The fluorescence intensity of the foci and single EGFP molecules was measured on a custom‐build inverted microscope IX83 (Olympus) equipped with a 100× objective lens (UPlanSApo NA 1.40; Olympus) and a 2× intermediate magnification lens (Olympus) (Lim et al., 2018 (link)). HILO illumination (Tokunaga et al., 2008 (link)) of a 488‐nm laser (OBIS 488 LS; Coherent; 100 mW) was used with the CellTIRF system (Olympus). Images were acquired at an exposure time of 32.55 ms using an EM‐CCD (C9100‐13; Hamamatsu Photonics). The EM gain was set to 180 to avoid the saturation of the foci intensity, and the two frames were averaged to increase the signal‐to‐noise ratio of the single‐molecule intensity. The fluorescence intensity was measured using ImageJ software. The single‐molecule intensity was determined using a circular area with a 7‐pixel diameter corresponding to the size of the airy disk, and the outer circular region with 2‐pixel thickness was used as the background. To convert the intensity of the foci into the number of molecules, the total intensities of the foci from 45 cells were divided by the averaged single‐molecule fluorescence from five extremely low‐expressing cells.
Uplansapo na 1
Manufactured by Olympus
The UPlanSApo NA 1.40 is a high-numerical aperture objective lens designed for use in optical microscopy. It features a numerical aperture of 1.40, which enables the collection of a large amount of light and provides a high resolution for imaging applications.
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4 protocols using uplansapo na 1
1
Quantifying Single Molecule Fluorescence
2
Single-Molecule Quantification of Fluorescent Foci
3
High-Resolution Magnetic Tweezers Setup
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The MT apparatus in this study was built on an inverted microscope (Olympus, IX73) similar to previously reported high-resolution MT setups (56 (link)–58 (link)). A pair of magnets (vertically aligned in opposite directions with a 1-mm gap) was placed above the stage holding a flow cell, and its vertical position and rotation were controlled by a translation stage (Physik Instrumente, M126) and a stepper motor (Autonics, A3K-S545W), respectively. The magnet axis was confirmed to be aligned to the imaging axis (within 1°) by following the motion of a free magnetic bead. Beads in a flow cell were illuminated by a red superluminescent diode (QPhotonics, QSDM-680-2) and imaged by a 100× oil-immersion objective (Olympus, UPlanSApo NA 1.40) and a high-speed complementary metal-oxide semiconductor (CMOS) camera (Mikrotron, EoSens MC-3082) grabbing 512 × 512 images at 4 kHz. The objective position was controlled by a piezo-controlled nanopositioner (Mad City Labs, Nano-F100S) to calibrate distances and to correct for drift. The images were recorded by a custom software written in LabVIEW (National Instruments), and the coordinates of beads were tracked in real time at up to 1.2 kHz. Unless necessary, measurements were performed at 100 Hz to reduce file size.
4
Quantifying Cellular EGFP Molecules
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The fluorescence intensity of the foci and single EGFP molecules were measured on a custombuild inverted microscope IX83 (Olympus) equipped with a 100× objective lens (UPlanSApo NA 1.40; Olympus) and a 2× intermediate magnification lens (Olympus) (Lim et al., 2018) . HILO illumination (Tokunaga et al., 2008) of a 488-nm laser (OBIS 488 LS; Coherent; 100 mW) was used with the CellTIRF system (Olympus). Images were acquired at an exposure time of 32.55 ms using an EM-CCD (C9100-13; Hamamatsu Photonics). The EM gain was set to 180 to avoid the saturation of the foci intensity, and the two frames were averaged to increase the signal-noise ratio of the single-molecule intensity. The fluorescence intensity was measured using ImageJ software. The single-molecule intensity was determined using a circular area with a 7-pixel diameter corresponding to the size of the Airy disk, and the outer circular region with 2-pixel thickness was used as the background. To convert the intensity of the foci into the number of molecules, the total intensities of the foci from 45 cells were divided by the averaged single-molecule fluorescence from 5 extremely low-expressing cells.
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