Force Measurement Software Module

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1 Microscopy from Carl Zeiss Force Measurement Software Module Optical tweezers are used to manipulate microscopic particles such as cells or sub-cellular structures. The Force Measurement (FM) module expands this functionality with the quantitative measurement of forces relevant to various disciplines of life science research. Forces in biology Optical tweezers are a widely used technique in life science research, enabling small dielectric objects such as cells, crystals or beads to be trapped and moved simply with the force of light. This technology exhibits precise force-exerting characteristics that are often used for mechanical force measurements in the pn-range, e. g. for single cell studies. Various biological processes such as intermolecular binding happen within this force regime and are therefore perfectly suited to be probed by optical tweezers. The broad force range (~ pN) and sensitivity of PALM MicroTweezers in combination with the ease of use of the Force Measurement software make this system an extremely powerful tool in life sciences. PALM MicroTweezers and Force Measurement

2 PALM MicroTweezers Optical tweezers are generally used to actively manipulate and position microscopic objects. This technology is rapidly gaining more importance in various scientific fields, especially biology. PALM MicroTweezers are typically applied to confine single cells as well as to organize, sort or modify them. Optical trapping PALM MicroTweezers use a continuous wave solid state laser with 1064nm wavelength and 3W output power. By focusing the laser beam with a high numerical aperture objective, optical traps are generated in the specimen allowing the non-invasive manipulation of microscopic particles ranging in size from approximately µm. The optical tweezers instrument by Carl Zeiss. PALM MicroTweezers & FM at a glance Ease of use with click & trap Contact-free 3D control of the trapped objects Perturbation-free handling of biological systems (working in medium) Robust and reliable setup No detector alignment necessary Calibrated and precise data Experimental freedom with high flexibility From manipulation to force measurement Safety with laser class 1M Attractive price-performance ratio Compatible with other Zeiss solutions such as PALM MicroBeam and Colibri PALM MicroTweezers can be used to trap and position cells non-invasively just with the force of light. Either the microscope stage or the laser beam itself can be used to move the trapped objects. 2

3 EXAMPLE Sperm Motility Sperm motility and sperm force are important parameters when comparing different sperm species and potentially in assessing sperm fertilizing capacity. PALM MicroTweezers and FM allow these measurements to be made easily. Sperm characterization Sperm motility and force were investigated using optical tweezers in combination with brightfield imaging. Motile sperms were optically trapped with a highly focused, near-infrared laser beam. The minimum power of a trapping laser beam at which sperm can escape from the laser trap became a direct measure of the sperm intrinsic escape force because the applied optical force is directly proportional to the cell force. In addition taking time lapse images of the freely moving sperm allowed measurement of sperm speed and directionality. Result Sperm of multi-mating partner primates were compared with sperm of single-mating partner primates. In multimating partner primates, sperm speed and force were significantly higher than in monogamous primates. More details are in Nascimento et al., J R Soc Interface. 2008, 5(20): The results foster the theory that faster and stronger sperm could have an advantage in the reproductive success of polygamous primates. Fesc (pn) VCL (µms 1 ) human gorilla chimp macaque human gorilla Sperm escape force (Fesc) and velocity (VCL). Graphs have been taken from Nascimento JM, Shi LZ, Meyers S, Gagneux P, Loskutoff NM, Botvinick EL, Berns MW. The use of optical tweezers to study sperm competition and motility in primates. J R Soc Interface. 2008, 5(20): Motile human sperm has been optically confined with PALM MicroTweezers. The sperm escapes the trap at a critical minimum laser power which corresponds to a force of 19pN. 3

4 PALM MircoTweezers From Manipulation to Quantitative Force Measurements The dedicated instrument for contact-free manipulation of microscopic particles combines high performance imaging functionality with optical trapping. The new FM software module allows you to make quantitative force measurements that are especially relevant in life science research. 4

5 The StarterKit FM helps you prepare your calibration or experimental sample quickly and easily. FluidCell is cell culture approved, can be sterilized and uses low-priced disposables. Various incubation systems can be combined with FluidCell. 5

6 EXAMPLE Plasma Membrane Properties The plasma membrane of a cell plays an important role in many cell functions including proliferation, differentiation, cell adhesion and motility and is specifically interesting in the field of tissue engineering. The FM software lets you characterize the mechanical properties of cell membranes. Tether force measurement Tether extraction is probably the most accurate method to quantitatively characterize the mechanical properties of the plasma membrane and its reservoir. Tether forces as well as tether lengths can directly be measured with PALM MicroTweezers and FM software. Force (pn) Elongation phase Membrane reservoir Typically, micron-sized beads coated with molecules (e. g. extracellular matrix proteins) are used as handles to grab the cell membrane. When the optically trapped bead is pulled away from the membrane, a thin hollow membrane tether is formed which exerts a force on the bead. Monitoring the displacement of the bead from the trap center measures the force once the trap stiffness has been calibrated. Experimental results In the initial process of tether force formation an increase in force is detected before it reaches a plateau. The force then stays constant over a large range of tether length, indicating a readily available membrane reservoir. Once this reservoir is depleted a critical tether length is reached, whereupon the force rapidly increases (data not shown). Membranecytoskeleton interactions have been investigated. Tether Length (µm) 5 0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5, Time (s) Tether Length (µm) Control Cyto-B Cyto-D Nocodazole Colchicine Taxol Membrane tether force and tether length. Experimental tether force data obtained with PALM MicroTweezers and FM software. Lower graph has been taken from Raucher D, Sheetz MP. Characteristics of a Membrane Reservoir Buffering Membrane Tension. Biophys J Oct;77(4): F tether F trap Membrane tether extraction of a NIH 3T3 fibroblast with PALM MicroTweezers. The force is calculated from the position of the bead center relative to the trap center. The tether length can also be measured. Membrane tether 6

7 Force Measurement This intuitive software is not just easy to use it also offers flexible functionality and experimental freedom ranging from simple manipulation to quantitative force measurement. The principle Microscopic particles can be confined with optical tweezers. The force applied on the object depends linearly on its displacement from the trap center. In other words the trap acts just like a linear spring where the restoring force F is proportional to the trap stiffness ktrap and displacement x. Once the trap stiffness is calibrated monitoring the displacement of the object directly results in a force measurement. x laser light trap center beam waist k trap F=k trap x Software features Automated trap stiffness calibration routines with optional correction terms for higher accuracy Easy operation FM Experiment Builder, offering all the flexibility you need to design your own experiments Position detection and force measurement capability Visual representation of calibration and experimental data Complete documentation with all images and associated data saved Optical trap stiffness calibration data for various laser powers. Trap calibration and experiment definition is easily realized with the Force Measurement software module by Carl Zeiss. 7

8 Application examples with the Force Measurement software module Cell elasticity measurements Binding / adhesion forces Cell-cell interactions Membrane tension Bacteria or sperm motility forces Carl Zeiss Microscopy GmbH Jena, Germany BioSciences microscopy@zeiss.com Information subject to change. Printed on environmentally friendly paper bleached without chlorine /e printed 03.09

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