PALM data was recorded on a custom-built three-camera RAMM frame microscope (ASI) using an Olympus 1.4 NA PLAPON 60× OSC objective, and a custom tube lens (LAO-300.0, Melles Griot), resulting in 100× overall magnification25 (link). A 2 mm thick quad-band excitation dichroic (ZT405/488/561/640rpc, Chroma), a 2 mm thick emission dichroic (T560lpxr, Chroma), and a band-pass emission filter (FF01-609/54-25, Semrock) filtered the emitted light. Dendra2 was photoconverted by 100 μs long excitation pulses of 405 nm (50 W/cm2) every 200 ms, which was ramped up to 1.2 ms long pulses every 200 ms during the course of image acquisition. Stroboscopic 405-nm excitation of the Stradus 405-100 laser (Vortran) was achieved using a NI-DAQ-USB-6363 acquisition board (National Instruments), Photoconverted Dendra2 molecules were excited with a 555-nm DPSS laser (CrystaLaser) at estimated sample power levels of 2 kW/cm2. Fluorescence was detected using μManager (v. 1.4.20)26 with a back-illuminated EMCCD camera (Andor Technology, Ixon Ultra DU-897_BV, 17 MHz EM amplifier, Gain 500, full-chip) at 20 frames/s.
Protocol detail
Quantitative Super-Resolution Imaging
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Variable analysis
independent variables
- Duration of 405 nm excitation pulses (ramped up from 100 μs to 1.2 ms during image acquisition)
- Intensity of 405 nm excitation pulses (ramped up from 50 W/cm^2 to unknown value during image acquisition)
- Photoconversion of Dendra2 molecules
- Microscope setup (3-camera RAMM frame, Olympus 1.4 NA PLAPON 60× OSC objective, custom tube lens, 100× overall magnification)
- Dichroic and emission filters (quad-band excitation dichroic, emission dichroic, band-pass emission filter)
- Laser sources (Stradus 405-100 laser for 405 nm excitation, 555-nm DPSS laser for excitation of photoconverted Dendra2)
- Imaging system (μManager software, Andor Technology EMCCD camera, 20 frames/s)
- Positive control: Photoconversion of Dendra2 molecules using 405 nm excitation
- Negative control: Not explicitly mentioned
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