Jochen Guck

The Optical Stretcher: A Novel Laser Tool to Micromanipulate Cells

When a dielectric object is placed between two opposed, nonfocused laser beams, the total force acting on the object is zero but the surface forces are additive, thus leading to a stretching of the object along the axis of the beams. Using this principle, we have constructed a device, called an optical stretcher, that can be used to measure the viscoelastic properties of dielectric materials, including biologic materials such as cells, with the sensitivity necessary to distinguish even between different individual cytoskeletal phenotypes. We have successfully used the optical stretcher to deform human erythrocytes and mouse fibroblasts. In the optical stretcher, no focusing is required, thus radiation damage is minimized and the surface forces are not limited by the light power. The magnitude of the deforming forces in the optical stretcher thus bridges the gap between optical tweezers and atomic force microscopy for the study of biologic materials.

Nuclear Architecture of Rod Photoreceptor Cells Adapts to Vision in Mammalian Evolution

We show that the nuclear architecture of rod photoreceptor cells differs fundamentally in nocturnal and diurnal mammals. The rods of diurnal retinas possess the conventional architecture found in nearly all eukaryotic cells, with most heterochromatin situated at the nuclear periphery and euchromatin residing toward the nuclear interior. The rods of nocturnal retinas have a unique inverted pattern, where heterochromatin localizes in the nuclear center, whereas euchromatin, as well as nascent transcripts and splicing machinery, line the nuclear border. The inverted pattern forms by remodeling of the conventional one during terminal differentiation of rods. The inverted rod nuclei act as collecting lenses, and computer simulations indicate that columns of such nuclei channel light efficiently toward the light-sensing rod outer segments. Comparison of the two patterns suggests that the conventional architecture prevails in eukaryotic nuclei because it results in more flexible chromosome arrangements, facilitating positional regulation of nuclear functions.

Viscoelastic properties of individual glial cells and neurons in the CNS

One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word “γλια” means “glue”) or provide a robust scaffold for them (“support cells”). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft (“rubber elastic”), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.

Oral Cancer Diagnosis by Mechanical Phenotyping

Oral squamous cell carcinomas are among the 10 most common cancers and have a 50% lethality rate after 5 years. Despite easy access to the oral cavity for cancer screening, the main limitations to successful treatment are uncertain prognostic criteria for (pre-)malignant lesions. Identifying a functional cellular marker may represent a significant improvement for diagnosis and treatment. Toward this goal, mechanical phenotyping of individual cells is a novel approach to detect cytoskeletal changes, which are diagnostic for malignant change. The compliance of cells from cell lines and primary samples of healthy donors and cancer patients was measured using a microfluidic optical stretcher. Cancer cells showed significantly different mechanical behavior, with a higher mean deformability and increased variance. Cancer cells (n approximately 30 cells measured from each patient) were on average 3.5 times more compliant than those of healthy donors [D(normal) = (4.43 +/- 0.68) 10(-3) Pa(-1); D(cancer) = (15.8 +/- 1.5) 10(-3) Pa(-1); P < 0.01]. The diagnosis results of the patient samples were confirmed by standard histopathology. The generality of these findings was supported by measurements of two normal and four cancer oral epithelial cell lines. Our results indicate that mechanical phenotyping is a sensible, label-free approach for classifying cancer cells to enable broad screening of suspicious lesions in the oral cavity. It could in principle be applied to any cancer to aid conventional diagnostic procedures.

Müller cells are living optical fibers in the vertebrate retina

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.

Real-time deformability cytometry: on-the-fly cell mechanical phenotyping

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

The regulatory role of cell mechanics for migration of differentiating myeloid cells

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytokeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.

Mechanical difference between white and gray matter in the rat cerebellum measured by scanning force microscopy

The mechanical properties of tissues are increasingly recognized as important cues for cell physiology and pathology. Nevertheless, there is a sparsity of quantitative, high-resolution data on mechanical properties of specific tissues. This is especially true for the central nervous system (CNS), which poses particular difficulties in terms of preparation and measurement. We have prepared thin slices of brain tissue suited for indentation measurements on the micrometer scale in a near-native state. Using a scanning force microscope with a spherical indenter of radius ∼20μm we have mapped the effective elastic modulus of rat cerebellum with a spatial resolution of 100μm. We found significant differences between white and gray matter, having effective elastic moduli of K=294±74 and 454±53Pa, respectively, at 3μm indentation depth (n(g)=245, n(w)=150 in four animals, p<0.05; errors are SD). In contrast to many other measurements on larger length scales, our results were constant for indentation depths of 2-4μm indicating a regime of linear effective elastic modulus. These data, assessed with a direct mechanical measurement, provide reliable high-resolution information and serve as a quantitative basis for further neuromechanical investigations on the mechanical properties of developing, adult and damaged CNS tissue.

Deformability-Based Flow Cytometry

Elasticity of cells is determined by their cytoskeleton. Changes in cellular function are reflected in the amount of cytoskeletal proteins and their associated networks. Drastic examples are diseases such as cancer, in which the altered cytoskeleton is even diagnostic. This connection between cellular function and cytoskeletal mechanical properties suggests using the deformability of cells as a novel inherent cell marker.

Mesenchymal Stem Cell Mechanics from the Attached to the Suspended State

Human mesenchymal stem cells (hMSCs) are therapeutically useful cells that are typically expanded in vitro on stiff substrata before reimplantation. Here we explore MSC mechanical and structural changes via atomic force microscopy and optical stretching during extended passaging, and we demonstrate that cytoskeletal organization and mechanical stiffness of attached MSC populations are strongly modulated over >15 population doublings in vitro. Cytoskeletal actin networks exhibit significant coarsening, attendant with decreasing average mechanical compliance and differentiation potential of these cells, although expression of molecular surface markers does not significantly decline. These mechanical changes are not observed in the suspended state, indicating that the changes manifest themselves as alterations in stress fiber arrangement rather than cortical cytoskeleton arrangement. Additionally, optical stretching is capable of investigating a previously unquantified structural transition: remodeling-induced stiffening over tens of minutes after adherent cells are suspended. Finally, we find that optically stretched hMSCs exhibit power-law rheology during both loading and recovery; this evidence appears to be the first to originate from a biophysical measurement technique not involving cell-probe or cell-substratum contact. Together, these quantitative assessments of attached and suspended MSCs define the extremes of the extracellular environment while probing intracellular mechanisms that contribute to cell mechanical response.