We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.
Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts' rheology.
Embryonic development is a highly reproducible process during which the cells and tissues deform to shape the future animal. Such deformations have been shown to require the active generation of forces at the cell level1,2. To understand morphogenetic processes during which cells and tissues change their shape, it is therefore key to assess the mechanical properties of the cells involved, and to measure the forces within the tissue during the process3,4. Epithelial layers, especially in Drosophila, have been widely studied due to their quasi-2D geometry and to their easy manipulation.
A number of techniques have thus been developed by us and others to assess epithelial mechanics in vivo during development. We will give a quick overview of the three main techniques used in epithelial tissues. Laser ablation, a widely used method, allows to reveal the local mechanical stress at cell junctions5,6,7,8 or at larger scale9,10,11 by performing local cuts that disrupt the mechanical integrity of the target. The dynamics of the opening following the cut provides information both on the stress prior ablation, and on the rheology of the tissue12,13. A drawback of laser ablation is that it is invasive, as it requires the local disruption of the cell cortex. Hence, one can only perform a limited number of ablations if one wants to preserve tissue integrity. Another drawback is that ablations only provide relative estimates of tension at cell contacts, since the opening velocity is dependent on viscous friction, which is generally not known. Magnetic manipulation has also been developed and used in Drosophila, involving either the use of ferrofluids14 or ultramagnetic liposomes15. They can provide absolute measurements16,17, but are also invasive in the sense that they require the injection of probes at the desired location. This can be very tricky depending on the system, which is not always amenable to precise injections. A third technique, fully non-invasive, is force inference18,19,20. Force inference relies on the assumption of mechanical equilibrium at triple points (tricellular junctions, or vertices), and allows to infer tensions at all cell-cell contacts (and possibly, pressures in all cells) by solving an inverse problem. For tensions, each vertex provides two equations (X and Y). This yields a large system of linear equations which can be inverted under some conditions to assess tension at all cell contacts. While this method is very attractive, as it only requires a segmented image and no extra experiment or setup, its accuracy is yet to determine, and again it only provides relative values, unless an absolute calibration measurement is performed.
To overcome some of these limitations, we introduce in this article a setup of optical tweezers coupled to a light sheet microscope to apply controlled forces at the cell scale in the embryonic epithelium of Drosophila melanogaster. Optical tweezers have been used for numerous biological applications including the measurements on single proteins and manipulation of organelles and cells21. Here, we report applied forces in the range of a few dozen pN, which is small yet sufficient to induce local deformations of cell contacts and perform mechanical measurements in vivo. Typically, we use perpendicular deflection of cell contacts, monitored through the analysis of kymographs, to relate force to deformation. Importantly, our setup does not require the injection of beads at the desired location in the tissue, as optical tweezers are able to directly exert forces on cell-cell contacts. The coupling of the optical tweezers to a light sheet microscope allows one to perform rapid imaging (several frames per second), which is very appreciable for a mechanical analysis at short time scales, and with reduced phototoxicity, since the illumination of the sample is limited to the plane of imaging22.
Overall, optical tweezers are a non-invasive way to apply controlled forces at cell contacts in vivo in the Drosophila embryo, and to extract mechanical information such as stiffness and tension at cell contacts23, rheological properties24, and gradient or anisotropy of tension23.
1. Setting-up the Light Sheet Microscope
2. Setting-up the Optical Tweezers Path
NOTE: Figure 1 gives a general scheme of the optical setup.
3. Interfacing the Instrument
NOTE: Figure 3 gives a general scheme of the National Instruments (NI) Card connections.
4. Calibration of the Optical Trap Position with Beads
5. Mounting Drosophila Embryos27
6. Trapping Experiment In Vivo
7. Mechanical Measurements
8. Calibration of the Trap Stiffness
NOTE: The determination of the absolute forces requires the knowledge of the trap stiffness on interfaces. This can be accessed using beads through a two-step procedure.
Figure 5 shows experimental data obtained by imposing a sinusoidal movement to the trap. The trap produces a deflection of the interface, as exemplified by the 3 snapshots showing 3 successive interface positions (Figure 5A)23. Recorded movies are used to generate a kymograph (Figure 5B) and are further analyzed to determine the position of the interface with subpixel resolution, using a Gaussian fit along the x direction for each frame. In the regime of small deformations, the trap and interface positions are proportional (Figure 5C). The amplitude of the interface deflection relative to that of the trap (Figure 5D) gives access to the interface tension or stiffness (see step 7.1).
