Fluorescence-guided surgical system using holographic display: from phantom studies to canine patients.

Significance: Holographic display technology is a promising area of research that can lead to significant advancements in cancer surgery. We present the benefits of combining bioinspired multispectral imaging technology with holographic goggles for fluorescence-guided cancer surgery. Through a series of experiments with 43D-printed phantoms, small animal models of cancer, and surgeries on canine patients with head and neck cancer, we showcase the advantages of this holistic approach. Aim: The aim of our study is to demonstrate the feasibility and potential benefits of utilizing holographic display for fluorescence-guided surgery through a series of experiments involving 3D-printed phantoms and canine patients with head and neck cancer. Approach: We explore the integration of a bioinspired camera with a mixed reality headset to project fluorescent images as holograms onto a see-through display, and we demonstrate the potential benefits of this technology through benchtop and in vivo animal studies. Results: Our complete imaging and holographic display system showcased improved delineation of fluorescent targets in phantoms compared with the 2D monitor display approach and easy integration into the veterinarian surgical workflow. Conclusions: Based on our findings, it is evident that our comprehensive approach, which combines a bioinspired multispectral imaging sensor with holographic goggles, holds promise in enhancing the presentation of fluorescent information to surgeons during intraoperative scenarios while minimizing disruptions.

Acousto-holographic reconstruction of whole-cell stiffness maps.

Accurate assessment of cell stiffness distribution is essential due to the critical role of cell mechanobiology in regulation of vital cellular processes like proliferation, adhesion, migration, and motility. Stiffness provides critical information in understanding onset and progress of various diseases, including metastasis and differentiation of cancer. Atomic force microscopy and optical trapping set the gold standard in stiffness measurements. However, their widespread use has been hampered with long processing times, unreliable contact point determination, physical damage to cells, and unsuitability for multiple cell analysis. Here, we demonstrate a simple, fast, label-free, and high-resolution technique using acoustic stimulation and holographic imaging to reconstruct stiffness maps of single cells. We used this acousto-holographic method to determine stiffness maps of HCT116 and CTC-mimicking HCT116 cells and differentiate between them. Our system would enable widespread use of whole-cell stiffness measurements in clinical and research settings for cancer studies, disease modeling, drug testing, and diagnostics.

Virtual Impactor-Based Label-Free Pollen Detection using Holography and Deep Learning.

Exposure to bio-aerosols such as pollen can lead to adverse health effects. There is a need for a portable and cost-effective device for long-term monitoring and quantification of various types of pollen. To address this need, we present a mobile and cost-effective label-free sensor that takes holographic images of flowing particulate matter (PM) concentrated by a virtual impactor, which selectively slows down and guides particles larger than 6 µm to fly through an imaging window. The flowing particles are illuminated by a pulsed laser diode, casting their inline holograms on a complementary metal-oxide semiconductor image sensor in a lens-free mobile imaging device. The illumination contains three short pulses with a negligible shift of the flowing particle within one pulse, and triplicate holograms of the same particle are recorded at a single frame before it exits the imaging field-of-view, revealing different perspectives of each particle. The particles within the virtual impactor are localized through a differential detection scheme, and a deep neural network classifies the pollen type in a label-free manner based on the acquired holographic images. We demonstrated the success of this mobile pollen detector with a virtual impactor using different types of pollen (i.e., bermuda, elm, oak, pine, sycamore, and wheat) and achieved a blind classification accuracy of 92.91%. This mobile and cost-effective device weighs ∼700 g and can be used for label-free sensing and quantification of various bio-aerosols over extended periods since it is based on a cartridge-free virtual impactor that does not capture or immobilize PM.

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.

Label-Free Study of the Global Cell Behavior during Exposure to Environmental Radiofrequency Fields in the Presence or Absence of Pro-Apoptotic or Pro-Autophagic Treatments.

