the presented method for real-time quantitative phase imaging.

Application of digital holographic tomography in antitumor effect of cantharides complex on 4T1 breast cancer cells.

The study focuses on a methodology providing noninvasive monitoring and evaluation of the antitumor effect of traditional Chinese medicine, cantharides complex (canth), on 4T1 breast tumor cells. Digital holographic tomography (DHT) and developed data post-processing algorithms were used for quantitative estimation of changes in optical and morphological parameters of cells. We calculated and compared data on the refractive index, thickness, and projected area of 4T1 breast tumor cells in control untreated specimens and those treated with doxorubicin hydrochloride (DOX), canth, and their combinations. Post-treatment changes in cellular morphology recorded by DHT demonstrated that the two drugs led to noticeably different morphological changes in cells that can be presumably associated with different pathways of their death, apoptosis, or necrosis. The effect of combined treatment with these two drugs strongly depended on their relative concentrations and could lead to changes characteristic either for DOX or for canth; however, being more profound than those obtained when using each drug solely. The results obtained by DHT are in a good correspondence with commonly used cell viability analysis and immunofluorescent analysis of changes in cellular cytoskeleton.

Long-term holographic phase-contrast time lapse reveals cytoplasmic circulation in dehydrating plant cells.

The intracellular dynamics of onion epidermal cells during the dehydration process is observed by holographic microscopy. Both the nucleus and cytoplasm are accurately revealed by quantitative phase imaging while dehydration takes place. Indeed, we notice that the contrast of phase images increases with the decrease in cellular water content. We foresee that such a dehydrating process can be effective for improving phase contrast, thus permitting better imaging of plant cells with the scope of learning more about cellular dynamics and related phenomena. Exploiting this concept, we observe intracellular cytoplasmic circulation and transport of biological material.

Combined high-speed holographic shape and full-field displacement measurements of tympanic membrane.

The conical shape of the tympanic membrane (TM or eardrum) plays an important role in its function, such that variations in shape alter the acoustically induced motions of the TM. We present a method that precisely determines both shape and acoustically induced transient response of the entire TM using the same optics and maintaining the same coordinate system, where the TM transient displacements due to a broadband acoustic click excitation (50-µs impulse) and the shape are consecutively measured within <200 ms. Interferograms gathered with continuous high-speed (>2 kHz) optical phase sampling during a single 100-ms wavelength tuning ramp allow precise and rapid reconstructions of the TM shape at varied resolutions (50 to 200 µm). This rapid acquisition of full-field displacements and shape is immune to slow disturbances introduced by breathing or heartbeat of live subjects. Knowledge of TM shape and displacements enables the estimation of surface normal displacements regardless of the orientation of the TM within the measurement system. The proposed method helps better define TM mechanics and provides TM structure and function information useful for the diagnosis of ear disease.

Testing of bioactive-nanovesicles on hepatotoxicity of atypical antipsychotics via digital holography.

Atypical antipsychotic drugs induce hepatic toxicity. Thus, it is of importance to eliminate the side effects of these drugs. Herein we describe the preparation of nanoemulsions with a dietary supplement; wheat germ oil (WGO), to ameliorate the liver damage induced by clozapine and olanzapine. THLE-2 cell line was used as a model to investigate the effects of these nanoemulsions on cell viability as well as antioxidative efficiency after antipsychotic insult. In this context, a conventional cell culture method; MTT was used along with a novel cellular imaging technique called digital holography (DH) to evaluate cell viability. Obtained data confirmed that both clozapine and olanzapine induced the liver damage in in vitro model and WGO nanoemulsions were found to be effective on cells and eliminate the cytotoxic effects of these drugs. Briefly, this study has some outputs as follows; it showed that different dietary supplements can be used in such formulations instead of their pristine forms to increase bioavailability. Also, DH was successfully applied for the monitoring of cell viability and it could be a promising approach as the reactive-free cytotoxicity test.

Design, fabrication, and in vitro testing of novel three-dimensionally printed tympanic membrane grafts.