Figure 6 shows experimental data obtained by performing pull release experiments. The optical trap is switched on approximately 1 µm away from the midpoint of the interface between two cells, which causes the interface to deflect towards the trap position (Figure 6A)24. The trap is then switched off after the desired amount of time. The position xm of the interface (Figure 6B) is obtained from kymographs (Figure 6C), again using Gaussian fits along the x direction for each frame. The resulting graph can be compared to hypothesized rheological models, for instance, a Maxwell model (Figure 6D).
Figure 1: Schematic of the optical tweezers (red path) setup combined with the light sheet microscope. This figure has been modified from Bambardekar, K. et al.23.The light sheet microscope, composed by the illumination unit and the detection unit, is described previously25. The optical tweezers correspond to the red part of the scheme: The infrared laser passes through an optical shutter and 2 galvanometers which control the position of the trap in the sample. A 1-fold telescope is placed between the 2 galvanometers to keep the conjugation between them. Then, a telescope increases the size of the beam by 2.5-fold and the periscope brings it to the height of the microscope. The infrared laser enters the detection objective of the microscope thanks to a dichroic mirror.Important distances between optical elements are given directly in the figure. Distance between the last lens (focal length 500 mm) and the back aperture of the objective is 500 mm. Please click here to view a larger version of this figure.
Figure 2: Homemade modified rail holding the dichroic mirror and the hot mirror. The dichroic mirror rail of the microscope has been cut on the left side to allow the entrance of the infrared laser. The dichroic mirror reflects the infrared light and transmits the visible light (fluorescence). Please click here to view a larger version of this figure.
Figure 3: Connection of the instruments to the NI Cards. AO: analog output, AI: analog input, AO0 and AI0 are connected to galvo1, AO1 and AI1 are connected to galvo2, PFI0 is connected to the fire of the camera, to AI2 and PFI1 is connected to the trigger in of the shutter and AI3 is connected to the trigger out of the shutter. Please click here to view a larger version of this figure.
Figure 4: Sample holder in the observation context. This figure has been modified from Chardès, C., et al.25. The embryos are immobilized on the glass coverslip. The slide is held by the sample holder. The sample holder is inserted in the glass cuvette, held by the holder fixed to the piezoelectric stage. The light sheet is horizontal and illuminates the embryos from the side. The detection objective lens is vertical, above the sample, and dips into the cuvette.
Figure 5: Interface deflection imposed by sinusoidal movement of the trap. This figure has been modified from Bambardekar, K. et al23. (A) The trap is moved sinusoidally perpendicular to the interface. The trap and interface positions are xt and xm, respectively. The right panels show three images of the interface at different positions. The laser trap position is marked by a yellow arrowhead. Interface are labelled with a membrane marker (GAP43::mcherry). (B) Kymograph along the axis defined by the direction of trap movement (perpendicular to the interface) (Period = 5 s). (C) Representative plot of deflection versus time showing both trap (red solid line) and interface positions (black solid line). (D) Interface position as a function of trap position during few cycles of laser oscillation (Amplitude = 0.5 µm, Period = 2 s). Please click here to view a larger version of this figure.
Figure 6: Interface deflection in pull-release experiments. This figure has been modified from Serge, A., et al.24. (A) The trap is switched on at a distance from the midpoint of the interface, then switched off again. (B) The trap and interface positions are xt and xm, respectively. Kymographs are generated along the x direction, perpendicular to the contact line's midpoint. (C) Kymograph of a pull-release experiment. Cell contacts are labelled with Utrophin::GFP. (D) Representative plot of deflection versus time showing both trap (dotted red/green lines) and interface position (black solid line). The solid red line shows a fit obtained using a Maxwell-like rheological model24. Please click here to view a larger version of this figure.
Supplementary Movie 1: Tweezing experiment. Pixel size = 194 nm, 10 fps, Trap oscillation period = 2 s, Labelling: Gap43::mCherry. Please click here to download this file.
Optical tweezers allow to perform absolute mechanical measurements directly in the developing embryonic epithelium in a non-invasive manner. In that sense, it presents advantages over other methods such as laser ablation, which are invasive and provide relative measurements, magnetic forces, which require injections, or force inference, which relies on strong assumptions and also provide relative measurements.