It remains controversial whether exposure to environmental radiofrequency signals (RF) impacts cell status or response to cellular stress such as apoptosis or autophagy. We used two label-free techniques, cellular impedancemetry and Digital Holographic Microscopy (DHM), to assess the overall cellular response during RF exposure alone, or during co-exposure to RF and chemical treatments known to induce either apoptosis or autophagy. Two human cell lines (SH-SY5Y and HCT116) and two cultures of primary rat cortex cells (astrocytes and co-culture of neurons and glial cells) were exposed to RF using an 1800 MHz carrier wave modulated with various environmental signals (GSM: Global System for Mobile Communications, 2G signal), UMTS (Universal Mobile Telecommunications System, 3G signal), LTE (Long-Term Evolution, 4G signal, and Wi-Fi) or unmodulated RF (continuous wave, CW). The specific absorption rates (S.A.R.) used were 1.5 and 6 W/kg during DHM experiments and ranged from 5 to 24 W/kg during the recording of cellular impedance. Cells were continuously exposed for three to five consecutive days while the temporal phenotypic signature of cells behavior was recorded at constant temperature. Statistical analysis of the results does not indicate that RF-EMF exposure impacted the global behavior of healthy, apoptotic, or autophagic cells, even at S.A.R. levels higher than the guidelines, provided that the temperature was kept constant.

Analyses of the Tympanic Membrane Impulse Response Measured with High-Speed Holography.

The Tympanic Membrane (TM) transforms acoustic energy to ossicular vibration. The shape and the displacement of the TM play an important role in this process. We developed a High-speed Digital Holography (HDH) system to measure the shape and transient displacements of the TM induced by acoustic clicks. The displacements were further normalized by the measured shape to derive surface normal displacements at over 100,000 points on the TM surface. Frequency and impulse response analyses were performed at each TM point, which enable us to describe 2D surface maps of four new TM mechanical parameters. From frequency domain analyses, we describe the (i) dominant frequencies of the displacement per sound pressure based on Frequency Response Function (FRF) at each surface point. From time domain analyses, we describe the (ii) rising time, (iii) exponential decay time, and the (iv) root-mean-square (rms) displacement of the TM based on Impulse Response Function (IRF) at each surface point. The resultant 2D maps show that a majority of the TM surface has a dominant frequency of around 1.5 kHz. The rising times suggest that much of the TM surface is set into motion within 50 µs of an impulsive stimulus. The maps of the exponential decay time of the IRF illustrate spatial variations in damping, the least known TM mechanical property. The damping ratios at locations with varied dominant frequencies are quantified and compared.

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.

Quasi-static and dynamic motions of the columellar footplate in ostrich (Struthio camelus) measured ex vivo.

The nature of the movement of the columellar footplate (CFP) in birds is still a matter of ongoing debate. Some sources claim that rocking motion is dominant, while others propose a largely piston-like motion. In this study, motions of the CFP are experimentally investigated in the ostrich using a post-mortem approach. For quasi-static loads, micro-CT scans of ostrich heads were made under positive and negative middle-ear pressures of 1 kPa. For dynamic loads, laser Doppler vibrometry was used to measure the velocity on multiple locations of the CFP as a function of excitation frequency from 0.125 to 4 kHz, and digital stroboscopic holography was used to assess the 1D full-field out-of-plane displacement of the CFP at different excitation frequencies. To expose the CFP in the experiments, measurements were made from the medial side of the CFP after opening and draining the inner ear. To determine the influence of the inner-ear load on CFP motions, a finite element model was created of the intact ostrich middle ear with inner-ear load included. For quasi-static loads, the CFP performed largely piston-like motions under positive ME pressure, while under negative ME pressure the difference between piston and rocking motion was smaller. For dynamic loads, the CFP motion was almost completely piston-like for frequencies below 1 kHz. For higher frequencies, the motions became more complicated with an increase of the rocking components, although they never exceeded the piston component. When including the inner-ear load to the model, the rocking components started to increase relative to the piston component when compared to the result of the model with unloaded CFP, but only at high frequencies above 1 kHz. In this frequency range, the motion could no longer be identified as purely piston-like or rocking. As a conclusion, the current results suggest that CFP motion is predominantly piston-like below 1 kHz, while at higher frequencies the motion becomes too complicated to be described as purely piston-like or rocking.

Standardized Polyalthia longifolia leaf extract (PLME) inhibits cell proliferation and promotes apoptosis: The anti-cancer study with various microscopy methods.