The tympanic membrane (TM) is an exquisite structure that captures and transmits sound from the environment to the ossicular chain of the middle ear. The creation of TM grafts by multi-material three-dimensional (3D) printing may overcome limitations of current graft materials, e.g. temporalis muscle fascia, used for surgical reconstruction of the TM. TM graft scaffolds with either 8 or 16 circumferential and radial filament arrangements were fabricated by 3D printing of polydimethylsiloxane (PDMS), flex-polyactic acid (PLA) and polycaprolactone (PCL) materials followed by uniform infilling with a fibrin-collagen composite hydrogel. Digital opto-electronic holography (DOEH) and laser Doppler vibrometry (LDV) were used to measure acoustic properties including surface motions and velocity of TM grafts in response to sound. Mechanical properties were determined using dynamic mechanical analysis (DMA). Results were compared to fresh cadaveric human TMs and cadaveric temporalis fascia. Similar to the human TM, TM grafts exhibit simple surface motion patterns at lower frequencies (400 Hz), with a limited number of displacement maxima. At higher frequencies (>1000 Hz), their displacement patterns are highly organized with multiple areas of maximal displacement separated by regions of minimal displacement. By contrast, temporalis fascia exhibited asymmetric and less regular holographic patterns. Velocity across frequency sweeps (0.2-10 kHz) measured by LDV demonstrated consistent results for 3D printed grafts, while velocity for human fascia varied greatly between specimens. TM composite grafts of different scaffold print materials and varied filament count (8 or 16) displayed minimal, but measurable differences in DOEH and LDV at tested frequencies. TM graft mechanical load increased with higher filament count and is resilient over time, which differs from temporalis fascia, which loses over 70% of its load bearing properties during mechanical testing. This study demonstrates the design, fabrication and preliminary in vitro acoustic and mechanical evaluation of 3D printed TM grafts. Data illustrate the feasibility of creating TM grafts with acoustic properties that reflect sound induced motion patterns of the human TM; furthermore, 3D printed grafts have mechanical properties that demonstrate increased resistance to deformation compared to temporalis fascia.

Response of the human tympanic membrane to transient acoustic and mechanical stimuli: Preliminary results.

The response of the tympanic membrane (TM) to transient environmental sounds and the contributions of different parts of the TM to middle-ear sound transmission were investigated by measuring the TM response to global transients (acoustic clicks) and to local transients (mechanical impulses) applied to the umbo and various locations on the TM. A lightly-fixed human temporal bone was prepared by removing the ear canal, inner ear, and stapes, leaving the incus, malleus, and TM intact. Motion of nearly the entire TM was measured by a digital holography system with a high speed camera at a rate of 42 000 frames per second, giving a temporal resolution of <24 µs for the duration of the TM response. The entire TM responded nearly instantaneously to acoustic transient stimuli, though the peak displacement and decay time constant varied with location. With local mechanical transients, the TM responded first locally at the site of stimulation, and the response spread approximately symmetrically and circumferentially around the umbo and manubrium. Acoustic and mechanical transients provide distinct and complementary stimuli for the study of TM response. Spatial variations in decay and rate of spread of response imply local variations in TM stiffness, mass, and damping.

Incubator proof miniaturized Holomonitor to in situ monitor cancer cells exposed to green tea polyphenol and preosteoblast cells adhering on nanostructured titanate surfaces: validity of the measured parameters and their corrections.

The in situ observation of cell movements and morphological parameters over longer periods of time under physiological conditions is critical in basic cell research and biomedical applications. The quantitative phase-contrast microscope applied in this study has a remarkably small size, therefore it can be placed directly into a humidified incubator. Here, we report on the successful application of this M4 Holomonitor to observe cancer cell motility, motility speed, and migration in the presence of the green tea polyphenol, epigallocatechin gallate, as well as to monitor the adhesion of preosteoblast cells on nanostructured titanate coatings, relevant for biomedical applications. A special mechanical stage was developed to position the sample into that range of the optical arrangement where digital autofocusing works with high reproducibility and precision. By in-depth analyzing the obtained single cell morphological parameters, we show that the limited vertical resolution of the optical setup results in underestimated single cell contact area and volume and overestimated single cell averaged thickness. We propose a simple model to correct the recorded data to obtain more precise single cell parameters. We compare the results with the kinetic data recorded by a surface sensitive optical biosensor, optical waveguide lightmode spectroscopy.

