We used a Nanowizard II AFM (JPK Instruments, Berlin, Germany) mounted on a Zeiss microscope (Carl Zeiss, Jena, Germany). This configuration allows to carry out AFM measurements and simultaneously observe the cells using phase contrast or fluorescence modes. This AFM is also equipped with the 'CellHesion' module (JPK Instruments, Berlin, Germany). This module enables a long-range vertical displacement of the stage up to 100 µm which makes force spectroscopy measurements possible including cell-cell interactions. In parallel, a vertical piezotranslator (PIFOC, Physik Instrumente, Karlsruhe, Germany) is mounted on the microscope objective to move the objective concurrently with the microscope stage and focus on cells while carrying out AFM measurements. All the measurements were carried out at 37°C using the Petri Dish Heater (JPK Instruments, Berlin, Germany).
Pifoc
Manufactured by Physik Instrumente
Sourced in Germany
The PIFOC is a high-precision piezo-driven objective nanopositioning system designed for microscopy and other optical applications. It provides fast and precise control of the position of an objective lens or other optical component with a travel range up to 400 μm and sub-nanometer resolution.
Automatically generated - may contain errors
Lab products found in correlation
Nanowizard 2 afm, by Bruker (1 mentions)
Cellhesion module, by Bruker (1 mentions)
Petri dish heater, by Bruker (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (1 mentions)
Axiophot, by Zeiss (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Csu10 spinning, by Yokogawa (1 mentions)
Labview, by National Instruments (1 mentions)
7 protocols using pifoc
1
Simultaneous AFM and Microscopy Imaging
2
Microscopy Imaging of GFP and mCherry
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Microscopy Images were captured with a ×100 magnification oil-immersion objective (1.46 numerical aperture) on a Leica DMI 6000B microscope (Leica Microsystems) equipped with a piezoelectric translator (PIFOC, Physik Instrumente), a ORCA-Flash 4.0 camera (Hamamatsu) an illumination system with leds (Lumencore) and rapid imaging software (Metamorph). Wavelengths of the leds used are 475nm (for GFP, 205mW), and/or 575nm (for mCherry, 300MW). Two-minute movies with a stack of 10 optical slices separated by 300nm every 338ms. Each slice was exposed for 30ms for a total of 338ms per stack. All microscopy was done in a temperature-controlled environment set to 25°C. The raw images were deconvolved using the Autoquant software. The movies were then tracking using ImageJ [40 (link)] with the Mosaic macro [41 (link)] to produce 3D+t trajectories. Further processing and analysis of the movies was done using Matlab.
3
Magnetic Tweezers for DNA Manipulation
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The custom made MT setup was previously described (27–30 (link)), and consisted of an inverted optical microscope equipped with an oil-immersion objective (NIKON 100x, NA = 1.25) mounted on a piezoelectric focusing system (PIFoc, Physik Instrumente, Bresso, Italy). The objective, coupled with a 15 cm focal-length lens, led to a 75x magnification. The magnetic field was generated by two permanent neodymium magnets placed above the flow chamber and two piezoelectric motors controlled the position of the magnets along the optical axis (z-direction) and the rotation around the same axis in order to apply a stretching force, or a torque, to the torsionally constrained DNA.
4
Compact Wide-Field Microscope for Live Imaging
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The compact wide-field microscope was equipped with a 473 nm, 50 mW diode laser (Spectra Physics Excelsior, USA) and a 561nm, 20 mW diode laser (Coherent Compass, Germany) for excitation. An acousto-optic tunable filter (‘AOTF’, AA OptoElectronic AOTF, France) was used to enable fast switching of the laser, to restrict illuminating the sample just during data acquisition, and to reduce photobleaching effects to a minimum. After passing through the AOTF, the excitation beam is coupled into a single mode fiber and then focused to the back focal plane of the objective lens (UPLSAPO60XO 60×, 1.35 NA oil objective, Olympus), which is held on a piezo-electric stage (PiFoc, Physik Instrumente, Germany) for fast displacement along the optical axis. The fluorescence signal was collected with the same objective lens and the emission was separated from the excitation source by a dichroic mirror (HC Dual Line—BSR488/561, Semrock, USA). The fluorescence signals were further separated by an additional dichroic mirror (HC BS 560i, Semrock, USA). The fluorescence images are focused on two industry-grade CMOS cameras (IDSμEye UI-3060CP-M-GL Rev.2) using a 180 mm tube lens and after passing additional emission filters (BP 520/77 (#87-749), BP 620/60 (#33-910), BP 591.5/49 (#67-034), all Edmund Optics), resulting in an overall projected pixel size of 97 nm.
