Evolutionary rescue of resistant mutants is governed by a balance between radial expansion and selection in compact populations
Serhii Aif,
Nico Appold,
Lucas Kampman,
Oskar Hallatschek,
Jona Kayser
Nature Communications
13
7916
(2022)
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Mutation-mediated treatment resistance is one of the primary challenges for modern antibiotic and anti-cancer therapy. Yet, many resistance mutations have a substantial fitness cost and are subject to purifying selection. How emerging resistant lineages may escape purifying selection via subsequent compensatory mutations is still unclear due to the difficulty of tracking such evolutionary rescue dynamics in space and time. Here, we introduce a system of fluorescence-coupled synthetic mutations to show that the probability of evolutionary rescue, and the resulting long-term persistence of drug resistant mutant lineages, is dramatically increased in dense microbial populations. By tracking the entire evolutionary trajectory of thousands of resistant lineages in expanding yeast colonies we uncover an underlying quasi-stable equilibrium between the opposing forces of radial expansion and natural selection, a phenomenon we term inflation-selection balance. Tailored computational models and agent-based simulations corroborate the fundamental nature of the observed effects and demonstrate the potential impact on drug resistance evolution in cancer. The described phenomena should be considered when predicting multi-step evolutionary dynamics in any mechanically compact cellular population, including pathogenic microbial biofilms and solid tumors. The insights gained will be especially valuable for the quantitative understanding of response to treatment, including emerging evolution-based therapy strategies.
Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system
Fidel-Nicolás Lolo,
Nikhil Walani,
Eric Seemann,
Dobryna Zalvidea,
Dácil María Pavón,
Gheorghe Cojoc,
Moreno Zamai,
Christine Varis de Lesegno,
Fernando Martínez de Benito, et al.
Nature Cell Biology
25
120-133
(2022)
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In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations—dolines—capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.
Immune Cell Deformability in Depressive Disorders: Longitudinal Associations Between Depression, Glucocorticoids and Cell Deformabilit
Andreas Walter,
Martin Kräter,
Clemens Kirschbaum,
Wei Gao,
Magdalena Wekenborg,
Marlene Penz,
Nicole Rothe,
Jochen Guck,
Lukas Daniel Wittwer, et al.
Background Cell deformability of all major blood cell types is increased in depressive disorders (DD). Furthermore, impaired glucocorticoid secretion is causally related to DD. Nevertheless, there are no longitudinal studies examining changes in glucocorticoid output and depressive symptoms regarding cell deformability in DD. Aim To investigate, whether changes in depressive symptoms or hair glucocorticoids predict cell deformability in DD. Methods In 136 individuals, depressive symptoms (PHQ-9) and hair glucocorticoids (cortisol and cortisone) were measured at timepoint one (T1), while one year later (T2) depressive symptoms and hair glucocorticoids were remeasured and additionally cell deformability of peripheral blood cells was assessed and DD status was determined by clinical interview. Results Depression severity at T1 predicted higher cell deformability in monocytes and lymphocytes over the entire sample. Subjects with continuously high depressive symptoms at T1 and T2 showed elevated monocyte deformability as compared to subjects with low depressive symptoms. Depression severity at T1 of subjects with a lifetime persistent depressive disorder (PDD) was associated with elevated monocyte, neutrophil, and granulo-monocyte deformability. Depression severity at T1 of subjects with a 12-month PDD was positively associated with monocyte deformability. Furthermore, increases in glucocorticoid concentrations from T1 to T2 tended to be associated with higher immune cell deformability, while strongest associations emerged for the increase in cortisone with elevated neutrophil and granulo-monocyte deformability in the 12-month PDD group. Conclusion Continuously elevated depressive symptomatology as well as an increase in glucocorticoid levels over one year are associated with higher immune cell deformability, particularly in PDD. These findings suggest, that persistent depressive symptomatology associated with increased glucocorticoid secretion may lead to increased immune cell deformability thereby compromising immune cell function and likely contributing to the perpetuation of PDD.
Viscoelastic properties of suspended cells measured with shear flow deformation cytometry
Richard Gerum,
Elham Mirzahossein,
Mar Eroles,
Jennifer Elsterer,
Astrid Mainka,
Andreas Bauer,
Selina Sonntag,
Alexander Winterl,
Johannes Bartl, et al.
Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 µm wide microfluidic channel. The fluid shear stress induces large, ear ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe [1] that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell-cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.
PNIPAAm microgels with defined network architecture as temperature sensors in optical stretchers
Nicolas Hauck,
Timon Beck,
Gheorghe Cojoc,
Raimund Schlüßler,
Saeed Ahmed,
Ivan Raguzin,
Martin Mayer,
Jonas Schubert,
Paul Müller, et al.
Materials Advances
3
6179-6190
(2022)
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Stretching individual living cells with light is a standard method to assess their mechanical properties. Yet, heat introduced by the laser light of optical stretchers may unwittingly change the mechanical properties of cells therein. To estimate the temperature induced by an optical trap, we introduce cell-sized, elastic poly(N-isopropylacrylamide) (PNIPAAm) microgels that relate temperature changes to hydrogel swelling. For their usage as a standardized calibration tool, we analyze the effect of free-radical chain-growth gelation (FCG) and polymer-analogous photogelation (PAG) on hydrogel network heterogeneity, micromechanics, and temperature response by Brillouin microscopy and optical diffraction tomography. Using a combination of tailor-made PNIPAAm macromers, PAG, and microfluidic processing, we obtain microgels with homogeneous network architecture. With that, we expand the capability of standardized microgels in calibrating and validating cell mechanics analysis, not only considering cell and microgel elasticity but also providing stimuli-responsiveness to consider dynamic changes that cells may undergo during characterization.
Quantitative phase imaging through an ultra-thin lensless fiber endoscope
Jiawei Sun,
Jiachen Wu,
Ruchi Goswami,
Salvatore Girardo,
Liangcai Cao,
Jochen Guck,
Nektarios Koukourakis,
Jürgen W. Czarske
Quantitative phase imaging (QPI) is a label-free technique providing both morphology and quantitative biophysical information in biomedicine. However, applying such a powerful technique to in vivo pathological diagnosis remains challenging. Multi-core fiber bundles (MCFs) enable ultra-thin probes for in vivo imaging, but current MCF imaging techniques are limited to amplitude imaging modalities. We demonstrate a computational lensless microendoscope that uses an ultra-thin bare MCF to perform quantitative phase imaging with microscale lateral resolution and nanoscale axial sensitivity of the optical path length. The incident complex light field at the measurement side is precisely reconstructed from the far-field speckle pattern at the detection side, enabling digital refocusing in a multi-layer sample without any mechanical movement. The accuracy of the quantitative phase reconstruction is validated by imaging the phase target and hydrogel beads through the MCF. With the proposed imaging modality, three-dimensional imaging of human cancer cells is achieved through the ultra-thin fiber endoscope, promising widespread clinical applications.
Long COVID: Association of Functional Autoantibodies against G-Protein-Coupled Receptors with an Impaired Retinal Microcirculation
Charlotte Szewczykowski,
Christian Mardin,
Marianna Lucio,
Gerd Wallukat,
Jakob Hoffmanns,
Thora Schröder,
Franziska Raith,
Lennart Rogge,
Felix Heltmann, et al.
International Journal of Molecular Sciences
23
(13)
7209
(2022)
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Long COVID (LC) describes the clinical phenotype of symptoms after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic and therapeutic options are limited, as the pathomechanism of LC is elusive. As the number of acute SARS-CoV-2 infections was and is large, LC will be a challenge for the healthcare system. Previous studies revealed an impaired blood flow, the formation of microclots, and autoimmune mechanisms as potential factors in this complex interplay. Since functionally active autoantibodies against G-protein-coupled receptors (GPCR-AAbs) were observed in patients after SARS-CoV-2 infection, this study aimed to correlate the appearance of GPCR-AAbs with capillary microcirculation. The seropositivity of GPCR-AAbs was measured by an established cardiomyocyte bioassay in 42 patients with LC and 6 controls. Retinal microcirculation was measured by OCT–angiography and quantified as macula and peripapillary vessel density (VD) by the Erlangen-Angio Tool. A statistical analysis yielded impaired VD in patients with LC compared to the controls, which was accentuated in female persons. A significant decrease in macula and peripapillary VD for AAbs targeting adrenergic β2-receptor, MAS-receptor angiotensin-II-type-1 receptor, and adrenergic α1-receptor were observed. The present study might suggest that a seropositivity of GPCR-AAbs can be linked to an impaired retinal capillary microcirculation, potentially mirroring the systemic microcirculation with consecutive clinical symptoms.