Applications of digital holographic microscopy in therapeutic evaluation of Chinese herbal medicines.

Therapeutic use of Chinese herbal medicines (CHMs) is a new approach to treat neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. The detection of soma volume and neurite outgrowth of living neurons is a highly relevant biomarker related to various application fields, including therapy efficacy and drug safety evaluation. Through the use of digital holographic microscopy (DHM), we may evaluate the therapeutic effect of CHMs in curing neurodegeneration. Panax ginseng has been used in traditional Chinese herbal medicine for centuries. In this study, DHM is applied to monitor the three-dimensional morphology change of retinoic acid-induced human neuroblastoma SH-SY5Y cells during Panax ginseng treatment. We demonstrate the capability of DHM to detect noninvasively SH-SY5Y cell apoptosis and rescue through the measurement of neuronal volume and neurite outgrowth regulation without any labeling reagent. Through DHM, we observed the phase images of the rapidly shrinking cells with decreasing soma volume and shortening neurite outgrowth during glutamate treatments. Then shrinkage in glutamate-induced cells is significantly alleviated during Panax ginseng treatment. The results through DHM are consistent with the result from MTT assay for assessing cell viability during Panax ginseng treatment. Thus, we suggest that application of DHM for measuring soma volume and neurite outgrowth of living neurons may be one appropriate therapeutic evaluation for CHMs.

Common-path configuration in total internal reflection digital holography microscopy.

Total Internal Reflection Digital Holographic Microscopy (TIRDHM) is recognized to be a powerful tool for retrieving quantitative phase images of cell-substrate interfaces, adhesions, and tissue structures close to the prism surface. In this Letter, we develop an improved TIRDHM system, taking advantage of a refractive index mismatch between the prism and the sample substrate, to allow phase-shifting DH with just a single-beam interferometric configuration. Instead of the traditional off-axis method, phase-shift method is used to retrieve amplitude and phase images in coherent light and TIR modality. Essentially, the substrate-prism interface acts like a beam splitter generating a reference beam, where the phase-shift dependence on the incident angle is exploited in this common-path configuration. With the aim to demonstrate the technique's validity, some experiments are performed to establish the advantage of this compact and simple configuration, in which the reference arm in the setup is avoided.

Faithful reconstruction of digital holograms captured by FINCH using a Hamming window function in the Fresnel propagation.

Recent advances in Fresnel incoherent correlation holography (FINCH) increase the signal-to-noise ratio in hologram recording by interference of images from two diffractive lenses with focal lengths close to the image plane. Holograms requiring short reconstruction distances are created that reconstruct poorly with existing Fresnel propagation methods. Here we show a dramatic improvement in reconstructed fluorescent images when a 2D Hamming window function substituted for the disk window typically used to bound the impulse response in the Fresnel propagation. Greatly improved image contrast and quality are shown for simulated and experimentally determined FINCH holograms using a 2D Hamming window without significant loss in lateral or axial resolution.

Measurements of three-dimensional shape and sound-induced motion of the chinchilla tympanic membrane.

UNLABELLED: Opto-electronic computer holographic measurements were made of the tympanic membrane (TM) in cadaveric chinchillas. Measurements with two laser wavelengths were used to compute the 3D-shape of the TM. Single laser wavelength measurements locked to eight distinct phases of a tonal stimulus were used to determine the magnitude and the relative phase of the surface displacements. These measurements were made at over 250,000 points on the TM surface. The measured motions contained spatial phase variations consistent with relatively low-order (large spatial frequency) modal motions and smaller magnitude higher-order (smaller spatial frequency) motions that appear to travel, but may also be explained by losses within the membrane. The measurement of shape and thin shell theory allowed us to separate the measured motions into those components orthogonal to the plane of the tympanic ring, and those components within the plane of the tympanic ring based on the 3D-shape. The predicted in-plane motion components are generally smaller than the out-of-plane perpendicular component of motion. Since the derivation of in-plane and out-of plane depended primarily on the membrane shape, the relative sizes of the predicted motion components did not vary with frequency. SUMMARY: A new method for simultaneously measuring the shape and sound-induced motion of the tympanic membrane is utilized to estimate the 3D motion on the membrane surface. This article is part of a special issue entitled "MEMRO 2012".