5
Widefield Fluorescence Microscopy of Nuclear Extensions
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Samples subjected to immunofluorescence were analyzed by widefield fluorescence microscopy (Axiophot, Carl Zeiss, Oberkochen, Germany). Images (40X and 100X magnifications) were acquired by a digital CCD monochromatic camera (CoolSnap HQ2, Photometrics Inc., Tucson, AZ, USA). To assess nuclear extensions through the fluorescent substrate, at list ten Z sections per sample field were obtained using a piezoelectric device (PIFOC, Physik Instrumente, Germany) coupled to the objective. The microscope and all devices were controlled by Metamorph Premier 7.6 software (Molecular Devices, Sunnyvale, CA, USA).
A total of 10 random cells were imaged per experimental group. Orthogonal projections and image restoration through deconvolution algorithms were carried out by Volocity software (PerkinElmer, Waltham, MA, USA).
Measurement of colocalization areas and particles analysis was determined using ImageJ public domain software (http://rsb.info.nih.gov/ij/ ). Colocalization analysis was carried out by the Linescan tool (Metamorph Premier 7.6 software), and the Image J plugins JaCop, Colocalization colormap and Colocalization threshold.
To evaluate nucleolus ADAMTS-1 staining, red channel was analyzed; a threshold area was determined and then measured. Control and siRNA groups nucleolus area were compared.
A total of 10 random cells were imaged per experimental group. Orthogonal projections and image restoration through deconvolution algorithms were carried out by Volocity software (PerkinElmer, Waltham, MA, USA).
Measurement of colocalization areas and particles analysis was determined using ImageJ public domain software (
To evaluate nucleolus ADAMTS-1 staining, red channel was analyzed; a threshold area was determined and then measured. Control and siRNA groups nucleolus area were compared.
6
High-Resolution Imaging of Fluorescent Cells
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Cells were also imaged and manipulated using an Olympus IX71 inverted microscope with a Yokogawa CSU10 spinning-disc scan head (Yokogawa Electric Corporation) equipped with a fast piezo objective z-positioner (PIFOC, Physik Instrumente GmbH & K.G.) and an Olympus UPlanSApo × 100/1.4 NA oil objective (Olympus). For cells expressing GFP and tdTomato/mCherry, we performed sequential imaging and images were acquired at a 2.1–4.7 s time intervals. For excitation, a sapphire 488 nm solid-state laser (75 mW; Coherent) and a Jive 561 nm solid-state laser (75 mW; Cobolt) were used for GFP and tdTomato/mCherry, respectively. The laser intensity was controlled using the acousto-optic tunable filter inside the Andor Revolution Laser Combiner (ALC, Andor Technology). The emission wavelength was selected using respective emission filters BL 525/30 (Semrock) and ET 605/70 (Chroma) mounted in a fast, motorized filter wheel (Lambda 10B, Sutter Instrument Company). The images have a xy-pixel size of 168 nm and the z-distance between optical sections was 500 nm. The system was controlled by Andor iQ software version 1.9.1 (Andor Technology).
7
Measuring Adhesive Contact of Nuclei
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To measure the adhesive contact radius and initial deformation of the nucleus we performed z-stacks through adhered HeLa nuclei that were fluorescently labelled. The sample was excited with a 488 nm laser and a stack of 150 images spaced by 166 nm was recorded for each nucleus. A custom written routine in LabVIEW (National Instruments, TX, USA) was used to control the objective scanner (PIFOC, Physik Instrumente, Germany) and to trigger frame acquisition on the camera (50 ms exposure time).
The z-stacks were post-processed using ImageJ and the DeconvolutionLab plugin49 to perform a 3D deconvolution. The point spread function was calculated using the Born and Wolf model50 . For the figures we used side-view projections through deconvolved z-stacks using the maximum intensity values.
The adhesion contact radius was quantified by manually plotting a circle around the nucleus to get the intersection with the supporting surface.
The z-stacks were post-processed using ImageJ and the DeconvolutionLab plugin49 to perform a 3D deconvolution. The point spread function was calculated using the Born and Wolf model50 . For the figures we used side-view projections through deconvolved z-stacks using the maximum intensity values.
The adhesion contact radius was quantified by manually plotting a circle around the nucleus to get the intersection with the supporting surface.
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