All optics were bolted to a 4′ × 8′ × 8″ optical table (Newport) in order to minimize external vibrations. A rapid automated modular microscope (Applied Scientific Instrumentation, RAMM-FULL-INVAR) served as the microscope body that housed sample, objectives (Olympus, PlanApo, NA 1.45 TIRF, for single cells, or UPLSAPO 60XS, NA 1.3 silicone oil, for zebrafish embryos samples), and automated XY stage equipped with Z axis piezo (Applied Scientific Instrumentation, PZ2300) that moved the axial position of the sample relative to the objective. For exciting fluorescence, two lasers were used: a 1W, 488 nm laser and a 0.5 W, 561 nm laser (Coherent, Genesis MX488-1000 STM and Genesis MX561-500 STM). Lasers were combined via a dichroic mirror (DC, Semrock, LPD01-488RU-25) and passed through an acousto-optical tunable filter (AOTF, AA Optoelectronic, Quanta Tech, AOTFnC-400.650-TN) for shuttering control. The intensity of each laser was maximized by tuning the rotation of half wave plates (Thorlabs, WPH10M-488 and AHWP05M-600) placed in front of each laser. After the AOTF, the beams were expanded 8.9x with a beam expander (Edmund, f = 45 mm, 64–837 and Thorlabs, f = 400, AC254-400-A), and passed through a converging microlens array (Amus, f = 1.86 mm, 1 mm thick, 25 mm diameter, antireflection coated over 400–650 nm, APO-Q-P222-F1.86), compensator plate (CVI Melles-Griot, PW1-2025-UV) and 6 mm thick dichroic mirror (TDC, Iridian Spectral Technologies, 488–561 DM). The compensator plate was used to cancel astigmatism that would otherwise arise when the focused beamlets passed through the tilted dichroic mirror. The resulting multifocal illumination was reimaged with a 1:1 telescope, consisting of 2 scan lenses (Scan lens 1 and 2, Special Optics, f = 190 mm, 55-S190-60-VIS) placed in a 4f configuration. An additional demagnification of 116.7x was achieved by reimaging the resulting excitation to the sample plane with tube lens (Edmund, f = 350 mm, NT49-289-INK) and objectives (f = 3 mm), also placed in a 4f configuration, and aligned so that the rear focal plane of the tube lens coincided with the front focal plane of Scan lens 2. Rotations of a 2-sided galvanometric mirror (Galvo, Nutfield Technology, QS-12 Galvo-Based Single-Axis Scan Set, P-PWR15 (power supply), S-0152 (Connector Kit), 10-2564 (mounting block); and Sierra Precision Optics, SPO9086 Rev B X-Mirror, double-coated) placed midway between each scan lens served to translate the multifocal array at the sample plane, thus covering the imaging field.
Several of these optics were reused for emission, as fluorescence was collected along the same path, through objective, tube lens, scan lenses 1 and 2, and galvanometric mirror, before reflection from the 6 mm thick dichroic mirror. Since the galvanometric mirror introduced an equal and opposite rotation angle on the return path (‘descanning’), the multiple fluorescence foci produced at the focus of scan lens 1 were stationary. A pinhole array, with pinhole spacing equivalent to the spacing between microlenses in the converging microlens array (Photosciences, Chrome on 0.090″ thick quartz, 222 μm pinhole spacing, 40 μm pinhole diameter) placed at the front focal plane of scan lens 1 served to reject out-of-focus fluorescence emission present around each fluorescent beamlet. The resulting filtered beamlets were relayed to a secondary microlens array via a 1:1 imaging telescope (Relay lenses, Thorlabs AC508-300-A-ML) and focused through a second microlens array (Amus, f = 0.93 mm, 1 mm thick, 25 mm diameter, antireflection coated over 400–650 nm, APO-Q-P222-F0.93) that locally contracted each fluorescent focus 2x, while preserving the orientation of each foci (see further discussion below andSupplementary Fig. 3 ). The locally scaled multifocal array thus produced was reimaged to a scientific grade complementary metal-oxide semiconductor camera (PCO-TECH, pco.edge) via additional scan lenses (Scan Lenses 3 and 4, Special Optics, f = 190 mm, 55-S190-60-VIS) placed in a 4f configuration. By placing the galvanometric mirror at the midpoint between scan lenses, fluorescence was rescanned onto the camera, producing a super-resolution, sectioned image of the sample plane. A filter wheel (Sutter, FG-LB10-BIQ and FG-LB10-NW) and notch filters (Semrock, NF03-488E-25 and NF03-561E-25) placed immediately before the cameras served to reject excitation laser light. These optical elements are shown in Supplementary Fig. 1 .
Our magnification of 116.7x resulted in an imaging pixel size of 55.5 nm. Excitation power varied between 5–50 W/cm2 depending on the particular sample.
Several of these optics were reused for emission, as fluorescence was collected along the same path, through objective, tube lens, scan lenses 1 and 2, and galvanometric mirror, before reflection from the 6 mm thick dichroic mirror. Since the galvanometric mirror introduced an equal and opposite rotation angle on the return path (‘descanning’), the multiple fluorescence foci produced at the focus of scan lens 1 were stationary. A pinhole array, with pinhole spacing equivalent to the spacing between microlenses in the converging microlens array (Photosciences, Chrome on 0.090″ thick quartz, 222 μm pinhole spacing, 40 μm pinhole diameter) placed at the front focal plane of scan lens 1 served to reject out-of-focus fluorescence emission present around each fluorescent beamlet. The resulting filtered beamlets were relayed to a secondary microlens array via a 1:1 imaging telescope (Relay lenses, Thorlabs AC508-300-A-ML) and focused through a second microlens array (Amus, f = 0.93 mm, 1 mm thick, 25 mm diameter, antireflection coated over 400–650 nm, APO-Q-P222-F0.93) that locally contracted each fluorescent focus 2x, while preserving the orientation of each foci (see further discussion below and
Our magnification of 116.7x resulted in an imaging pixel size of 55.5 nm. Excitation power varied between 5–50 W/cm2 depending on the particular sample.
