GATTA-PAINT nanoruler slide samples (HiRes 20R, GattaQuant DNA Technologies) were used as purchased. Imaging was done on an Olympus IX71 inverted wide field fluorescence microscope setup as described previously41 (link). Fluorescence excitation of the sample was done using a 642 nm laser diode (HL6366DG, Thorlabs). The laser beam was collimated and passed through a multi-mode fiber (P1-488PM-FC-2, Thorlabs), before being focused on the back focal plane of a 1.45 NA oil objective (UAPON 150XOTIRF, Olympus America Inc.). TIRF excitation of the sample was achieved by translating the laser close to the edge of the objective back aperture. Fluorescence emission collected from the nanoruler sample was passed through a quad band dichroic/emission filter set (LF405/488/561/635-A; Semrock, Rochester, NY) and a band pass filter (685/45, Brightline) before being detected using an EM CCD camera (iXon 897, Andor Technologies). A total of 100,000, 256 × 256 pixel frames were collected using a 100 ms exposure time. Data collection on the microscope was controlled by custom-written MATLAB instrument control software (MIC)34 (link). The raw super-resolution data of DNA rulers was processed by a single-emitter fitting algorithm42 (link) and thresholded by p-value and localization uncertainty43 (link). Localizations from the same binding events were combined using a frame connection algorithm.
Uapon 150xotirf
Manufactured by Olympus
The UAPON 150XOTIRF is a high-performance objective lens designed for use in optical tweezers and other advanced microscopy techniques. It features a high numerical aperture of 1.45 and a magnification of 150X, providing excellent resolution and light-gathering capabilities. The lens is optimized for total internal reflection fluorescence (TIRF) microscopy, allowing for the selective excitation of fluorophores near the coverslip surface.
Automatically generated - may contain errors
Lab products found in correlation
Imagem c9100 13, by Hamamatsu Photonics (4 mentions)
Metamorph software, by Molecular Devices (3 mentions)
P35gc 0 10 c, by MatTek (2 mentions)
Ff01 624 40 25 bandpass filter, by IDEX Corporation (2 mentions)
Plapon, by Olympus (2 mentions)
Delta vision elite microscope system, by GE Healthcare (2 mentions)
Ix3 zdc2, by Olympus (2 mentions)
Autoquant x3, by Media Cybernetics (2 mentions)
Stage top incubator, by Tokai Hit (2 mentions)
Lf405 488 561 635 a, by IDEX Corporation (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Dichroic mirror, by Olympus (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Nanolog spectrofluorometer, by Horiba (1 mentions)
Lambda 1050 uv vis nir spectrophotometer, by PerkinElmer (1 mentions)
Ix70 inverted microscope, by Olympus (1 mentions)
Uplansapo, by Olympus (1 mentions)
8 protocols using uapon 150xotirf
1
Super-resolution Imaging of DNA Nanorulers
2
Single-Tube PL Imaging of SWCNTs
3
Rapamycin-Induced Fluorescence Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast cells were grown to the mid-log phase and treated with rapamycin for indicated times. Cells were then concentrated by centrifugation and mounted on an agarose gel pad (SD-N containing 3.5% [wt/vol] agarose; Young et al., 2011 (link); Graef et al., 2013 (link)). Fluorescence microscopy was performed at room temperature using an inverted fluorescence microscope (IX83; Olympus) equipped with ×150 objective lens (UAPON 150XOTIRF; Olympus). A 488-nm blue laser (50 mW; Coherent) and 588-nm yellow laser (50 mW; Coherent) were used for the excitation of mNeonGreen and mCherry, respectively. Fluorescence was filtered with a dichroic mirror reflecting 405-, 488-, and 588-nm wavelengths (Olympus), separated into two channels using the DV2 multichannel imaging system (Photometrics) equipped with a Di02-R594-25x36 dichroic mirror (Semrock), and then passed through a TRF59001-EM ET bandpass filter (Chroma) for the mNeonGreen channel and FF01-624/40-25 bandpass filter (Semrock) for the mCherry channel. Images were acquired using an electron-multiplying CCD camera (ImagEM C9100-13; Hamamatsu Photonics K.K.) and MetaMorph software (Molecular Devices) and processed using Fiji (Image J; Schneider et al., 2012 (link)).
