Photoactivated localization microscopy (PALM) was conducted on an ELYRA P.1 microscope (Carl Zeiss) equipped with an oil-immersion objective (×100, 1.46 NA). The fluorescence images (100 nm per pixel) were acquired by using light sources (405 nm and 488 nm), a band pass filter (495–590 nm), and a detection camera (EM-CCD at 295 K). To obtain wide-field and super-resolved images, total 10,000 frames of the images were collected and analyzed with Zeiss Zen software. The wide-field image was reconstructed from the default value of the software. The super-resolved image was reconstructed by analyzing all frames, in which fluorescent spots between 70 and 200 nm were specifically selected to discard the unreasonable patterns. The super-resolved image (10 nm per pixel) was rendered by selecting the fluorescent spots, whose localization precision was from 10 to 50 nm, to exclude the poorly localized fluorescent probes. ImageJ plugin was used to analyze the Fourier ring correlation (FRC) of the localizations.
Elyra p 1 microscope
Manufactured by Zeiss
The Elyra P.1 microscope is a high-performance imaging system designed by Zeiss for advanced microscopy applications. It combines structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM) techniques to enable super-resolution imaging. The Elyra P.1 provides researchers with the ability to achieve sub-diffraction-limited resolution, allowing for detailed analysis of cellular structures and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Plan apochromat, by Zeiss (2 mentions)
Zen 2012, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Uv 3600 pc spectrophotometer, by Shimadzu (1 mentions)
Tecnai g2t20 electron microscope, by Zeiss (1 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Ixon 897 back thinned emccd camera, by Oxford Instruments (1 mentions)
Tetraspeck bead, by Thermo Fisher Scientific (1 mentions)
9 protocols using elyra p 1 microscope
1
Super-resolution Imaging of Fluorescent Samples
2
Super-resolution Imaging of Chromatin Spreads
3
Super-resolution Imaging of Telomeres and Centromeres
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SMLM and DNA-PAINT images
were acquired using a Zeiss Elyra P.1 microscope equipped with 405
nm (100 mW), 561 nm (100 mW), and 642 nm (100 mW) lasers, a 100×/1.46
oil immersion objective, and an Andor EM-CCD camera (iXon DU897).
SMLM images of the telomere probes were obtained using 405 and 642
nm excitation with a 655 nm longpass filter. SMLM images of the centromere
probes were obtained using 405 and 561 nm excitation with a 570–630
nm bandpass filter. DNA-PAINT images of the telomere probes were obtained
using 642 nm excitation with a 655 nm longpass filter. DNA-PAINT images
of the centromere probes were obtained using 561 nm excitation with
a 570–630 nm bandpass filter. The exposure time for SMLM was
50 ms, and that for DNA-PAINT was 150 ms.
Widefield TIRFM images
of the DAPI dye was obtained using 405 nm excitation with a 420–480
nm bandpass filter; the exposure time for each frame was 150 ms. For
each super-resolved SMLM or DNA-PAINT images, 5000 or 10,000 frames
were collected for reconstruction. The super-resolved images were
reconstructed using Zeiss ZEN 2012 software integrated with the microscope
using the Gaussian fitting of each blinking event. The parameters
were kept constant for different samples.
were acquired using a Zeiss Elyra P.1 microscope equipped with 405
nm (100 mW), 561 nm (100 mW), and 642 nm (100 mW) lasers, a 100×/1.46
oil immersion objective, and an Andor EM-CCD camera (iXon DU897).
SMLM images of the telomere probes were obtained using 405 and 642
nm excitation with a 655 nm longpass filter. SMLM images of the centromere
probes were obtained using 405 and 561 nm excitation with a 570–630
nm bandpass filter. DNA-PAINT images of the telomere probes were obtained
using 642 nm excitation with a 655 nm longpass filter. DNA-PAINT images
of the centromere probes were obtained using 561 nm excitation with
a 570–630 nm bandpass filter. The exposure time for SMLM was
50 ms, and that for DNA-PAINT was 150 ms.
Widefield TIRFM images
of the DAPI dye was obtained using 405 nm excitation with a 420–480
nm bandpass filter; the exposure time for each frame was 150 ms. For
each super-resolved SMLM or DNA-PAINT images, 5000 or 10,000 frames
were collected for reconstruction. The super-resolved images were
reconstructed using Zeiss ZEN 2012 software integrated with the microscope
using the Gaussian fitting of each blinking event. The parameters
were kept constant for different samples.
4
Super-resolution Imaging of Cellular Samples
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Super-resolution imaging was performed on a Zeiss Elyra P.1 microscope, equipped with an oil-immersion objective (alpha “Plan-Apochromat” 100X/1.46 Oil DIC) and total internal fluorescence (TIRF) illumination. Emitted fluorescence was collected by the same objective and captured by an Andor iXon 897 back-thinned EMCCD camera. Integration time per frame was 33 ms at full laser power for the 488 channel, and 50 ms for the 561 and 647 channels. The laser power of 561 nm and 647 nm required adjustment according to the staining condition. Typically 10,000 frames were collected, which corresponded to a measurement duration of 5–10 min for each channel. XY drift and channel misalignment was corrected by localizing 0.2-μm TetraSpeckTM beads (Invitrogen, USA) immobilized on the sample coverslip. For super-resolution data analysis, the raw data was processed using Zeiss Zen software to detect single-molecule events above the background noise (more details are described in18 (link)). After reconstruction, a super-resolution image and a table containing the x-y coordinates of all the single-molecule events were obtained. In the post-processing step, events which were above the 20 nm localization limit were discarded. A super-resolution (SR) image was generated by fitting each event with the Gaussian function. The exported SR images were then processed in ImageJ.
