Elyra p 1 tirf microscope

Images were acquired on a Zeiss Elyra P.1 TIRF microscope equipped with a Zeiss alpha Plan-Apochromat TIRF 100x/1.6 NA oil objective; tube lens for an additional factor of 1.6x magnification; and quad-band dichroic (405/488/561/642). For both the CF568 and Alexa647 dyes, an Andor iXon+ EMCCD camera captured a sequential time-series of 20,000 frames each at a gain setting of 100 with an integration time of 18ms. Image size was 256X256 pixels, with a pixel size of 100 nm xy. Alexa-647 molecules were ground-state depleted and imaged with a 100mW 642 laser at 100% AOTF transmission in ultra-high-power mode (condensed field of illumination), corresponding to approximately 1.4W/cm². Emission light passed through a LP 655 filter. CF-568 molecules were ground-state depleted and imaged with a 200mW 561 laser at 100% AOTF transmission in ultra-high-power mode, corresponding to approximately 2.5W/cm². Emission light was passed through a BP 570–650 + LP 750 filter. For each dye, ground-state return was elicited by continuous illumination with a 50mW 405 laser at 0.01 to 0.1% AOTF transmission.

Samples for dSTORM and SIM imaging were prepared as previously described.^{26 (link)} Images were acquired on a Zeiss Elyra P.1 TIRF microscope equipped with a Zeiss alpha Plan-Apochromat TIRF 100x/1.6 NA oil objective; tube lens for an additional factor of 1.6x magnification; and quad-band dichroic (405/488/561/642). For both the CF568 and Alexa 647 dyes, an Andor iXon+ EMCCD camera captured a sequential time-series of 20,000 frames each at a gain setting of 100 with an integration time of 18ms. Image size was 256X256 pixels, with a pixel size of 100 nm xy. Alexa 647 molecules were ground-state depleted and imaged with a 100mW 642 laser at 100% AOTF transmission in ultra-high-power mode (condensed field of illumination), corresponding to approximately 1.4W/cm². Emission light passed through an LP 655 filter. CF-568 molecules were ground-state depleted and imaged with a 200mW 561 laser at 100% AOTF transmission in ultra-high-power mode, corresponding to approximately 2.5W/cm². Emission light was passed through a BP 570–650 + LP 750 filter. For each dye, ground-state return was elicited by continuous illumination with a 50mW 405 laser at 0.01 to 0.1% AOTF transmission.