We performed atomic force microscopy (AFM) on live and fixed cells cultured on glass bottom dishes using a BioScope Resolve BioAFM in PeakForce QNM mode and a PFQNM-LC-A-CAL cantilever (Bruker, Santa Barbara, CA). We did not perform AFM on cells cultured on EM grids due to the delicate and porous nature of the carbon thin films. There were no discernable differences in cell morphology on micropatterned glass and EM grids (Fig. 3A -B , 2A -B , S2 -S4 , and S6 ). We measured cell-cell contact heights of cells cultured on blanket coated and micropatterned glass bottom dishes with the height sensor channel using Nanoscope Analysis 1.9 (Fig. S6 ). The AFM was mounted on a Zeiss Axio Observer Z1 inverted epifluorescent microscope outfitted with a 20x objective.
Pfqnm lc a cal cantilever
Manufactured by Bruker
Sourced in United States
The PFQNM-LC-A-CAL cantilever is a laboratory equipment component used for various analytical and measuring applications. It functions as a probe for atomic force microscopy (AFM) techniques, enabling high-resolution surface characterization of samples. The cantilever is designed for use with the Bruker PFQNM-LC-A-CAL system.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Bioscope resolve bioafm, by Bruker (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Zen black software, by Zeiss (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Cell tak, by Merck Group (1 mentions)
Nanowizard 4bio afm, by Bruker (1 mentions)
Microscope slide, by Avantor (1 mentions)
Sylgard 184, by Dow (1 mentions)
4 protocols using pfqnm lc a cal cantilever
1
Atomic Force Microscopy of Live Cells
2
Microscopic Imaging and AFM Analysis
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Read Probest reagent (R37112, Thermo-Fisher Scientific) at 1 drop/mL to stain F-actin. Images in Fig. 2A-B were acquired using an inverted Zeiss LSM 780 confocal microscope with a 40X/1.3 NA C-Apo water objective and using Zen Black software (Carl Zeiss). Images used for Fig. 1B,2C, and S1 were acquired on an inverted Nikon Ti-E microscope equipped with a Heliophor light engine (89 North) and an Andor sCMOS Neo camera using a 20x Plan Apo Lambda air objective lens. Nuclear-junctional distances were measured using ImageJ (S6,S7) (17) . Atomic force microscopy. We performed atomic force microscopy (AFM) on live and fixed cells cultured on glass bottom dishes using a BioScope Resolve BioAFM in PeakForce QNM mode and a PFQNM-LC-A-CAL cantilever (Bruker, Santa Barbara, CA). We did not perform AFM on cells cultured on EM grids due to the delicate and porous nature of the carbon thin films. There were no discernable differences in cell morphology on micropatterned glass and EM grids (Fig. 3A-B, 2A-B, S2-4). We measured cell-cell contact heights of cells cultured on blanket coated and micropatterned glass bottom dishes with the height sensor channel using Nanoscope Analysis 1.9. The AFM was mounted on a Zeiss Axio Observer Z1 inverted epifluorescent microscope outfitted with a 20x objective.
3
Atomic Force Microscopy of Immobilized Cells
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Cells were immobilized onto clean glass slides using CellTak (Merck, Germany) and imaged in NanoWizard 4Bio AFM (Bruker, USA) using Quantitative Imaging mode under ambient temperature and buffered with 150 mM KCl and 10 mM Tris.Cl pH 7.5. PFQNM-LC-A-CAL cantilevers (Bruker, USA) with tip length of 17 µm were calibrated using the method by Sader et al. (62 (link)) yielding a spring constant k = 0.033 N·m−1 and a resonant frequency f0 = 39.8 kHz in air and f0 = 20.1 kHz in the buffer. The 6 × 6 µm2 topography images were scanned with a resolution of 256 × 256 pixels2 at a setpoint of 80 pN and the tip vertical speed was 83.3 µm s−1.
4
Measuring Mycobacterial Cell Elasticity
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To estimate Young’s modulus, M. smegmatis cultures were grown in 7H9-OADC-Tween 80 medium. Two milliliters of bacterial culture were centrifuged (6000 rpm for 5 min), washed twice with 7H9-OADC (to remove the Tween 80) and resuspended in 50 µl 7H9-OADC. microscope slides were prepared as previously described42 (link) by mixing polydimethylsiloxane (PDMS; Sylgard 184; Dow Corning) at a ratio of 15:1 (elastomer:curing agent). Air bubbles were removed from the mixture under negative pressure for 20 min. The PDMS mixture was dropped onto the microscope slides (VWR), which were spin-coated and baked at 80 °C for 10 min before use. A concentrated bacterial pellet was deposited on the surface of a PDMS-coated slide and incubated for 10 min. Then, the PDMS-coated slide was washed with 7H9-OADC to remove non-immobilized cells. AFM measurements were conducted in 7H9-OADC medium. The Young’s modulus was estimated using the PeakForce QNM scanning mode (scan rate, 1 Hz; amplitude, 100 nm) with PFQNM-LC-A-CAL cantilevers (resonant frequency, 45 kHz; spring constant, 0.1 N/m; Bruker). Young’s modulus was measured at five points of each cell using the NanoScope Analysis 1.9 software, and then the mean modulus value was calculated for M. smegmatis WT and Δlsr2 cells.
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