In vivo assessment of mechanical properties during axolotl development and regeneration using confocal Brillouin microscopy
In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behaviour are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such an increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.
Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens
Shada Abuhattum,
Petra Kotzbeck,
Raimund Schlüßler,
Alexandra Harger,
Angela Ariza de Schellenberger,
Kyoohyun Kim,
Joan-Carles Escolano,
Torsten Müller,
Jürgen Braun, et al.
Scientific Reports
12
10325
(2022)
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Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders.
Amoeboid-like migration ensures correct horizontal cell layer formation in the developing vertebrate retina
Migration of cells in the developing brain is integral for the establishment of neural circuits and function of the central nervous system. While migration modes during which neurons employ predetermined directional guidance of either preexisting neuronal processes or underlying cells have been well explored, less is known about how cells featuring multipolar morphology migrate in the dense environment of the developing brain. To address this, we here investigated multipolar migration of horizontal cells in the zebrafish retina. We found that these cells feature several hallmarks of amoeboid-like migration that enable them to tailor their movements to the spatial constraints of the crowded retina. These hallmarks include cell and nuclear shape changes, as well as persistent rearward polarization of stable F-actin. Interference with the organization of the developing retina by changing nuclear properties or overall tissue architecture hampers efficient horizontal cell migration and layer formation showing that cell-tissue interplay is crucial for this process. In view of the high proportion of multipolar migration phenomena observed in brain development, the here uncovered amoeboid-like migration mode might be conserved in other areas of the developing nervous system.
Wie mikrobielle Modellsysteme helfen, Tumorevolution zu entschlüsseln
While cellular evolution is one of the most fundamental concepts of life, its consequences are among the most pressing issues of modern health care, including cancer and the emergence of therapy resistance. We currently still lack the ability to accurately predict evolutionary trajectories, especially in spatially dense, pathogenic cellular populations such as microbial biofi lms or solid tumors. Here, we discuss the conceptual framework of evolution in dense populations and the potential of tailored microbial model systems to systematically study the underlying mechanisms.
Best practices for reporting throughput in biomedical research
Maik Herbig,
Akihiro Isozaki,
Dino Di Carlo,
Jochen Guck,
Nao Nitta,
Robert Damoiseaux,
Shogo Kamikawaji,
Eigo Suyama,
Hirofumi Shintaku, et al.
mRNA Subtype of Cancer-Associated Fibroblasts Significantly Affects Key Characteristics of Head and Neck Cancer Cells
Barbora Peltanová,
Hana Holcová Polanská,
Martina Raudenská,
Jan Balvan,
Jiri Navrátil,
Tomás Vicar,
Jaromir Gumulec,
Barbora Cechová,
Martin Kräter, et al.
Cancers / Molecular Diversity Preservation International (MDPI)
14
(9)
2286
(2022)
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Head and neck squamous cell carcinomas (HNSCC) belong among severe and highly complex malignant diseases showing a high level of heterogeneity and consequently also a variance in therapeutic response, regardless of clinical stage. Our study implies that the progression of HNSCC may be supported by cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) and the heterogeneity of this disease may lie in the level of cooperation between CAFs and epithelial cancer cells, as communication between CAFs and epithelial cancer cells seems to be a key factor for the sustained growth of the tumour mass. In this study, we investigated how CAFs derived from tumours of different mRNA subtypes influence the proliferation of cancer cells and their metabolic and biomechanical reprogramming. We also investigated the clinicopathological significance of the expression of these metabolism-related genes in tissue samples of HNSCC patients to identify a possible gene signature typical for HNSCC progression. We found that the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes can support the growth of primary cancer cells or metastases. Specifically, during coculture, cancer cell colony supporting effect and effect of CAFs on cell stiffness of cancer cells are driven by the mRNA subtype of the tumour from which the CAFs are derived. The degree of colony-forming support is reflected in cancer cell glycolysis levels and lactate shuttle-related transporters.
Depressive disorders are associated with increased peripheral blood cell deformability: a cross-sectional case-control study (Mood-Morph)
Andreas Walther,
Anne Mackens-Kiani,
Julian Eder,
Maik Herbig,
Christoph Herold,
Clemens Kirschbaum,
Jochen Guck,
Lucas Wittwer,
Katja Beesdo-Baum, et al.
