Single capture bright field and off-axis digital holographic microscopy: publisher's note.

This publisher's note contains corrections to Opt. Lett.48, 876 (2023)10.1364/OL.478674.

Single capture bright field and off-axis digital holographic microscopy.

We report on a single capture approach for simultaneous incoherent bright field (BF) and laser-based quantitative phase imaging (QPI). Common-path digital holographic microscopy (DHM) is implemented in parallel with BF imaging within the optical path of a commercial optical microscope to achieve spatially multiplexed recording of white light images and digital off-axis holograms, which are subsequently numerically demultiplexed. The performance of the proposed multimodal concept is firstly determined by investigations on microspheres. Then, the application for label-free dual-mode QPI and BF imaging of living pancreatic tumor cells is demonstrated.

Massive ordering and alignment of cylindrical micro-objects by photovoltaic optoelectronic tweezers.

Optical tools for manipulation and trapping of micro- and nano-objects are a fundamental issue for many applications in nano- and biotechnology. This work reports on the use of one such method, known as photovoltaic optoelectronics tweezers, to orientate and organize cylindrical microcrystals, specifically elongated zeolite L, on the surface of Fe-doped LiNbO3 crystal plates. Patterns of aligned zeolites have been achieved through the forces and torques generated by the bulk photovoltaic effect. The alignment patterns with zeolites parallel or perpendicular to the substrate surface are highly dependent on the features of light distribution and crystal configuration. Moreover, dielectrophoretic chains of zeolites with lengths up to 100 µm have often been observed. The experimental results of zeolite trapping and alignment have been discussed and compared together with theoretical simulations of the evanescent photovoltaic electric field and the dielectrophoretic potential. They demonstrate the remarkable capabilities of the optoelectronic photovoltaic method to orientate and pattern anisotropic microcrystals. The combined action of patterning and alignment offers a unique tool to prepare functional nanostructures with potential applications in a variety of fields such as nonlinear optics or plasmonics.

Synchronization in pairs of rotating active biomotors.

Although synchronization is a well-known physical phenomenon, experimental studies of its emergence in living bacterial cells are still scarce. The difficulty in generating a controlled scenario to detect synchronization has limited the experimental outcomes so far. We present a realization based on holographic optical tweezers in which adhered pairs of self-propelled bacteria rotate in a plane. The separation distance between the bacteria determines the strength of the hydrodynamic coupling. Despite the noisy environment and autonomous dynamics of the living bacteria, we find evidence of phase locking and frequency entrainment in their rotation. The observation of higher order frequency synchronization is also discussed.

Biolens behavior of RBCs under optically-induced mechanical stress.

In this work, the optical behavior of Red Blood Cells (RBCs) under an optically-induced mechanical stress was studied. Exploiting the new findings concerning the optical lens-like behavior of RBCs, the variations of the wavefront refracted by optically-deformed RBCs were further investigated. Experimental analysis have been performed through the combination of digital holography and numerical analysis based on Zernike polynomials, while the biological lens is deformed under the action of multiple dynamic optical tweezers. Detailed wavefront analysis provides comprehensive information about the aberrations induced by the applied mechanical stress. By this approach it was shown that the optical properties of RBCs in their discocyte form can be affected in a different way depending on the geometry of the deformation. In analogy to classical optical testing procedures, optical parameters can be correlated to a particular mechanical deformation. This could open new routes for analyzing cell elasticity by examining optical parameters instead of direct but with low resolution strain analysis, thanks to the high sensitivity of the interferometric tool. Future application of this approach could lead to early detection and diagnosis of blood diseases through a single-step wavefront analysis for evaluating different cells elasticity. © 2017 International Society for Advancement of Cytometry.

Optical assembly of bio-hybrid micro-robots.