The protocol includes a few critical steps. First, as the objective lens may show chromatic aberrations and that the laser trap pushes the object "downstream", it is important to check that the IR laser traps the bead in the imaging plane, and eventually correct for it (step 2.18). Second, the method relies on the measurements of the cell contacts' position. It is thus crucial to use a high-contrast fluorescent marker.
The quality of the objective lens and the laser beam are critical to effective trapping. The numerical aperture of the objective lens should exceed 1.0. If trapping is ineffective, make sure that the laser beam fills the rear aperture of the objective lens.
Our method comes with several limitations. First, it is not clear what exactly provides the support for optical trapping. Although a mismatch of refractive index is detected, its origin remains to be determined. Second, the calibration process, if one is interested in absolute measurements, can be a bit tedious, as it requires passive microrheology experiments, and calibration of the trap stiffness on beads. It is important to recognize that the calibration of the stiffness on cell contacts is subject to experimental uncertainty: it relies on measurements of the trap stiffness on beads, which can be measured only in the cytosol, but not near cell contacts. Third, it is unclear how versatile optical tweezers can be. Although they are able to deform the cells in the Drosophila embryo, other tissues might present higher tension or more difficult access (such as going through a cuticle) and therefore be less amenable to optical tweezing.
Optical forces are small (<few tens of pN) and may be thus insufficient to deform stiff or highly tensed structures. Magnetic tweezers on large particles would probably be more effective in this case.
We described here the coupling of optical tweezers to a light sheet microscope, but optical tweezers can be coupled to other types of microscopes, such as an epifluorescence or a confocal spinning disk microscope. The introduction of the IR trapping laser into the microscope depends on the microscope configuration. It mainly requires the possibility to add a dichroic mirror to combine the paths for imaging and optical manipulation. Most microscopy companies propose modular illumination systems, with a two-layer module, which allow this combination.
Several directions exist to improve or upgrade the technique. A possibility is to split the laser residence time among several positions or to use more advanced holographic techniques, to produce several traps. This could allow to create more complex force patterns on target cells or cell contacts. Another improvement could be to design a real-time feedback between the deflection caused and the position of the trap. This could allow proper creep experiments in which the force applied is maintained constant throughout the experiment.
The authors have nothing to disclose.
This work was supported by an FRM Equipe Grant FRM DEQ20130326509, Agence Nationale de la Recherche ANR-Blanc Grant, Morfor ANR-11-BSV5-0008 (to P.-F.L.). We acknowledge France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04-01, «Investments for the future»). We thank Brice Detailleur and Claude Moretti from the PICSL-FBI infrastructure for technical assistance.
Ytterbium Fiber Laser LP, 10 W, CW | IPG Laser | YLM-10-LP-SC | including collimator LP : beam D=1.6 mm and red guide laser |
Ø1/2" Optical Beam Shutter | Thorlabs | SH05 | |
Small Beam Diameter Galvanometer Systems | Thorlabs | GVS001 | 1 for X displacement, 1 for Y displacement |
1D or 2D Galvo System Linear Power Supply | Thorlabs | GPS011 | galvanometers power supply |
2 lenses f = 30mm | Thorlabs | LB1757-B | relay telescope between 2 galva |
Lens f = 200mm | Thorlabs | LB1945-B | 2.5X telescope |
Lens f = 500mm | Thorlabs | LB1869-B | 2.5X telescope |
Right-Angle Kinematic Elliptical Mirror Mount with Tapped Cage Rod Holes | Thorlabs | KCB1E | Periscope |
Laser Safety Glasses, Light Green Lenses, 59% Visible Light Transmission, Universal Style | Thorlabs | LG1 | |
45° AOI, 50.0mm Diameter, Hot Mirror | Edmund Optics | #64-470 | |
Multiphoton-Emitter HC 750/S | AHF | HC 750/SP | |
CompactDAQ Chassis | National Instruments | cDAQ-9178 | |
C Series Voltage Output Module | National Instruments | NI-9263 | Analog output module |
C Series Voltage Input Module | National Instruments | NI-9215 | Analog input module |
FluoSpheres Carboxylate-Modified Microspheres, 0.5 µm, red fluorescent (580/605), 2% solids | ThermoFisher Scientific | F8812 | calibration beads |
C++ (Qt) home made optical tweezers software | developed by Olivier Blanc and Claire Chardès. Alternative solution: labview |