Reduction of the influence of parasitic interferences and coherence induced disturbances in digital holography-based quantitative phase microscopy by modulation of the object illumination with an electrically focus tunable lens

Kemper, Björn; Barroso, Álvaro; Eder, Kai; Marzi, Anne; Ritz, Sabrina; Schnekenburger, Jürgen; Ketelhut, Steffi

doi:10.1117/12.2577807

Cited by 3 publications

(6 citation statements)

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“…Initially selected field of views (FOVs) at the beginning of the imaging process were observed during the whole experimental period of 24 h. FOVs at t = 0 were selected individually with regard to representative cell confluencies for each cell line and to achieve cell numbers > 100 cells within 12 FOVs for each individual experiment, in accordance with earlier studies on the reproducibility of cell analysis with DHM [ 42 ]. For each measurement and time point, one bright-field image and 15 holograms were captured while the object illumination wave was modulated by an electrically tunable lens (ETL) [ 43 ]. The reconstruction of quantitative phase images from the digitally captured holograms was performed numerically by a variant of the Fourier transformation method as described before [ 44 ], utilizing custom-built software implemented in python 3.7.…”

Section: Methodsmentioning

confidence: 99%

“…Optional numerical refocusing was applied, utilizing a convolution approach [ 24 ] to propagate the object wave to the image plane if cells were not optically in focus during hologram recording. Subsequently, after reconstruction, unwrapped quantitative phase images for every position and time point were averaged to reduce coherence-induced image disturbances [ 43 ]. Irregular spatial phase background fluctuations, such as caused by optical thickness variations of the bottom of the applied polymer well chambers used, were compensated by the ImageJ feature “Subtract background” based on the rolling ball algorithm [ 45 ] with identical parameters for all conducted experiments.…”

Section: Methodsmentioning

confidence: 99%

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

Eder¹,

Marzi²,

Barroso³

et al. 2022

Cells

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Eder¹,

Marzi²,

Barroso³

et al. 2022

Cells

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“…As explained in detail in Eder et al [28], digitally captured off-axis holograms from the DHM QPI measurements were reconstructed by a variant of the Fourier transformation method as described before [37] and, if required, numerically refocused by using a convolution approach [36]. The obtained QPI images for every position and time point were averaged to reduce coherence-induced image disturbances [38] and subsequently processed by the ImageJ (version 2.14.0/1.54f) feature "Subtract background" based on the rolling ball algorithm [39] to compensate for irregular spatial phase background fluctuations. From the QPI images, the dry mass (dm) [40] of the whole cell population within the observed FOV (S FOV = 405 µm × 538 µm) was calculated at the beginning of the measurement (t = 0) and after 24 h (t = 24), as described in Equation ( 1).…”

Section: Dhm Qpi Cell Proliferation Assaymentioning

confidence: 99%

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

Marzi,

Eder,

Barroso

et al. 2024

Cells

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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.

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“…2B. For each measurement and time point, one bright-field image and 7 digital off-axis holograms were captured with exposure times in millisecond or sub-millisecond range while the object illumination wave was modulated by an electrically tuneable lens [43]. Subsequent processing of the acquired holograms was performed as illustrated in Fig.…”

Section: Utilized Dhm Systems and Generation Of Qpi Imagesmentioning

confidence: 99%

“…First, quantitative phase images were reconstructed from each series of digitally captured holograms numerically utilizing a previously described variant of the Fourier transformation method [44] and optional numerical refocussing [45]. Then, QPI images for every position and time point were averaged to reduce coherence induced image disturbances [43]. Prior the experimental investigations, the QPI image quality of each DHM system was analysed utilizing a 3D-printed phase test chart as shown in supplementary material, Fig.…”

Section: Utilized Dhm Systems and Generation Of Qpi Imagesmentioning

confidence: 99%

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

Marzi¹,

Eder²,

Barroso³

et al. 2022

Drug Deliv. and Transl. Res.

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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. Graphical abstract

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Reduction of the influence of parasitic interferences and coherence induced disturbances in digital holography-based quantitative phase microscopy by modulation of the object illumination with an electrically focus tunable lens

Cited by 3 publications

References 0 publications

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

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

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

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

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