Single-particle tracking PALM (sptPALM) was performed on a Nikon Eclipse Ti-E N-STORM system equipped with a Nikon 100× Apo TIRF oil immersion objective (NA 1.49) and perfect focus system. Photoactivation and excitation were performed with the 405 nm, 488 nm, 561 nm, and 647 nm excitation lasers within the MLC400B laser box (Agilent technologies) under TIRF or HiLo illumination through a quad-band polychroic mirror (Nikon 97335). An Ixon3 EMCCD (Andor) was used for detection, resulting in an effective pixel size of 160 nanometer. The microscope was fitted with a temperature-controlled stage that was adapted to fit the Nikon microscope. A pump was used to continuously flow cold water through the heat sink with 120 mL/min, to buffer the residual heat from the Peltiers. The Peltiers were controlled via Meerstetter Engineering TEC controllers and software. Additionally, the objective was cooled using a fitted copper collar with a fluid channel in the center that allowed a continuous flow of ~100 mL/min of cooled water through a peristaltic pump from a cold water bath of approximately 2.5 °C (see also Fig. 1A and SI Appendix, Fig. S1 for the full experimental setup). This allowed control of the sample temperature with <0.1° centigrade precision.
Apotirf oil immersion objective
Manufactured by Nikon
Sourced in United Kingdom
The ApoTIRF oil immersion objective from Nikon is a high-performance lens designed for total internal reflection fluorescence (TIRF) microscopy. It provides a numerical aperture of 1.49, enabling efficient light collection and high-resolution imaging of samples. This objective is optimized for use with oil immersion, providing a clear and detailed view of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Ti e microscope, by Nikon (2 mentions)
Nis elements ar 3, by Nikon (2 mentions)
Ixon3 emccd, by Oxford Instruments (1 mentions)
Mlc 400 b laser box, by Agilent Technologies (1 mentions)
Flash 4.0 v2, by Hamamatsu Photonics (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (1 mentions)
Mosaic digital micromirror device, by Oxford Instruments (1 mentions)
Coolsnap hq2 ccd, by Teledyne (1 mentions)
Metamorph automation and image analysis software, by Molecular Devices (1 mentions)
Csu x confocal scanhead, by Yokogawa (1 mentions)
Tecnai 10, by Thermo Fisher Scientific (1 mentions)
Plan fluor air objective, by Nikon (1 mentions)
Sem feg xl 30, by Philips (1 mentions)
Eclipse ti, by Nikon (1 mentions)
N sim microscope, by Nikon (1 mentions)
Ixon 897 emccd camera, by Oxford Instruments (1 mentions)
Nis elements ar, by Nikon (1 mentions)
9 protocols using apotirf oil immersion objective
1
Single-Particle Tracking PALM for Precise Temperature Control
2
Multicolor SMLM Imaging of Insulin and Cytoskeleton
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-molecule localization microscopy (SMLM) of fixed INS-1E cells was performed using a Nikon Ti-E microscope with a 100× Apo TIRF oil immersion objective (NA 1.49) and a Perfect Focus System 3. Excitation was performed through a custom illumination pathway with a Lighthub-6 (Omicron) laser containing a 638 nm laser (BrixX 500 mW multimode, Omicron), a 488 nm laser (Luxx 200 mW, Omicron), and a 405 nm laser (Luxx 60 mW, Omicron) and optics to tune the incident angle for evanescent wave or oblique illumination. Signal was detected with a sCMOS camera (Hamamatsu Flash 4.0v2). For imaging of actin and insulin, first a widefield image of insulin was obtained. Then, a low concentration of LifeAct–mNeonGreen was added such that single molecules could be observed and ∼25,000 frames were acquired at 60 ms exposure to reconstruct a super resolved image (Schätzle et al., 2018 (link); Tas et al., 2018 (link)). For co-imaging of paxillin, RIM and actin, first sequential DNA-PAINT was performed with Imager strands I2-560 and I1-650 (Ultivue) to image RIM and paxillin, respectively, with 100 ms exposure time and subsequently LifeAct-mNeonGreen was added to image actin similar to the imaging of actin and insulin described above. Images were reconstructed using ‘Detection of Molecules’ ImageJ plugin v.1.2.2 (https://github.com/ekatrukha/DoM_Utrecht ; Chazeau et al., 2016 (link)).