Translational Psychiatry
12
150
(2022)
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Pathophysiological landmarks of depressive disorders are chronic low-grade inflammation and elevated glucocorticoid output. Both can potentially interfere with cytoskeleton organization, cell membrane bending and cell function, suggesting altered cell morpho-rheological properties like cell deformability and other cell mechanical features in depressive disorders. We performed a cross-sectional case-control study using the image-based morpho-rheological characterization of unmanipulated blood samples facilitating real-time deformability cytometry (RT-DC). Sixty-nine pre-screened individuals at high risk for depressive disorders and 70 matched healthy controls were included and clinically evaluated by Composite International Diagnostic Interview leading to lifetime and 12-month diagnoses. Facilitating deep learning on blood cell images, major blood cell types were classified and morpho-rheological parameters such as cell size and cell deformability of every individual cell was quantified. We found peripheral blood cells to be more deformable in patients with depressive disorders compared to controls, while cell size was not affected. Lifetime persistent depressive disorder was associated with increased cell deformability in monocytes and neutrophils, while in 12-month persistent depressive disorder erythrocytes deformed more. Lymphocytes were more deformable in 12-month major depressive disorder, while for lifetime major depressive disorder no differences could be identified. After correction for multiple testing, only associations for lifetime persistent depressive disorder remained significant. This is the first study analyzing morpho-rheological properties of entire blood cells and highlighting depressive disorders and in particular persistent depressive disorders to be associated with increased blood cell deformability. While all major blood cells tend to be more deformable, lymphocytes, monocytes, and neutrophils are mostly affected. This indicates that immune cell mechanical changes occur in depressive disorders, which might be predictive of persistent immune response.
Changes in Blood Cell Deformability in Chorea-Acanthocytosis and Effects of Treatment With Dasatinib or Lithium
Felix Reichel,
Martin Kräter,
Kevin Peikert,
Hannes Glaß,
Philipp Rosendahl,
Maik Herbig,
Alejandro Rivera Prieto,
Alexander Kihm,
Giel Bosman, et al.
Frontiers in Physiology
13
852946
(2022)
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Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of five ChAc patients compared to healthy controls during up to 1-year individual off-label treatment with the tyrosine kinase inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results showed that blood cell deformability–including both RBCs and leukocytes - in general was altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observed alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hinted at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.
Unbiased retrieval of frequency-dependent mechanical properties from noisy time-dependent signals
Shada Abuhattum,
Hui-Shun Kuan,
Paul Mueller,
Jochen Guck,
Vasily Zaburdaev
Biophysical Reports
2
(3)
100054
(2022)
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The mechanical response of materials to dynamic loading is often quantified by the frequency-dependent complex modulus. Probing materials directly in the frequency domain faces technical challenges such as a limited range of frequencies, long measurement times, or small sample sizes. Furthermore, many biological samples, such as cells or tissues, can change their properties upon repetitive probing at different frequencies. Therefore, it is common practice to extract the material properties by fitting predefined mechanical models to measurements performed in the time domain. This practice, however, precludes the probing of unique and yet unexplored material properties. In this report, we demonstrate that the frequency-dependent complex modulus can be robustly retrieved in a model-independent manner directly from time-dependent stress-strain measurements. While applying a rolling average eliminates random noise and leads to a reliable complex modulus in the lower frequency range, a Fourier transform with a complex frequency helps to recover the material properties at high frequencies. Finally, by properly designing the probing procedure, the recovery of reliable mechanical properties can be extended to an even wider frequency range. Our approach can be used with many state-of-the-art experimental methods to interrogate the mechanical properties of biological and other complex materials.
An explicit model to extract viscoelastic properties of cells from AFM force-indentation curves
Shada Abuhattum Hofemeier,
Dominic Mokbel,
Paul Müller,
Despina Soteriou,
Jochen Guck,
Sebastian Aland
Atomic force microscopy (AFM) is widely used for quantifying the mechanical properties of soft materials such as cells. AFM force-indentation curves are conventionally fitted with a Hertzian model to extract elastic properties. These properties solely are, however, insufficient to describe the mechanical properties of cells. Here, we expand the analysis capabilities to describe the viscoelastic behavior while using the same force-indentation curves. Our model gives an explicit relation of force and indentation and extracts physically meaningful mechanical parameters. We first validated the model on simulated force-indentation curves. Then, we applied the fitting model to the force-indentation curves of two hydrogels with different crosslinking mechanisms. Finally, we characterized HeLa cells in two cell cycle phases, interphase and mitosis, and showed that mitotic cells have a higher apparent elasticity and a lower apparent viscosity. Our study provides a simple method, which can be directly integrated into the standard AFM framework for extracting the viscoelastic properties of materials.
