We used an Olympus IX83 inverted microscope with an autofocus system. The excitation path was composed of three laser lines: 637, 532, and 405 nm (Errol lasers) and a TIRF module (Errol lasers) used in combination with a matched 390/482/532/640 multiband filter (LF405/488/532/635‐A‐000, Semrock). The fluorescence was collected through an Olympus x100 1.49 NA oil immersion objective lens. The detection path was composed of a SAFe module (Abbelight) and a Flash 4 v3 (Hamamatsu). The pixel size in the object was 100 nm.
Lf405 488 532 635 a 000
Manufactured by IDEX Corporation
Sourced in United States
The LF405/488/532/635-A-000 is a laser module that can produce laser beams at wavelengths of 405 nm, 488 nm, 532 nm, and 635 nm. It is designed for use in various laboratory and scientific applications. The laser module provides a compact and stable source of coherent light at these specific wavelengths.
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Lab products found in correlation
Ti s microscope, by Nikon (4 mentions)
Zyla 5.5 scmos camera, by Oxford Instruments (3 mentions)
Led4d120, by Thorlabs (3 mentions)
Ix83 inverted microscope, by Olympus (2 mentions)
Flash 4 v3, by Hamamatsu Photonics (2 mentions)
1.2 na water immersion objective lens, by Nikon (2 mentions)
Perfect focus system, by Nikon (1 mentions)
Lf405 488 561 635 a 000, by IDEX Corporation (1 mentions)
Apo tirf 100 1.49 na, by Nikon (1 mentions)
Ixon3, by Oxford Instruments (1 mentions)
5.5 scmos camera, by Oxford Instruments (1 mentions)
Sp5 inverted confocal point scanning microscope, by Leica (1 mentions)
7 protocols using lf405 488 532 635 a 000
1
Super-Resolution Microscopy Setup
2
Live Cell Fluorescence Imaging Protocol
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Conventional widefield epifluorescence imaging was performed on an inverted Nikon Ti-S microscope configured with a 60 × 1.2 NA water-immersion objective lens (Nikon, Melville, NY, USA), a light emitting diode source (LED4D120, Thorlabs, Newton, NJ, USA), a multiband filter set (LF405/488/532/635-A-000, Semrock, Rochester, NY, USA) and images were captured with a Zyla 5.5 sCMOS camera (Andor, Windsor, CT, USA). The samples were illuminated 470 nm light at an intensity of ~2 W/cm2 and with 200 ms exposures. For live cell experiments, samples were incubated at 37°C with Gibco CO2 Independent Medium containing 50 µM DFHBI for 10 minutes prior to imaging. Time lapse movies were acquired over a period of 5 minutes with a 200 ms exposure every 5 seconds. For fixed cell imaging, samples were incubated at room temperature (~22°C) in PBS containing 50 µM DFHBI for 10 minutes prior to imaging.
3
Multicolor single-molecule imaging setup
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A schematic of the optical setup used is presented in Fig. 1a . We used a Nikon Eclipse Ti inverted microscope with a Nikon Perfect Focus System. The excitation was performed thanks to five different lasers: 637 nm (Obis 637LX, 140 mW, Coherent), 561 nm (Genesis MX 561 STM, 500 mW), 532 nm (Verdi G5, 5 W, Coherent), 488 nm (Genesis MX 488 STM, 500 mW, Coherent), and 405 nm (Obis 405LX, 100 mW, Coherent). The corresponding 390/482/532/640 or 390/482/561/640 multiband filters (LF405/488/532/635-A-000 and LF405/488/561/635-A-000, Semrock) were used. The fluorescence was collected through a Nikon APO TIRF ×100 1.49 NA oil immersion objective lens, sent in the DAISY module and recorded on two halves of a 512 × 512-pixel EMCCD camera (iXon3, Andor). The camera was placed at the focal plane of the module of magnification 1.67 and the optical pixel size was ~100 nm. Finally, the imaging paths were calibrated in intensity to compensate the non-ideality of the 50–50 beam splitter, as well as the reflection on the cylindrical lens surface (this measurement was performed for each fluorescence wavelength). The object focal plane of the EPI path was typically at the coverslip (z = 0 nm) and the UAF path had two focal lines, at z = 0 nm and z = 800 nm for the y and x axes, respectively.
4
Widefield Epifluorescence Imaging of Live and Fixed COS-7 Cells
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Conventional widefield epifluorescence imaging of cultured live COS-7 cells (Supplementary Movie 1 and Supplementary Movie 2 ) and fixed COS-7 cells (Supplementary Fig. 4 ) was performed on an inverted Nikon Ti-S microscope configured with a 60x/1.2 NA water-immersion objective lens (Nikon), a multiband filter set (LF405/488/532/635-A-000, Semrock), and a Zyla 5.5 sCMOS camera (Andor). Micro-Manager software with MM Studio and MMCore were used for acquisition. For widefield epifluorescence microscopy of fixed COS-7 cells expressing mFAP2a or mFAP2b targeted to the endoplasmic reticulum (ER) (Supplementary Fig. 4 ), samples were labeled with either 40.0 µM DFHBI or 40.0 µM DFHBI-1T in 1x PBS for at least 10 min before imaging. Cells were rinsed three times with 1.00 mL of 1x PBS. Samples were illuminated with 470 nm light at an intensity of ~2 W cm−2. Exposure times were 200 ms and current was 500 mA. For time-lapse widefield epifluorescence microscopy of live COS-7 cells expressing mFAP2a or mFAP2b targeted to the ER (Supplementary Movie 1 , Supplementary Movie 2 ), cells were labeled with 40.0 µM DFHBI in 1x PBS. The time-lapse movies were acquired using 200 ms exposure times every 5 s for 25 total frames, with 100 mA excitation current. The total acquisition duration per movie was just over 2 min, and movie playback speeds adjusted to 5 frames s−1.
5
Epifluorescence and Confocal Microscopy
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Conventional epifluorescence microscopy was performed with an inverted Nikon Ti-S microscope fitted with a 4 × 0.2 NA air objective lens (Nikon, Melvill, NY, USA), a 10 × 0.25 NA air objective lens (Nikon), or a 20 × 0.45 NA air objective lens (Nikon). Illumination was achieved using a four-channel light emitting diode (LED4D120, Thorlabs, Newton, NJ, USA) with a multiband filter set LF405/488/532/635-A-000, Semrock, Rochester, NY, USA) and images were captured using a Zyla 5.5 sCMOS camera (Andor, Windsor, CT, USA). Confocal microscopy was performed on a Leica SP5 inverted confocal point scanning microscope at the UW Biology Imaging Core using a 10 × 0.4 NA air objective lens (Leica, Nussloch, Germany), 20 × 0.7 NA air objective (Leica), and a 63 × 1.2 NA water immersion objective (Leica).
6
Live Cell Fluorescence Imaging Protocol
7
Super-Resolution Microscopy Setup
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We used an Olympus IX83 inverted microscope with an autofocus system. The excitation path was composed of three laser lines: 637 nm, 532 nm, and 405 nm (Errol lasers) and a TIRF module (Errol lasers) used in combination with a matched 390/482/532/640 multiband filter (LF405/488/532/635-A-000, Semrock). The fluorescence was collected through an Olympus x100 1.49 NA oil immersion objective lens. The detection path was composed of a SAFe module (Abbelight) and a Flash 4 v3 (Hamamatsu). The pixel size in the resulting image was 100 nm.
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