We demonstrate that the optical stretcher, a fully automated dual-beam laser trap for probing single-cell mechanics, can also be used to trap pairs of cells and manipulate them. More specifically, we can press cells against each other and tear them apart again, enabling us to measure cell adhesion / dissociation dynamics.
We show that we can see differences in adhesion behaviour between cell lines and we see single-molecule dissociation processes. We calculate the forces which we exert on the cells, which are in the pN range.
This "optical micromanipulator" provides high-throughput adhesion measurements with about 50 cell pairs per hour, while allowing for full optical inspection of the cell dissociation process. The method can be combined with other characterization methods readily available in the stretcher such as fluorescence imaging and cell rheology evaluation.
Even minute alterations in a cell's intracellular scaffolds, i.e. the cytoskeleton, which organize a cell, result in significant changes in a cell's elastic strength since the cytoskeletal mechanics nonlinearly amplify these alterations. Light has been used to observe cells since Leeuwenhoek's times and novel techniques in optical microscopy are frequently developed in biological physics. In contrast, with the optical stretcher we use the forces caused by light described by Maxwell's surface tensor to feel cells. Thus, the stretcher exemplifies the other type of biophotonic devices that do not image but manipulate cells. The optical stretcher uses optical surface forces to stretch cells between two opposing laser beams, while optical gradient forces, which are used in optical tweezers, play a minor role and only contribute to a stable trapping configuration. The combination of the optical stretcher's sensitivity and high throughput capacity make a cell's "optical stretchiness" an extremely precise parameter to distinguish different cell types. This avoids the use of expensive, often unspecific molecular cell markers. This technique applies particularly well to cells with dissimilar degrees of differentiation, as a cell's maturation correlates with an increase in cytoskeletal strength. Because malignant cells gradually dedifferentiate during the progression of cancer, the optical stretcher should allow, the direct staging from early dysplasia to metastasis of a tumor sample obtained by MRI-guided fine needle aspirations or cytobrushes. With two prototypes of a microfluidic optical stretcher at our hands, we prepare preclinical trials to study its potential in resolving breast tumors' progression towards metastasis. Since the optical stretcher represents a basic technology for cell recognition and sorting, an abundance of further biomedical applications can be envisioned.
Confocal and multiphoton microscopy are essential tools in modern life sciences. They allow fast and highly resolved imaging of a steadily growing number of fluorescent markers, ranging from fluorescent proteins to quantum dots and other fluorophores, used for the localization of molecules and the quantitative detection of molecular properties within living cells and organisms. Up to now, only one physical limitation seemed to be unavoidable. Both confocal and multiphoton microscopy rely on lasers as excitation sources, and their monochromatic radiation allows only a limited number of simultaneously usable dyes, which depends on the specific number of laser lines available in the used microscope. We have overcome this limitation by successfully replacing all excitation lasers in a standard confocal microscope with pulsed white light ranging from 430 to 1300 nm generated in a tapered silica fiber. With this easily reproducible method, simultaneous confocal and multiphoton microscopy was demonstrated. By developing a coherent and intense laser source with spectral width comparable to a mercury lamp, we provide the flexibility to excite any desired fluorophore combination.
Understanding and controlling neuronal growth are basic objectives in neuroscience, biology, biophysics, and biomedicine, and are vital for the formation of neural circuits in vitro, as well as for nerve regeneration in vivo. All molecular stimuli for neuronal growth eventually address the polymeric cytoskeleton, which advances a neurite's leading edge also known as the growth cone. We have shown that optical forces of a highly focused infrared laser beam influence the motility of a growth cone by biasing the polymerization-driven intracellular machinery. In actively extending growth cones, a laser spot placed at specific areas of the neurite's leading edge affects the growth speed, the direction taken by a growth cone, and the splitting of a growth cone. This novel optical tool manipulates a natural biological process, the cytoskeleton driven morphological changes in growth cones, with potential applications in the formation of neuronal networks and in understanding growth cone motility. The current apparatus combines optical tweezers, phase contrast and fluorescence imaging, and real-time shape detection. Automated and dynamically readjusted irradiation of the growth cone is used to examine and to influence structural and morphological changes of neuronal growth.