Infrared macro-fingerprint analysis-through-separation for holographic chemical characterization of herbal medicine.

Although herbal medicine (HM) behaves as a complex system having high-potential for treating chronic/life-threatening diseases, compatible characterization metrics with hierarchical approaches to integrate molecular-level information into the whole system are lacking. Herein, we report a high-throughput methodology (holographic infrared (IR) spectroscopy) harmonizing with the character of HM, providing hierarchical infrared fingerprints (entirety, parts and single ingredients), and working as a "GPS" to navigate a comprehensive chemical characterization of HM circularly from system to molecular level by step-by-step HM analysis-through-separation of IR spectra indicative and vice versa by reconstitution-through-combination of adducting spectrum without a complete understanding of the chemical constituents, and demonstrate holographic chemical charaterization of a Chinese herb Danshen. Global chemical fingerprints of species at each hierarchical level and ingredient profile variations among multilevel species of Danshen are integratively interpreted with fast estimation of their relative contents of active compounds. Finally, integral dynamic information of Danshen separation process is disclosed straightforward by spectral retrieving technique.

Toward a scalable holographic word-form representation.

Phenomena in a variety of verbal tasks--for example, masked priming, lexical decision, and word naming--are typically explained in terms of similarity between word-forms. Despite the apparent commonalities between these sets of phenomena, the representations and similarity measures used to account for them are not often related. To show how this gap might be bridged, we build on the work of Hannagan, Dupoux, and Christophe, Cognitive Science 35:79-118, (2011) to explore several methods of representing visual word-forms using holographic reduced representations and to evaluate them on their ability to account for a wide range of effects in masked form priming, as well as data from lexical decision and word naming. A representation that assumes that word-internal letter groups are encoded relative to word-terminal letter groups is found to predict qualitative patterns in masked priming, as well as lexical decision and naming latencies. We then show how this representation can be integrated with the BEAGLE model of lexical semantics (Jones & Mewhort, Psychological Review 114:1-37, 2007) to enable the model to encompass a wider range of verbal tasks.

Exact complex-wave reconstruction in digital holography.

We address the problem of exact complex-wave reconstruction in digital holography. We show that, by confining the object-wave modulation to one quadrant of the frequency domain, and by maintaining a reference-wave intensity higher than that of the object, one can achieve exact complex-wave reconstruction in the absence of noise. A feature of the proposed technique is that the zero-order artifact, which is commonly encountered in hologram reconstruction, can be completely suppressed in the absence of noise. The technique is noniterative and nonlinear. We also establish a connection between the reconstruction technique and homomorphic signal processing, which enables an interpretation of the technique from the perspective of deconvolution. Another key contribution of this paper is a direct link between the reconstruction technique and the two-dimensional Hilbert transform formalism proposed by Hahn. We show that this connection leads to explicit Hilbert transform relations between the magnitude and phase of the complex wave encoded in the hologram. We also provide results on simulated as well as experimental data to validate the accuracy of the reconstruction technique.

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy.

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

Digital holographic microscopy by use of surface plasmon resonance for imaging of cell membranes.

A technique called surface plasmon resonance digital holographic microscopy (SPRDHM) for optical imaging of cell membranes is proposed. The intensity and phase distributions of the reflected light that is modulated by the cell membrane in surface plasmon resonance can be simultaneously obtained. The imaging principle and capability are theoretically analyzed and demonstrated by experiments. In addition, the technique is compared with total internal reflection digital holographic microscopy (TIRDHM) in theory and experiment, respectively. The results show that the SPRDHM technique is better in spatial resolution and phase sensitivity than the TIRDHM technique for imaging of cell membranes.

Transport of intensity phase imaging in a volume holographic microscope.

We demonstrate a method for single-shot quantitative phase imaging based on the transport of intensity equation (TIE) in a volume holographic microscope (VHM). The VHM system uses a multiplexed volume hologram to laterally separate images from different focal planes. This axial intensity information is then used to solve the TIE and recover object phase quantitatively. Further, we show improved phase recovery by using five multiplexed gratings in one hologram.

Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy.

For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.