5
Super-resolution Imaging of Chromatin Spreads
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After NIH2/4 cells were fixed and quenched, cells were treated with
Trypsin (GIBCO) and re-suspended with culture medium. Chromatin spreads were
prepared according to the methods previously described33 (link). Super-resolution imaging was performed on a Zeiss Elyra
P.1 microscope equipped with an oil-immersion objective, and images were
analysed according to the procedures previously described33 (link).
Trypsin (GIBCO) and re-suspended with culture medium. Chromatin spreads were
prepared according to the methods previously described33 (link). Super-resolution imaging was performed on a Zeiss Elyra
P.1 microscope equipped with an oil-immersion objective, and images were
analysed according to the procedures previously described33 (link).
6
Spectroscopic and Microscopic Characterization
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Extinction
spectra were recorded by using
a Shimadzu UV-3600 PC spectrophotometer with quartz cuvettes of a
1 cm path length. Photoluminescence emission spectra were recorded
by using an Edinburg FLS 920 spectrofluorometer. Transmission electron
microscopy (TEM) images were obtained with an FEI Tecnai G2T20 electron
microscope operating at 200 kV. Fluorescence and SMLM images were
acquired using a Zeiss Elyra P.1 microscope equipped with 405 (50
mW), 488 (100 mW), 561 (100 mW), and 640 nm (150 mW) lasers. Fluorescence
images were recorded using a 100×/1.46 oil immersion objective
and an Andor EM-CCD camera (iXon DU897). Imaging data were analyzed
using the Zeiss Zen 2012 software.
spectra were recorded by using
a Shimadzu UV-3600 PC spectrophotometer with quartz cuvettes of a
1 cm path length. Photoluminescence emission spectra were recorded
by using an Edinburg FLS 920 spectrofluorometer. Transmission electron
microscopy (TEM) images were obtained with an FEI Tecnai G2T20 electron
microscope operating at 200 kV. Fluorescence and SMLM images were
acquired using a Zeiss Elyra P.1 microscope equipped with 405 (50
mW), 488 (100 mW), 561 (100 mW), and 640 nm (150 mW) lasers. Fluorescence
images were recorded using a 100×/1.46 oil immersion objective
and an Andor EM-CCD camera (iXon DU897). Imaging data were analyzed
using the Zeiss Zen 2012 software.
7
Super-resolution TIRF Microscopy
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Super-resolution imaging was performed on a Zeiss Elyra P.1 microscope, equipped with an oil-immersion objective (alpha “Plan-Apochromat” 100X/1, 46 Oil DIC) and Total internal fluorescence (TIRFM) illumination. TIRFM illumination was achieved by using lasers with motorized TIRFM angle adjustment. The resulting illuminated area was 51.1 × 51.1 μm (with alpha “Plan-Apochromat” 100×/1.46 Oil DIC, full chip recording). Excitation was provided by a 488 nm laser line (100 mW) with AOTF-based intensity control. Emitted fluorescence was collected by the same objective and captured by an Andor iXon 897 back-thinned EMCCD camera. Integration time per frame was 50 ms at full laser power. Typically 10,000 frames were collected, which corresponded to measurement duration of ~10 min. XY drift and alignment differences between different channels were corrected by localizing 0.2-μm Tetraspeck beads (Invitrogen, USA) immobilized on the sample coverslip.
8
Immobilizing FRET Nanoprobes and GQDs
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The FRET nanoprobes and GQDs
were immobilized onto the bottom of cell culture dishes as follows.
First, the bottom of the cell culture dish was modified with PEI (0.1%)
for 20 min to introduce amino groups. Then, excess PEI was washed
away with water. FRET nanoprobes (4 μL) were added into 120
μL of deionized water. After ultrasonication for 10 s, the diluted
FRET nanoprobes were added into the cell culture dishes and incubated
for 20 min to allow electrostatic adsorption of the nanoprobes. After
washing away excess FRET nanoprobes with water, 120 μL of imaging
buffer was added into the cell culture dish, which was subsequently
imaged using a Zeiss Elyra P.1 microscope. The immobilization of GQDs
followed the same method, as described above.
were immobilized onto the bottom of cell culture dishes as follows.
First, the bottom of the cell culture dish was modified with PEI (0.1%)
for 20 min to introduce amino groups. Then, excess PEI was washed
away with water. FRET nanoprobes (4 μL) were added into 120
μL of deionized water. After ultrasonication for 10 s, the diluted
FRET nanoprobes were added into the cell culture dishes and incubated
for 20 min to allow electrostatic adsorption of the nanoprobes. After
washing away excess FRET nanoprobes with water, 120 μL of imaging
buffer was added into the cell culture dish, which was subsequently
imaged using a Zeiss Elyra P.1 microscope. The immobilization of GQDs
followed the same method, as described above.
9
Visualizing DNA Origami Nanostructures via TIRF Microscopy
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Images were acquired using a Zeiss Elyra P.1 microscope using total internal reflection fluorescence (TIRF) with a Zeiss Alpha Plan Apochromat 100x NA 1.46 oil objective. First, to locate DNA origami structures, a time series of images of the 3/4mer ATTO565 labelled origami nanotubes was taken (0.5 kW/cm 2 , 100 ms, 500 frames). This was then repeated for the Alexa488 labelled origami nanotubes with a single docking strand for 500 frames with an integration time of 100 ms/frame under 0.004 kW/cm 2 488 nm laser illumination with a TIRF angle of 66.90°. For DNA-PAINT imaging, 642 nm (0.075 kW/cm 2 ) laser illumination was used with an integration time of 300 ms for 50,000 frames with a TIRF angle of 66.90°. All images were 512x512 pixels with a pixel size of 97 nm.
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