Over the years a number of microscopy methods have been developed to assess the changes in cells. Some non-invasive techniques such as holographic digital microscopy (HDM), which although does not destroy the cells, but helps to monitor the events that leads to initiation of apoptotic cell death. In this study, the apoptogenic property and the cytotoxic effect of P. longifolia leaf methanolic extract (PLME) against the human cervical carcinoma cells (HeLa) was studied using light microscope (LM), holographic digital microscopy (HDM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The average IC50 value of PLME against HeLa cells obtained by MTT and CyQuant assay was 22.00µg/mL at 24h. However, noncancerous Vero cells tested with PLME exhibited no cytotoxicity with the IC50 value of 51.07µg/mL at 24h by using MTT assay. Cytological observations showed nuclear condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, disappearance of microvilli and filopodia, narrowing of lamellipodia, holes, formation of numerous smaller vacuoles, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by HDM, LM, SEM and TEM. In conclusion, PLME was able to produce distinctive morphological features of HeLa cell death that corresponds to apoptosis.

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.

A single-ossicle ear: Acoustic response and mechanical properties measured in duck.

To date, the single-ossicle avian middle ear (ME) is poorly understood, despite its striking resemblance to the design of many currently used ossicular replacement prostheses. This study aims to improve comprehension of this system. The acoustic response and the mechanical properties of the mallard middle ear were studied by means of optical interferometry experiments and finite element (FE) simulations. A finite element model was constructed based on µCT data and validated using the experimental results. Stroboscopic holography was used to measure the full-field displacement of the tympanic membrane (TM) under acoustic stimulation, and the transfer function was obtained with laser Doppler vibrometry. A sensitivity analysis concluded that the most influential parameters for ME mechanics are the elasticity of the TM, the extracolumella (the cartilaginous part of the columella) and the annular ligament of the columellar footplate. Estimates for the Young's modulus of the TM were obtained by iteratively updating the FE model to match experimental data. A considerable inter-individual variability was found for the TM's elasticity. Comparison of the experimental results and the optimized FE model shows that, similar to the human middle ear, damping needs to be present in the TM to describe the specific spatial and frequency dependent vibrations of the TM. In summary, our results indicate which mechanical parameters are essential to the good functioning of the avian ME and provide a first estimation of their values.

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.

Lorentz microscopy sheds light on the role of dipolar interactions in magnetic hyperthermia.

Monodispersed Fe3O4 nanoparticles with comparable size distributions have been synthesized by two different synthesis routes, co-precipitation and thermal decomposition. Thanks to the different steric stabilizations, the described samples can be considered as a model system to investigate the effects of magnetic dipolar interactions on the aggregation states of the nanoparticles. Moreover, the presence of magnetic dipolar interactions can strongly affect the nanoparticle efficiency as a hyperthermic mediator. In this paper, we present a novel way to visualize and map the magnetic dipolar interactions in different kinds of nanoparticle aggregates by the use of Lorentz microscopy, an easy and reliable in-line electron holographic technique. By exploiting Lorentz microscopy, which is complementary to the magnetic measurements, it is possible to correlate the interaction degrees of magnetic nanoparticles with their magnetic behaviors. In particular, we demonstrate that Lorentz microscopy is successful in visualizing the magnetic configurations stabilized by dipolar interactions, thus paving the way to the comprehension of the power loss mechanisms for different nanoparticle aggregates.

Evaluation of a method based on coherence in aqueous systems and resonance-based isotherapeutic remedy in the treatment of chronic psoriasis vulgaris.

Psoriasis is a chronic skin disorder that exhibits three main features: lymphocytic infiltration into the dermis and epidermis, uncontrolled proliferation and abnormal differentiation of keratinocytes. In this study we have evaluated the effect of treatment with WHITE Holographic Bioresonance Method and a resonance-based isotherapeutic remedy on patients affected by chronic psoriasis vulgaris. The WHITE Holographic Bioresonance Method is based on the principles of electrodynamic coherence. By exploiting the phenomenon of bio-resonance, it uses a transfer plate to produce resonance- and light-based isotherapeutic coherent acqueous remedies and gels that emit coherent oscillations which "imprint" the area of psoriasis-affected skin. Levels of proinflammatory cytokines have been evaluated in the plasma of psoriatic patients treated with isotherapeutic remedies. The obtained results demonstrate a positive effect on the natural course of the disease and matched the results obtained by psoriatic patients treated with narrow band UVB. A significant reduction in plasma levels of cytokines involved in pathogenesis of psoriasis has been observed. Our findings may suggest that WHITE Holographic Bioresonance method used in combination with resonance-based isotherapeutic remedy could well be a new useful treatment option for patients with limited psoriatic plaques.

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.