The combination of micro synthetic structures with bacterial flagella motors represents an actual trend for the construction of self-propelled micro-robots. The development of methods for fabrication of these bacteria-based robots is a first crucial step towards the realization of functional miniature and autonomous moving robots. We present a novel scheme based on optical trapping to fabricate living micro-robots. By using holographic optical tweezers that allow three-dimensional manipulation in real time, we are able to arrange the building blocks that constitute the micro-robot in a defined way. We demonstrate exemplarily that our method enables the controlled assembly of living micro-robots consisting of a rod-shaped prokaryotic bacterium and a single elongated zeolite L crystal, which are used as model of the biological and abiotic components, respectively. We present different proof-of-principle approaches for the site-selective attachment of the bacteria on the particle surface. The propulsion of the optically assembled micro-robot demonstrates the potential of the proposed method as a powerful strategy for the fabrication of bio-hybrid micro-robots.

Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment.

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.

Three-dimensional exploration and mechano-biophysical analysis of the inner structure of living cells.

A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fields, are combined with self-interference digital holographic microscopy. This biophotonic holographic workstation enables non-contact, minimally invasive, flexible, high-precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a first model experiment, internalized polystyrene microspheres with 1 µm diameter are three-dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.

Durable 3D murine ex vivo retina glaucoma models for optical coherence tomography.

Durable and standardized phantoms with optical properties similar to native healthy and disease-like biological tissues are essential tools for the development, performance testing, calibration and comparison of label-free high-resolution optical coherence tomography (HR-OCT) systems. Available phantoms are based on artificial materials and reflect thus only partially ocular properties. To address this limitation, we have performed investigations on the establishment of durable tissue phantoms from ex vivo mouse retina for enhanced reproduction of in vivo structure and complexity. In a proof-of-concept study, we explored the establishment of durable 3D models from dissected mouse eyes that reproduce the properties of normal retina structures and tissue with glaucoma-like layer thickness alterations. We explored different sectioning and preparation procedures for embedding normal and N-methyl-D-aspartate (NMDA)-treated mouse retina in transparent gel matrices and epoxy resins, to generate durable three-dimensional tissue models. Sample quality and reproducibility were quantified by thickness determination of the generated layered structures utilizing computer-assisted segmentation of OCT B-scans that were acquired with a commercial HR-OCT system at a central wavelength of 905 nm and analyzed with custom build software. Our results show that the generated 3D models feature thin biological layers close to current OCT resolution limits and glaucoma-like tissue alterations that are suitable for reliable HR-OCT performance characterization. The comparison of data from resin-embedded tissue with native murine retina in gels demonstrates that by utilization of appropriate preparation protocols, highly stable samples with layered structures equivalent to native tissues can be fabricated. The experimental data demonstrate our concept as a promising approach toward the fabrication of durable biological 3D models suitable for high-resolution OCT system performance characterization supporting the development of optimized instruments for ophthalmology applications.

Label-Free Digital Holographic Microscopy for In Vitro Cytotoxic Effect Quantification of Organic Nanoparticles.

Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots® nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.

Interlaboratory evaluation of a digital holographic microscopy-based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers.

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.

[Contribution of computed tomography in patients with lung metastases of differentiated thyroid carcinoma not apparent on plain radiography who were treated with radioiodine]. / Contribuição da tomografia computadorizada em pacientes com metástases pulmonares de carcinoma diferenciado de tireóide não-aparentes na radiografia e tratados com radioiodo.

Computed tomography (CT or CAT Scan) of the chest is more sensitive than radiography in the detection of lung metastases of differentiated thyroid cancer (DTC), but little information is available regarding the aggregated value of this method. The present study evaluated the response of patients with lung metastases of DTC not apparent on radiography to treatment with 131I and the value of CT in these cases. Twenty-five patients with lung metastases not apparent on radiography, who initially received 100-200 mCi I151, were evaluated and those presenting pulmonary uptake on post-therapy WBS were submitted to a new treatment after 6 to 12 months, and so on. The chance of detection of pulmonary uptake on post-therapy WBS did not differ between patients with negative and positive CT (100% versus 91.5%). Mean serum Tg levels were higher in patients with positive CT (108 ng/ml versus 52 ng/ml). Negative post-therapy WBS was achieved in 82% of patients with positive CT and in 92.3% with negative CT and the cumulative I131 activity necessary to achieve this outcome did not differ between the two groups (mean=300 mCi). Stimulated Tg was undetectable in 47% of patients with negative CT at the end of treatment, but in none of the patients whose CT continued to be positive. In patients with elevated Tg, the CT result apparently did not change the indication of therapy or the I131 activity to be administered. In cases with lung metastases, the persistence of micronodules on CT was associated with the persistence of detectable Tg in patients presenting negative post-therapy WBS.