An exception to the rule? Regeneration of the injured spinal cord in the spiny mouse
The capacity for long-distance axon regeneration and functional recovery after spinal cord injury in the adult has long been thought to be a unique feature of certain non-mammalian vertebrates. However, in this issue of Developmental Cell, Nogueira-Rodrigues et al. report an astonishingly high regenerative ability in the spiny mouse.
Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition
Kyoohyun Kim,
Vamshidhar Gade,
Teymuras V. Kurzchalia,
Jochen Guck
Biophysical Journal
121
(7)
1219-1229
(2022)
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Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva, which can then further survive harsh desiccation in an anhydrobiotic state. We have previously identified the genetic and biochemical pathways essential for survival—but without detailed knowledge of their material properties, the mechanistic understanding of this intriguing phenomenon remains incomplete. Here we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. ODT revealed that the properties of the dauer larvae undergo a dramatic transition upon harsh desiccation. Moreover, mutants that are sensitive to desiccation displayed structural abnormalities in the anhydrobiotic stage that could not be observed by conventional microscopy. Our advance opens a door to quantitatively assessing the transitions in material properties and structure necessary to fully understand an organism on the verge of life and death.
Nonlinear microscopy using impulsive stimulated Brillouin scattering for high-speed elastography
Benedikt Krug,
Nektarios Koukourakis,
Jochen Guck,
Jürgen Czarske
Optics Express
30
(4)
4748-4758
(2022)
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The impulsive stimulated Brillouin microscopy promises fast, non-contact measurements of the elastic properties of biological samples. The used pump-probe approach employs an ultra-short pulse laser and a cw laser to generate Brillouin signals. Modeling of the microscopy technique has already been carried out partially, but not for biomedical applications. The nonlinear relationship between pulse energy and Brillouin signal amplitude is proven with both simulations and experiments. Tayloring of the excitation parameters on the biologically relevant polyacrylamide hydrogels outline sub-ms temporal resolutions at a relative precision of <1%. Brillouin microscopy using the impulsive stimulated scattering therefore exhibits high potential for the measurements of viscoelastic properties of cells and tissues.
Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues
Maik Herbig,
Karen Tessmer,
Martin Nötzel,
Ahmad Ahsan Nawaz,
Tiago Santos-Ferreira,
Oliver Borsch,
Sylvia J. Gasparini,
Jochen Guck,
Marius Ader
Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells’ properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.
Machine learning assisted real-time deformability cytometry of CD34+ cells allows to identify patients with myelodysplastic syndromes
Maik Herbig,
Angela Jacobi,
Manja Wobus,
Heike Weidner,
Anna Mies,
Martin Kräter,
Oliver Otto,
Christian Thiede,
Marie-Theresa Weickert, et al.
Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts.
Mechanical spinal cord transection in larval zebrafish and subsequent whole-mount histological processing
Nora John,
Julia Kolb,
Daniel Wehner
STAR Protocols
3
(1)
101093
(2022)
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Zebrafish regenerate their spinal cord after injury, both at larval and adult stages. Larval zebrafish have emerged as a powerful model system to study spinal cord injury and regeneration due to their high optical transparency for in vivo imaging, amenability to high-throughput analysis, and rapid regeneration time. Here, we describe a protocol for the mechanical transection of the larval zebrafish spinal cord, followed by whole-mount tissue processing for in situ hybridization and immunohistochemistry to elucidate principles of regeneration.
Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity
Raimund Schlüßler,
Kyoohyun Kim,
Martin Nötzel,
Anna Taubenberger,
Shada Abuhattum,
Timon Beck,
Paul Müller,
Shovamaye Maharana,
Gheorghe Cojoc, et al.
Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples − so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epifluorescence imaging for explicitly measuring the Brillouin shift, RI, and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample − a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.
Contact
Cell Physics Division Prof. Vahid Sandoghdar Acting Division Head
Max Planck Institute for the Science of Light Staudtstr. 2 91058 Erlangen, Germany