4
Fluorescence Microscopy of Yeast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast cells were analyzed using two different fluorescence microscopy systems, as described previously (Mochida et al., 2020) (link). The images in Fig. 1D, 5B, S1A, S3C, and S4 were acquired using an inverted microscope (IX81; Olympus) equipped with an electron-multiplying CCD camera (ImagEM C9100-13; Hamamatsu Photonics), a 150× objective lens (UAPON 150XOTIRF, NA/1.45; Olympus), a Z drift compensator (IX3-ZDC2; Olympus), and appropriate lasers and filters. For time-lapse imaging, cells were grown in the glass-bottom dish and kept at 30°C using a stage top incubator (TOKAI HIT). The images in Fig. S3C were deconvoluted by AutoQuant X3 software (Media Cybernetics). All other fluorescence microscopy images were acquired using a Delta Vision Elite microscope system (GE Healthcare) equipped with a scientific CMOS camera (pco.edge 5.5; PCO AG) and a 60× objective lens (PLAPON, NA/1.42; Olympus).
Images acquired by a Delta Vision were deconvoluted using SoftWoRx software. All acquired images were analyzed using Fiji software (Schindelin et al., 2012) (link).
Images acquired by a Delta Vision were deconvoluted using SoftWoRx software. All acquired images were analyzed using Fiji software (Schindelin et al., 2012) (link).
5
Multicolor Fluorescence Microscopy Setup
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence microscopy was performed using an inverted fluorescence microscope (IX83; Olympus) equipped with an electron-multiplying CCD camera (ImagEM C9100-13; Hamamatsu Photonics), and a 150× objective lens (UAPON 150XOTIRF, NA/1.45; Olympus). GFP and mCherry were excited using a 488 nm blue laser (50 mW; Coherent) and a 588 nm yellow laser (50 mW; Coherent), respectively. Fluorescence was filtered with a dichroic mirror reflecting 405 nm, 488 nm, and 588 nm wavelengths (Olympus), separated into two channels using the DV2 multichannel imaging system (Photometrics) equipped with a Di02-R594-25×36 dichroic mirror (Semrock), and further filtered with the TRF59001-EM ET bandpass filter (Chroma) for the GFP channel and the FF01-624/40-25 bandpass filter (Semrock) for the mCherry channel. Images were acquired using MetaMorph software (Molecular Devices) and processed using Fiji (ImageJ) (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)).
6
Imaging and Spectroscopy of (6,5)-SWCNTs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reactions
were monitored in situ using a NanoLog spectrofluorometer (HORIBA
Jobin Yvon). The samples were excited with a 450 W xenon source dispersed
by a double-grating monochromator. The slit width of the excitation
and emission beams was 10 nm. Excitation–emission maps and
single excitation PL spectra were collected using a liquid-N2 cooled linear InGaAs array detector. Absorption spectra were measured
using a Lambda 1050 UV-vis-NIR spectrophotometer (PerkinElmer) equipped
with both a photomultiplier tube and an extended InGaAs detector.
For single tube PL imaging, a small aliquot of (6,5)-SWCNT-C6H13 solution in 1% wt/v sodium deoxycholate (Sigma-Aldrich,
> 99%) was deposited on polyd -lysine coated glass slides
(part no. P35GC-0-10-C, MatTek Corporation). The imaging was performed
using a custom-built microscope that integrates a volume Bragg grating
system (Photon etc) and an oil immersion objective (UAPON 150XOTIRF,
NA = 1.45, Olympus).42 (link) The nanotubes were
excited by a 730 nm diode laser at a power density of 0.5 kW/cm2, and the PL emission was collected using a liquid-N2 cooled 2D InGaAs detector array (Cougar 640, Xenics) with an integration
time of 16 s.