Is adjuvant therapy useful in patients with papillary carcinoma smaller than 2 cm?

To evaluate tumor recurrence after total thyroidectomy in patients with single papillary carcinoma with size 0.5 mIU/L in >or=50% of the measurements in all patients. Complete remission (stimulated thyroglobulin (Tg) 0.05). Six patients who still had stimulated Tg > 1 ng/mL (<5 ng/mL) showed a >50% decrease in comparison with Tg measured 12-24 months earlier. In conclusion, we suggest a more conservative approach with respect to central-compartment neck dissection, postoperative (131)I, and suppressive therapy in patients with small tumors restricted to the thyroid.

Management of low-risk patients with thyroid carcinoma and detectable thyroglobulin on T4 after thyroidectomy and ablation with iodine-131.

OBJECTIVE: To evaluate the positive predictive value of detectable Tg during T4 therapy (Tg on T4) in patients with thyroid cancer after total thyroidectomy and remnant ablation, discussing the work-up in this situation and the empirical indication of 131I. PATIENTS AND METHODS: Initially, 234 low-risk patients [tumor < 5cm, completely resected, no extensive extrathyroid invasion (pT4)] submitted to total thyroidectomy and ablation with 131I (3.7-5.5 GBq) who presented no ectopic uptake on RxWBS were studied. Of these, 23 patients with detectable Tg on T4 (> 1ng/ml) during the first year after initial therapy were selected. RESULTS: Metastases were detected by neck US in 7 patients, by chest CT in 2 and by US and CT in 3. Four of five patients with lung metastases upon CT had a positive RxWBS. Eleven patients with negative US and CT received a new 131I dose (without DxWBS), and RxWBS showed ectopic uptake in 3 patients. Among the patients with negative RxWBS, 7 remained free of apparent disease and Tg was declining (5 with undetectable Tg on T4 at the end of the study). One patient presented an increase in Tg and FDG-PET was positive for lymph node and bone metastases. CONCLUSIONS: All patients with Tg on T4 > 5ng/ml presented apparent disease. In these cases, even when US and CT are negative, the administration of a therapeutic dose of 131I (without DxWBS) and FDG-PET are recommended. Among patients with detectable Tg on T4

Testicular function after radioiodine therapy in patients with thyroid cancer.

Our aim was to assess testicular function in patients treated with high-dose radioiodine. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were determined in 52 men with thyroid carcinoma before and 6, 12, and 18 months after radioiodine therapy (3.7-5.5 GBq (131)I; mean, 4.25 GBq (131)I) (group 1) and were also determined before and 18 months after the last radioiodine therapy in 22 patients who received high cumulative activities (13-27.7 GBq; mean, 20.3 GBq (131)I) (group 2). FSH levels were increased 6 months after therapy in all patients of group 1, while a decline was observed after 12 months, with 37 of 52 (71%) subjects presenting normal values. FSH values returned to normal after 18 months in all patients. In group 2, 12 of 22 (54.5%) patients presented elevated FSH and 8 (66%) of these individuals had oligospermia. Six months after radioiodine, increased LH levels were observed in only 5 of 52 (9.6%) patients of group 1, which returned to normal after 12 months, and in 5 of 22 (22%) of group 2. All patients showed normal testosterone levels. We conclude that 131I therapy may cause impairment of testicular function. A generally transient increase in FSH is highly common but is usually reversed within 18 months. Oligospermia was common (one third) after high cumulative (131)I activities. Becausee we did not perform a spermiogram before therapy, we cannot state that high cumulative (131)I activities cause permanent infertility. We recommend the routine use of sperm banks in the cases of men who still wish to have children and who will undergo therapy with (131)I activities of 14 GBq or more or in the case of patients with pelvic metastases.

