Fast S TED Microscopy: Experimental Implemen-

Figure 2.4 shows schematically the setup of the beam-scanning STED

microscope used in this thesis. The main distinguishing element to the STEDmicroscopes built previously is a resonant beam scanner. Its oper-ation at 16 kHz allowed acquiring a whole line of the image in 31 µs, us-ing forward and backward scans (bidirectional scannus-ing). This allowed recording movies of living cells at 28 fps and of nano-beads crystallizing into colloidal crystals at 200 fps. Bidirectional scanning also along the second lateral axis was used for these movies.

To ease sample handling, the setup was built around an inverted mi-croscope stand (IRB, Leica, Heidelberg, Germany). A slight modification of the microscope stand gave access to its optical path from the bottom.

Internal mirrors (of questionable stability) were not used. Excitation and de-excitation of the fluorescent dyes were performed with synchro-nized pulsed lasers (except for the CW experiments in Sec. 4.1.3). A diode laser (Exc Laser red in Fig. 2.4, LDH-P-635, PicoQuant, Berlin, Germany) provided excitation pulses of 120 ps duration at a wavelength of 635 nm. An amplified diode laser (Exc Laser blue, Pico TA 110–

2V0 00018, PicoQuant) provided excitation pulses at a wavelength of 490 nm. Liquid crystal variable retarders (VR1/VR2, Meadowlark Op-tics, Frederick, CO, USA) followed by Glan Thompson polarizers (GT1/

GT2, Bernhard Halle Nachfl. GmbH, Berlin, Germany) allowed adjust-ing of the power. The spatial mode profile of the laser beam was cleaned by single-mode fibers (SMF, Sch ¨after + Kirchhoff GmbH, Hamburg, Ger-many). The spectrum was cleaned by dielectric filters (F1, z488/10 and F2, z633/10, AHF analysentechnik AG, T ¨ubingen, Germany). A mode-locked Ti:Sapphire laser (STED Laser, Mai Tai, Spectra Physics,

Moun-E x c

Figure 2.4: Setup of the Fast STED microscope. Abbreviations:

APD: Avalanche Photo Diode; DM: Dichroic Mirror; Exc: Excitation; F: Fil-ter; FI: Faraday Isolator, FS: Fiber SplitFil-ter; GL: Glass Rod; GT: Glan Thompson Polarizer; L: Lens; M: Mirror; MMF: Multi Mode Fiber;

OBJ: Objective; PM: Phase Mask; PMT: Photomultiplier Tube; R: Retarder Wave Plate; SM: Scan Mirror; SMF: Single-Mode Fiber (polarization main-taining); SMP: Sample; VR: Variable Retarder.

tain View,CA,USAand Chameleon, Coherent, Santa Clara,CA,USA⁽⁵⁾) operating at 750 nm or 760 nm provided the de-excitation pulses. Its pulses were stretched to≈300 ps by a 30 cm rod of SF6 glass (GL) and 100 m of polarization maintaining single-mode fiber [SMF, (610–750) nm, cutoff 675 nm Thorlabs]. The glass rod is needed for stretching the pulses to avoid nonlinear effects in the fiber. A half-wave plate (R1, Thorlabs) was used to match the polarization axis with the fast axis of the fiber. A Faraday isolator (FI, FR 500/1100, 5 mm aperture, Linos, G¨ottingen, Germany) prevented back reflections into the laser cavity.

A dichroic mirror (DM1, AHF) combined the two excitation beams, an-other dichroic mirror (DM3, AHF) combined the depletion beam with the excitation beams. In the detection path, the dichroic mirror DM2 (AHF) separated the fluorescence from the excitation beams. Note that thick substrates (6 mm) are necessary to preserve the wave front of the reflected beam.

Shaping the wavefront of the de-excitation beam by a vortex phase plate (PM, VPP-A1, RPC Photonics, NY, USA) in the collimated de-exci-tation beam path generated the toroidal focus. The phase plate was im-aged onto the 16 kHz resonant beam scanner (SM,SC-30, EOPC, Glen-dale,NY,USA) by two achromats (L7, L8, Linos) in 4f configuration. The original scan and tube lens of the microscope in 4f configuration imaged the scan mirror onto the aperture of the objective (OBJ, NA = 1.4, oil immersion, HCX PL AP, Leica). In front of the objective’s aperture, a quarter-wave plate (R2,WPH05M-670 achromatic (690–1200) nm, Thor-labs, Newton,NJ,USA) polarized the beams circularly.

A fast photo diode (S5973-01, Hamamatsu, Herrsching am Ammer-see, Germany) recorded the 80 MHz pulses of the Ti:Sapphire laser, cap-turing a reflection from the Faraday isolator. These pulses were thresh-old detected, converted intoNIM⁽⁶⁾ signals, delayed and used to trigger the excitation diode laser. Proper adjustment of the electronic delay en-sured that the de-excitation pulses followed the excitation pulse imme-diately. A custom-built delay with a time resolution of 10 ps was used.

The fluorescence was collected by the objective and after de-scanning it was separated from the incoming beams by dichroic mirrors (DM2,

(5)The laser was changed once during this thesis

(6)Nuclear Instrumentation Module Standard

DM3, AHF). A bandpass filter (F4, HQ675/60, AHF, T ¨ubingen, Ger-many) removed scattered light. Confocal detection was used: the ob-ject plane was imaged onto a variable pinhole (PH, Leica) or onto the aperture of a multimode fiber with an opening diameter of 0.7 times the back-projected Airy disc of a diffraction-limited spot. Either an APD⁽⁷⁾

(APD, SPCM-AQR13, later SPCM-AQRH13, Perkin Elmer, Fremont, CA,

USA) or a PMT⁽⁸⁾ (PMT, H7422PA-40 select, Hamamatsu) detected the fluorescence. To combine the high quantum efficiency of an APD with high dynamic range, the fluorescence was divided with a 1:4 fiber split-ter (FS,F002197, Fiber Optic Network Technology, Surrey,BC, Canada) onto fourAPDs in some experiments.

A custom-builtFPGA⁽⁹⁾board allowed photon counting and data pre-processing. It was connected viaUSBto aPC, which was used for further data manipulation and storage. Due to the sinusoidal movement of the resonant scanner, a correction of the image brightness and dwell times was necessary: the primary data were collected with a pixel dwell time that was maximally half of the dwell time in the final image. The bright-ness and pixel sizes were then corrected for the sinusoidal movement of the scanner. This led to fractional photon counts in the images.

While the resonant beam scanner was used to scan the first lateral axis, a piezo stage (733-3DD and E-710, Physik Instrumente GmbH, Karlsruhe, Germany) with a digital controller and an internal feedback loop performed the scanning along the second lateral axis and the axial scanning. A custom-built cross table, equipped with piezo motors (Pico-motor, New Focus, San Jose,CA,USA) allowed the coarse positioning of the samples.

The optomechanical parts were purchased from Linos and Owis (Staufen, Germany). All Mirror mounts were of type “Suprema”, pur-chased from Newport (Irvine, CA, USA). All lenses were achromats (Linos), all mirrors dielectric (LBSM-VIS, LBSM-NIR, Linos). Fiber cou-plers were purchased from Point Source, Hamble,UK.

(7)Avalanche Photo Diode

(8)Photomultiplier Tube

(9)Field Programmable Gate Array