3
TIRF Microscopy of Live Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A Nikon Eclipse Ti inverted microscope equipped with a Nikon TI-TIRF Illuminator unit (Nikon Corporation) and a 60X Apo-TIRF oil-immersion objective (numerical aperture, 1.49) were used to capture images. The cells were kept in a humid, live-cell chamber set at 37°C and 5% CO2 throughout the experiment. Images were recorded using a Nikon Digital Sight DS-U3 CCD camera and processed using Nikon NIS-Elements AR 3.22 software and Fiji software. The TIRF microscope was able to excite fluorophores only ~100 nm from the coverslip.
4
Live-cell Imaging of MSC-BCC Invasion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSCs-GFP and BCCs were co-plated (6 × 104 cells each) into 35-mm microscopy dishes containing coverslips (0.17 mm in diameter) (MatTek, Ashland, MA), and incubated overnight at 37oC prior the beginning of the experiment. Nikon Eclipse Ti inverted microscope equipped with a Nikon TI-TIRF Illuminator unit (Melville, NY) and a 60X Apo-TIRF oil-immersion objective (NA 1.49) was used to capture images. Cells were kept in a humid, live-cell chamber set at 37oC and 5% CO2 throughout the experiment. A 40 mW Argon ion laser tuned to 488 nm and a solid-state 561 nm laser were used to excite GFP and DsRed or β-actin-mCherry, respectively. Images were recorded using a Nikon Digital Sight DS-U3 CCD camera and processed using Nikon NIS Elements AR 3.22 software and Fiji. When indicated, 3 μg/μl anti-CD9 Ab (Abcam clone MEM-61) was added to the dish. Since the TIRF microscope excites fluorophores only ~100 nm from the coverslip, overlapping green and red fluorescence signified entry of the BCC into the MSC rather than cell stacking. Entries of BCCs were considered invasions if ≥50% of the cell was inside of, or passed through, a MSC.
5
TIRF Microscopy at 37°C
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TIRF imaging was performed at 37°C using a VisiTech Infinfity 3 array-scanning confocal microscope (VisiTech International Ltd.; Sunderland, United Kingdom) equipped with a 100× APO TIRF objective oil-immersion (Nikon, NA 1.49). MetaMorph v7.71 image acquisition software was used to process the data.
6
TIRF Microscopy at 37°C
7
Optogenetic Recruitment Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glass coverslips were placed in a Chamlide magnetic chamber (Live Cell Instrument, Seoul, Korea) in culture media supplemented with 10 mM HEPES and 30 μl ml−1 Oxyrase (Oxyrase Inc., Mansfield, OH) and maintained at 37 °C. Cells were imaged on an inverted Nikon Ti-E microscope (Nikon, Melville, NY) with a Yokogawa CSU-X confocal scanhead (Yokogawa Electric, Tokyo, Japan) and laser merge module containing 491, 561 and 642 nm laser lines (Spectral Applied Research, Ontario, Canada). Images were collected on either a CoolSNAP HQ2 CCD (Roper Scientific, Trenton, NJ) or Zyla 4.2 sCMOS Camera (Andor, Belfast, UK). Local recruitment using the optogenetic probe was performed using a 405 nm laser coupled to a Mosaic digital micromirror device (Andor). Images were collected using a 60 × 1.49 NA ApoTIRF oil immersion objective (Nikon). All hardware was controlled using the MetaMorph Automation and Image Analysis Software (Molecular Devices, Sunnyvale, CA).
