All TIRF imaging data were collected using an inverted microscope (IX71; Olympus) equipped with a 150×/1.45 NA oil-immersion, TIRF objective (U-APO; Olympus). A 637-nm laser diode (HL63133DG; Thorlabs) was used for AF647 excitation, and a 488-nm laser (Cyan Scientific; Spectra-Physics) was used for fluorescence excitation of mNG. A quad-band dichroic and emission filter set (LF405/488/561/635-A; Semrock) was used for sample illumination and emission. Emission light was separated onto different quadrants of an electron-multiplying charge-coupled device (EMCCD) camera (iXon 897; Andor Technologies), using a custom-built two-channel splitter with a 655-nm dichroic (Semrock) and 584/20-nm, 690/20-nm additional emission filters. Images had a pixel size of 0.106 μm and were acquired at 10 frames per second (100-ms exposure time).
Lf405 488 561 635 a
Manufactured by IDEX Corporation
The LF405/488/561/635-A is a laser module designed for laboratory equipment. It provides laser light at four different wavelengths: 405 nm, 488 nm, 561 nm, and 635 nm. The device is capable of delivering stable, high-quality laser output for various research and analytical applications.
Automatically generated - may contain errors
4 protocols using lf405 488 561 635 a
1
TIRF Microscopy for Protein Dynamics
2
Multi-color Single-molecule Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All data was collected using an inverted
microscope (IX71; Olympus) equipped with a 150×, 1.45 NA oil-immersion
total internal reflection fluorescence objective (U-APO; Olympus).
A 637 nm laser diode (HL63133DG, Thorlabs) was used for MG excitation,
and a 488 nm laser (Cyan Scientific; Spectra-Physics) was used for
AF488 and QD585 excitation. A quad-band dichroic and emission filter
set (LF405/488/561/635-A; Semrock) set was used for sample illumination
and emission. Emission light was separated onto different quadrants
of an electron-multiplying charge-coupled device (EMCCD) camera (iXon
897; Andor Technologies), using either a four channel image splitter
(QV2; Photometrics) with additional emission filters (692/40 and 525/30
nm; Semrock) or a custom-built two channel splitter with a 635 nm
dichroic (Semrock) and additional emission filters (584/20 692/40,
525/30 nm; Semrock). Images were 256 × 256 pixels, with a pixel
size of 0.106 μm and acquired at 20 frames/s (50 ms exposure
time).
microscope (IX71; Olympus) equipped with a 150×, 1.45 NA oil-immersion
total internal reflection fluorescence objective (U-APO; Olympus).
A 637 nm laser diode (HL63133DG, Thorlabs) was used for MG excitation,
and a 488 nm laser (Cyan Scientific; Spectra-Physics) was used for
AF488 and QD585 excitation. A quad-band dichroic and emission filter
set (LF405/488/561/635-A; Semrock) set was used for sample illumination
and emission. Emission light was separated onto different quadrants
of an electron-multiplying charge-coupled device (EMCCD) camera (iXon
897; Andor Technologies), using either a four channel image splitter
(QV2; Photometrics) with additional emission filters (692/40 and 525/30
nm; Semrock) or a custom-built two channel splitter with a 635 nm
dichroic (Semrock) and additional emission filters (584/20 692/40,
525/30 nm; Semrock). Images were 256 × 256 pixels, with a pixel
size of 0.106 μm and acquired at 20 frames/s (50 ms exposure
time).
3
Super-resolution Imaging of DNA Nanorulers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
4
TIRF Microscopy for Tracking Single Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TIRF imaging data were collected using an inverted microscope (IX71; Olympus) equipped with a 150×/1.45 NA oil-immersion, TIRF objective (U-APO; Olympus). A 637-nm laser diode (HL63133DG; Thorlabs) was used for AF647 excitation, and a 488-nm laser (Cyan Scientific; Spectra-Physics) was used for fluorescence excitation of mNG. A quad-band dichroic and emission filter set (LF405/488/561/635-A; Semrock) was used for sample illumination and emission. Emission light was separated onto different quadrants of an EMCCD camera (iXon 897; Andor Technologies), using a custom-built two-channel splitter with a 655-nm dichroic (Semrock) and 584/20-nm and 690/20-nm additional emission filters. Images had a pixel size of 0.106 μm and were acquired at 10 frames/second (100 ms exposure time).
The analysis algorithms and parameters used to track individual mNG molecules were previously described in Schwartz et al. (2017) (link). Briefly, tracks were analyzed using home-built MATLAB code that localized single particles per frame and determined connections between frames. The collected tracks were fitted to a two-component distribution to determine the off-rates of Syk at the membrane (Schwartz et al., 2017 (link)).
The analysis algorithms and parameters used to track individual mNG molecules were previously described in Schwartz et al. (2017) (link). Briefly, tracks were analyzed using home-built MATLAB code that localized single particles per frame and determined connections between frames. The collected tracks were fitted to a two-component distribution to determine the off-rates of Syk at the membrane (Schwartz et al., 2017 (link)).
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!