Positive predictive value of detectable stimulated tg during the first year after therapy of thyroid cancer and the value of comparison with Tg-ablation and Tg measured after 24 months.

This study evaluated the positive predictive value (PPV) of detectable stimulated thyroglobulin during the first year after treatment of thyroid carcinoma (Tg-1) and the value of comparison with Tg-ablation and measured after 24 months (Tg-2). Forty-two consecutive patients undergoing total thyroidectomy and ablation with detectable Tg-1 (>1ng/mL) were selected. The patients had well-differentiated tumors, which were completely resected, and there was no ectopic uptake on whole body scan after 3.7-5.5GBq I(131). Imaging methods during follow-up revealed metastases in 10 patients (24%) (15% if Tg-1 10 ng=mL). Tg-ablation (cutoff of 10 ng/mL) presented a negative predictive value (NPV) of 91% and PPV of 42%. Comparing Tg-ablation with Tg-1, the PPV of an increase was 100%, whereas the NPV of a decrease was 88%. Thirty-six patients presented negative imaging results upon first assessment and Tg-1 was compared to Tg-2. Metastases were detected in all patients who presented an increase in Tg (n=4), whereas patients without variation (n=4) or with a decrease (n=28) showed no apparent disease. Among disease-free patients (n=32), 50% presented undetectable Tg and 40% showed a >50% decrease after 2 years. In conclusion, most patients with detectable stimulated Tg during the first year after therapy had no metastases, and evaluation of the slope of Tg helped discriminate cases with apparent disease.

[Follow-up of high-risk patients with differentiated thyroid cancer without persistent disease after initial therapy]. / Seguimento de pacientes de alto risco com carcinoma diferenciado de tireóide sem doença persistente após a terapia inicial.

This study evaluated the follow-up of high-risk patients with thyroid cancer after initial therapy. A total of 125 high-risk patients (tumor >4 cm and/or extrathyroid invasion and/or lymph node metastases, and age >45 years), with complete resection of the tumor, were selected. All patients underwent total thyroidectomy and ablation with (131)I[3.7-5.5 GBq (100-150 mCi)]. Eighteen patients (14.8%) presenting metastases on post-dose whole-body scan (RxWBS) were excluded. The negative predictive value of stimulated Tg < or =1 ng/ml in combination with neck US during first assessment (612 mo. after ablative therapy) was 96.2% for the absence of recurrence up to 5 years. This value increased to 98.7% when adding WBS performed with 185 MBq (5 mCi) (131)I (DxWBS). The positive predictive value (PPV) of stimulated Tg >1 ng/ml was 52% for the detection of the presence of metastases up to 5 years; however, considering only patients with initially negative DxWBS and US, the PPV was 19% (9% if Tg of 110 ng/ml vs. 40% if Tg >10 ng/ml). Tg levels decreased spontaneously in patients with stimulated Tg >1 ng/ml during first assessment, negative US and DxWBS, and no recurrence during follow-up, with Tg being undetectable in half these patients at the end of 5 years. Twenty patients presented uptake in the thyroid bed upon DxWBS during the first year after ablative therapy, with stimulated Tg and US being negative, and were not treated with 131I; these patients did not relapse and no uptake on DxWBS was observed in 60% after 5 years. Recurrence after 5 years was only 1.3% in patients without apparent disease (negative US and DxWBS) and stimulated Tg <1 ng/ml. An algorithm for the follow-up of high-risk patients after initial therapy is presented in this study.

Malformations in the offspring of women with thyroid cancer treated with radioiodine for the ablation of thyroid remnants.

