The isoSTED microscope for live cell imaging

path lengths and therefore interfere at the common focus of both objective lenses.

In section 3.3, a closer look at the cavity and its components is provided.

The fluorescence emitted from the sample is collected by both objective lenses in-coherently, de-scanned and separated from the laser light by the dichoric mirrors.

Additional filters suppress residual laser light in the detection path. A multimode fiber coupled to a single-photon counting module (SPCM-AQRH-13-FC, Excelitas Technologies, Waltham, MA, USA) acts as a confocal pinhole. A removable pelli-cle beam splitter and a photo-multiplier tube (PMT,Hamamatsu, Japan) allow for measurements in reflection mode, which are needed for system alignment.

In each laser beam path a fast shutter (Uniblitz LS6ZM2-NL, Vincent Associates, Rochester, NY, USA) is implemented to block the lasers separately and thus allow-ing to switch between confocal and STED imagallow-ing.

For electronic component and microscope control, as well as for data acquisition, a multifunction National Instruments Data Acquisition (NIDAQ) card (PCIe-6363, National Intruments, Austin, TX, USA) in combination with the acquisition soft-ware Imspector (Abberior Instruments GmbH, Göttingen, Germany) is used.

While this configuration performs at its best with fixed samples, it has some limi-tations for live cell imaging. When living cells are under analysis, they have to be embedded in an aqueous medium with nutrients. This results in a refractive index difference between the embedding medium and the immersion medium. Focusing into a mismatched medium causes spherical aberrations that increase with increas-ing sample depth [47]. The influence of the cover glass is typically corrected by the objective lenses. Therefore, a variant of the presented setup, adapted for live cell imaging is described in the following.

3.2. The isoSTED microscope for live cell imaging

The isoSTED setup for live cell imaging is presented in figure 3.2. The implemented adaptations are highlighted in green boxes.

Objective lenses: In order to minimize the aberrations due to refractive index differences, the oil-immersion objective lenses are replaced by 60x water-immersion objective lenses (UPLSAPO 60XW, Olympus, Japan).

STED laser system: Since STED microscopy uses high laser powers, the fluo-rescent molecules have an increased chance of bleaching. This bleaching scales nonlinearly with the applied STED intensity [48, 49]. STED pulses impinging on molecules in T₁ can effectively pump them into higher triplet states T_n, which are known as starting points for bleaching reactions [29, 44]. With a repetition rate

3. The isoSTED microscope SPCM ... counting module PMT ... tube

objektive lens 1 sample objektive lens 2 dichroic

Figure 3.2.: Adaption of the isoSTED microscope for live cell imaging. Changes in the setup are highlighted in green boxes. The objective lenses are changed to water-immersion objectives (UPLSAPO 60XW, NA 1.2, Olympus). STED laser systems with a repetition rate of 30 MHz and acousto-optical modulators that allow to switch off pixelwise reduce the effect of photobleaching. For improving the image quality the detection fiber is replaced by a fiber bundle.

3.2. The isoSTED microscope for live cell imaging of 80 MHz, a fluorophore in its triplet state will experience on average 80 high in-tensity pulses before it relaxes back to S₀ (triple lifetime of 1 µs [29]). Therefore reducing the repetition rate and thus decreasing the number of laser pulses that hit the molecules while they are in T₁, reduces the probability of photobleaching.

This method is called triplet relaxation (T-Rex) STED. In extreme cases even lasers with only 250 kHz repetition rate are used [29]. In our setup two independent fiber lasers (PFL-P-30-775-B1R, MPB Communications Inc., Quebec, Canada) de-livering pulses with a temporal width of approximately 900 ps and a wavelength of 775 nm at a repetition rate of 30 MHz are used. This reduces the probability of photobleaching without substantially increasing the data acquisition time.

Acousto-optic modulators (AOM): In order to reduce photobleaching even fur-ther, the ability to switch the lasers pixelwise on and off is beneficial. Techniques as RESCue-STED [50] reduce photobleaching by switching the lasers off when enough or no fluorescence is detected within a certain time. This reduces the amount of state transitions as well as the average time the dye molecules stays in the off-state. The so far used shutters have a maximum continuous frequency of 20 Hz, and thus only allow switching from line to line or frame to frame. Faster switching is realized by inserting acousto-optic modulators into the STED beam paths ( AA-MT110-A1.5-IR, AA Opto-Electronic, Orsay, France) and the excitation beam path (AA-MT110-A1.5-VIS, AA Opto-Electronic, Orsay, France). Those modulators uti-lize the acousto-optical effect to generate a refractive index grating within a glass or crystal by applying sound waves. Incident light is diffracted by this grating. With these AOMs switching of the laser beams on the single pixel level is possible.

To handle the increased amount of electronic signals, e.g. the switching signals for the AOMs, the previously used NIDAQ card is upgraded to an FPGA card (PCIe-7852R, National Intruments, Austin, TX, USA) and a patch panel (Abberior Instruments GmbH, Göttingen, Germany) is used to simplify the communication between Imspector and the hardware.

Detection: For improving the image quality (see section 4.2.3) the detection path is also changed. The multimode fiber, used as a detection pinhole, is exchanged with a customized 1-7-fan-out fiber (Thorlabs, NJ, USA) and three additional single-photon counting modules are implemented (SPCM-AQRH-13-FC, Excelitas Tech-nologies, Waltham, MA, USA).

3. The isoSTED microscope