Unless otherwise stated, cells were imaged in the 561 channel every 20 s for 45 min, with the first 15 min used to determine the steady state of the system, the second 15 min to perform local recruitment and the final 15 min to record any recovery. During recruitment, a local region drawn in MetaMorph was illuminated by the 405 nm laser for 960 ms at a power <1 μJ s−1 immediately before the acquisition of each 561 image.
Unless otherwise stated, cells were imaged in the 561 channel every 20 s for 45 min, with the first 15 min used to determine the steady state of the system, the second 15 min to perform local recruitment and the final 15 min to record any recovery. During recruitment, a local region drawn in MetaMorph was illuminated by the 405 nm laser for 960 ms at a power <1 μJ s−1 immediately before the acquisition of each 561 image.
8
Characterization of Micro-Particles and Encapsulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The dimensions of the rough-smooth dumbbells
and spherical particles were determined by analyzing TEM images taken
using a FEI Tecnai 10 transmission electron microscope. The size distribution
of the microspheres was obtained from the analysis of optical microscopy
images using the program ImageJ.12 ,13 (link) The surface roughness of the microspheres and dumbbells was investigated
using a scanning electron microscope (SEM XL FEG 30, Philips). A Malvern
ZetaSizer Nano-ZS was used to measure both the polymer size of the
depletant with dynamic light scattering (DLS) and the zeta potential
of both the microspheres and dumbbells using laser doppler electrophoresis.
Encapsulation of the microspheres with dumbbell particles was studied
using a Nikon Eclipse Ti optical microscope with a Nikon Plan Fluor
air objective (NA = 0.75, 40× magnification). A Nikon Apo TIRF
oil immersion objective (NA = 1.49, 100× magnification) was used
to study the encapsulation of microspheres by the smaller spherical
particles. All images were acquired with an additional 1.5× magnification.
Image acquisition was performed using a Hamamatsu Digital Camera ORCA-Flash4.0
C11440 and the NIS-Elements Imaging Software.
and spherical particles were determined by analyzing TEM images taken
using a FEI Tecnai 10 transmission electron microscope. The size distribution
of the microspheres was obtained from the analysis of optical microscopy
images using the program ImageJ.12 ,13 (link) The surface roughness of the microspheres and dumbbells was investigated
using a scanning electron microscope (SEM XL FEG 30, Philips). A Malvern
ZetaSizer Nano-ZS was used to measure both the polymer size of the
depletant with dynamic light scattering (DLS) and the zeta potential
of both the microspheres and dumbbells using laser doppler electrophoresis.
Encapsulation of the microspheres with dumbbell particles was studied
using a Nikon Eclipse Ti optical microscope with a Nikon Plan Fluor
air objective (NA = 0.75, 40× magnification). A Nikon Apo TIRF
oil immersion objective (NA = 1.49, 100× magnification) was used
to study the encapsulation of microspheres by the smaller spherical
particles. All images were acquired with an additional 1.5× magnification.
Image acquisition was performed using a Hamamatsu Digital Camera ORCA-Flash4.0
C11440 and the NIS-Elements Imaging Software.
9
Bruchpilot Morphology Analysis via SIM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SIM images were acquired on an N-SIM microscope (Nikon) equipped with a 100× (1.49 NA) Apo TIRF oil-immersion objective (Nikon) and an iXon 897 EMCCD camera (Andor Technology). Images were reconstructed and analyzed using NIS-Elements Ar (Nikon) and Fiji software. For analysis of Bruchpilot morphology, z-stacks were flattened using the Maximum Intensity Z-projection function and background subtracted using the rolling ball method in Fiji. For each NMJ image, ∼20 central Bruchpilot punctae in planar orientation were selected, blind to genotype, for analysis. For each active zone, a fluorescence intensity profile plot was generated in Fiji along a 1-µm line drawn along the longest axis through the center of the Bruchpilot spot.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!