RATIONALE: Since ovarian function is only temporarily compromised by radioiodine therapy, many women with thyroid cancer treated with radioiodine can become pregnant. The present study evaluated the evolution of these pregnancies and the consequences for the offspring. PATIENTS AND METHODS: We retrospectively analyzed 78 pregnancies of 66 women submitted to total thyroidectomy, followed by radioiodine therapy 3.75-5.5 GBq (131)I, mean 4.64 GBq). In all patients, conception occurred one year after ablative therapy (mean of 30 months). Age ranged form 19 to 36 years (mean of 30.6 years) at the time of radioiodine treatment and from 23 to 39 years (mean of 32.8 years) at the time of conception. RESULTS: Four (5.1%) of the 78 pregnancies resulted in spontaneous abortions. Three (4%) of the 74 deliveries were preterm and there was no case of stillbirth. The birthweight was > 2500 g in 94.6% of the children (+/- SD: 3350 +/- 450 g) and only one infant (1.3%) presented an apparent malformation at birth (intraventricular communication). No difference in the age at the time of radioiodine therapy or conception or in radioiodine dose was observed between pregnancies with an unfavorable outcome and those with a favorable outcome. CONCLUSION: We conclude that pregnancies that occur 12 months after ablative therapy are safe.

Managing thyroid cancer without thyroxine withdrawal.

Thyroxine (T4) withdrawal or recombinant TSH is used for the stimulation of thyroglobulin (Tg), whole-body scanning (WBS) and iodine-131 treatment in patients with thyroid carcinoma. This study evaluated the T4 dose reduction protocol as an alternative for patients' preparation. Fifty-one patients were submitted to total T4 withdrawal for WBS and Tg measurement. T4 treatment was then resumed and maintained until TSH reached levels < 0.3 mIU/l. The T4 dose was then decreased to 0.8 microg/kg/day and TSH was measured weekly. Tg was assayed when TSH was > 30 mIU/l. Patients diagnosed with the disease upon initial evaluation were treated. We also evaluated the clinical and laboratory changes observed for both preparations. Using the reduced dose protocol, TSH levels > 30 mIU/l were reached within 6 and 8 weeks in 84.6 and 100% of the patients, respectively. T4 withdrawal was associated with more common symptoms of hypothyroidism and elevation of creatine kinase (CK) and LDL cholesterol. The T4 dose reduction protocol proved to be useful for Tg stimulation and ablative therapy, without the complication of severe hypothyroidism or the cost of recombinant TSH.

Spatiotemporally Resolved Tracking of Bacterial Responses to ROS-Mediated Damage at the Single-Cell Level with Quantitative Functional Microscopy.

Herein we report on the implementation of photofunctional microparticles in combination with optical tweezers for the investigation of bacterial responses to oxidative stress by means of quantitative functional microscopy. A combination of a strongly hydrophobic axially substituted Si(IV) phthalocyanine adsorbed onto silica microparticles was developed, and the structural and photophysical characterization was carried out. The microparticles are able to produce reactive oxygen species under the fluorescence microscope upon irradiation with red light, and the behavior of individual bacteria can be consequently investigated in situ and in real time at the single cell level. For this purpose, a methodology was introduced to monitor phototriggered changes with spatiotemporal resolution. The defined distance between the photoactive particles and individual bacteria can be fixed under the microscope before the photosensitization process is started, and the photoinduced damage can be monitored by tracing the time-dependent fluorescence turn-on of a suitable marker. The results showed a distance-dependent photoinduced death time, defined as the onset of the incorporation of propidium iodide. Our methodology constitutes a new tool for the in vitro design and evaluation of photosensitizers for the treatment of cancer and infectious diseases with the aid of functional optical microscopy, as it enables a quantitative response evaluation of living systems toward oxidative stress. More generally, it provides a way to understand the response of an ensemble of living entities to reactive oxygen species by analyzing the behavior of a set of individual organisms.