were monitored in situ using a NanoLog spectrofluorometer (HORIBA
Jobin Yvon). The samples were excited with a 450 W xenon source dispersed
by a double-grating monochromator. The slit width of the excitation
and emission beams was 10 nm. Excitation–emission maps and
single excitation PL spectra were collected using a liquid-N2 cooled linear InGaAs array detector. Absorption spectra were measured
using a Lambda 1050 UV-vis-NIR spectrophotometer (PerkinElmer) equipped
with both a photomultiplier tube and an extended InGaAs detector.
For single tube PL imaging, a small aliquot of (6,5)-SWCNT-C6H13 solution in 1% wt/v sodium deoxycholate (Sigma-Aldrich,
> 99%) was deposited on poly
(part no. P35GC-0-10-C, MatTek Corporation). The imaging was performed
using a custom-built microscope that integrates a volume Bragg grating
system (Photon etc) and an oil immersion objective (UAPON 150XOTIRF,
NA = 1.45, Olympus).42 (link) The nanotubes were
excited by a 730 nm diode laser at a power density of 0.5 kW/cm2, and the PL emission was collected using a liquid-N2 cooled 2D InGaAs detector array (Cougar 640, Xenics) with an integration
time of 16 s.
7
Single-molecule Imaging of TMR-Halo-TfR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Individual TMR-Halo-TfR molecules located on the basal PM were observed at 37 ± 1°C using the TIR illumination mode of a home-built objective lens-type TIRF microscope (based on an Olympus IX70 inverted microscope), which was modified and optimized for the camera system developed in the companion paper. A 532-nm laser (Millennia Pro D2S-W, 2W, Spectra-Physics) was attenuated with neutral density filters, circularly polarized, and then steered into the edge of a high numerical aperture (NA) oil immersion objective lens (UAPON 150XOTIRF, NA = 1.45, Olympus), focused on the back-focal plane of the objective lens. The TIR illumination intensities at the sample plane were 14 and 0.16 µW/µm2 for the camera frame rates of 6 kHz (Fig. 8 ) and 60 Hz (Fig. S4 ), respectively. Right before recording the images of single TMR-Halo-TfR molecules, the mGFP-paxillin image was obtained in the same view field by using the TIR illumination (0.063 µW/µm2 at the specimen using a Spectra-Physics Cyan-PC5W 488-nm laser using a frame rate of 60 Hz, averaged over 10 s).
8
Yeast Fluorescence Microscopy Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For fluorescence microscopy, yeast cells were grown in SD+CA medium containing appropriate supplements unless otherwise indicated. Cells were analyzed using two different fluorescence microscopy systems, as described previously (Mochida et al., 2020 (link)). The images in Figs. 1 D , S1 A , S5 B , and S6 were acquired using an inverted microscope (IX81; Olympus) equipped with an electron-multiplying charge-coupled device camera (ImagEM C9100-13; Hamamatsu Photonics), a 150× objective lens (UAPON 150XOTIRF, NA/1.45; Olympus), a Z drift compensator (IX3-ZDC2; Olympus), appropriate lasers and filters, and MetaMorph software (Molecular Devices). For time-lapse imaging, cells were grown in the glass-bottom dish and kept at 30°C using a stage-top incubator (TOKAI HIT). The images in Fig. S5 B were deconvoluted by AutoQuant X3 software (Media Cybernetics). All other fluorescence microscopy images were acquired using a Delta Vision Elite microscope system (GE Healthcare) equipped with a scientific complementary metal-oxide-semiconductor camera (pco.edge 5.5; PCO AG), a 60× objective lens (PLAPON, NA/1.42; Olympus), a 100× objective lens (UPlanSApo, NA/1.40; Olympus), and SoftWoRx software. Images acquired by a Delta Vision were deconvoluted using SoftWoRx software. All acquired images were analyzed using Fiji.
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!