In this section, the experimental conditions for the experiments reported in the following sections are reported. In particular, the confocal microscope and the epi-fluorescence microscope, employed in the FFS experiments, will be described.
Following the setup descriptions, the data acquisition and analysis will be illus-trated.
3.4.1 Confocal microscopy
The setup used is based on a modified inverted microscope (Olympus IX73, Olympus Europa SE & CO. KG, Hamburg, Germany). The excitation light is provided by two diode pulsed lasers (Cobolt Samba-532 100mW and Cobolt Calypso-491 25mW, Cobolt AB, Solna, Sweden) inserted into a laser combiner box (C-Flex, Cobolt AB, Solna, Sweden). After exiting the optical fiber, the laser light passes through a clean-up filter (HC Laser Clean-Up MaxLine 491/1.9, AHF Analysentechnik, Tübingen, Germany HC Laser Clean-up MaxLine 532/2).
The laser beam is expanded by a factor of 10 using a 10X objective (Olympus UPLFLN10XP, NA=0.30) and a f = 200 mm lens (Qioptiq Photonics GmbH KG, Göttingen, Germany) in order to illuminate the full back aperture of the microscope objective. The laser intensity is attenuated with a neutral density filter (OD = 6, Qioptiq Photonics) before being deflected by a dichroic mirror (DualLine zt488/532rpc, AHF Analysentechnik AG, Tübingen, Germany) into the microscope. The laser beam is focused onto the sample using a 60X water immersion objective (UPlanApo, NA = 1.2, Olympus). The fluorescence light is
Figure 3.7: Schematic representation of the confocal microscopy setup.
then focused using a f = 200 mm lens to the pinhole (diameter 50 µm, Qioptiq Photonics) cutting off the out off focus light contribution. After the emission filter (razor Edge Long Pass Filter 488 or RazorEdge LP Edge Filter 532, AHF analy-sentechnik AG) the light is collimated using a f = 50 mm lens and directed to the active area of two different avalanche photo diodes (τ-SPAD, Picoquant GmbH, Berlin, Germany and CountBlue Count50, Laser Components GmbH Olching, Germany), using a 50:50 beam splitter (Thorlabs BSW10R, Thorlabs Inc. New-ton, New Jersey, USA). A scheme of the optical path is shown in Figure 3.7.
Theτ-SPADs are connected either to a digital correlator card (ALV-7004 USB, ALV-Laser Vertriebsgesellschaft mbH, Langen, Germany) used for autocorrelation measurements, or to an acquisition card (NI-6602, National Instruments, Austin, USA) to access directly the raw photon arrival times. The digital correlator can calculate four correlation functions simultaneously: the autocorrelation functions of the two distinct APDs and the forward and backward cross-correlation func-tions between the two detector signals. In principle, it is also possible to measure
2-color cross-correlation functions, if the 50:50 beam splitter is replaced by a dichroic mirror. The digital correlator card is directly connected to the PC to store and analyze the data. To access the data from the acquisition card, we use a custom-written code written using on a python interface [123]. The photon arrival times are measured relative to an arbitrary start point with an internal clock of 10 MHz. All the experimental data are then analyzed using self-written Python code (Python Software Foundation, https://www.python.org/). To access differ-ent positions in the sample, an automated sample stage (Prior Scidiffer-entific, Inc., Rockland, MA, USA) is used.
3.4.2 Epi-fluorescence microscopy
The same microscope described above can also be used for epi-fluorescence mi-croscopy. A mirror in the second deck of the microscope body allows us to switch between the two microscope configurations. The excitation light comes from a mercury arc lamp (X-Cite 120 PC Q, Excelitas Technologies) and it is guided onto a fluorescence filter cube (filter sets available: DAPI, GFP, Cy3, TxRed and Cy5, all from AHF analysentechnik AG, Tübingen) which selects the wavelength of the excitation and filters the emission light. Images are acquired using a CCD-camera (Hamamatsu Orca R-2, Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany) controlled by Micro-Manager [124].
3.4.3 Data acquisition and analysis Settings for microfluidic experiments
Flow measurements are performed using the microfluidic devices described in Sec-tion 3.3. Polyethylene tubings (inner diameter 0.38 mm, outer diameter 1.09 mm, Intramedic Clay Adams Brand, Becton Dickinson and Company, Sparks, USA) are connected to Hamilton Gastight glass syringes (Bonaduz, Switzerland) using disposable needles. For a precise control of the flow, syringe pumps (neMESYS, Cetoni GmbH, Korbußen, Germany) are employed to regulate the flow rates of the syringes connected to all the inlets. In the microfluidic experiments where vimentin assembly is studied, Chapter 4.1, the starting vimentin concentration is 0.003 g/L and the assembly is initiated by adding 100 mM KCl by diffusive mix-ing. Flow rates in these experiments are12µL/h (central inlet), 10µL/h(sheath inlets) and190µL/h (side inlets).
In the experiments, where SVs interactions under flow are investigated, in Chapter 4.2.3, the total inflow velocity used is 3 mm/s and it is divided over the three inlets. In the central channel a constant velocity of 0.1 mm/s is employed, while in the side inlets a periodic rectangular anti-synchronous flow velocity profile is applied. As shown in Figure 4.38, the velocity profile of the side inlets has a maximum flow velocity of 1 mm/s, a minimum of 0.1 mm/s and a width of 10 s.
The velocities in the two side inlets are shifted in time to achieve a constant flow velocity in the center of the channel.
FCS experiments
Stationary FCS or PCH measurements are performed using 300µL of sample placed in an eight-well glass slide (Nunc Lab-Tek chamber slides, Thermo Fisher Scientific, Pittsburg PA). Before every measurement on the confocal microscope, the setup is aligned and the observation volume is measured. The calibration pro-cedure is fundamental to determine the absolute value of the diffusion coefficients [125] obtained during the measurements. The diameter of the observation volume is calculated measuring the autocorrelation function of dyes with a known diffu-sion coefficient. For measurements at 532 nm, rhodamine 6G (Thermo Fisher, D = 414 ± 5µm2/s at 25 ◦C [126]) is used. For measurements at 491 nm, Atto 488 (AttoTech GmbH, Siegen, Germany ,D = 400± 10µm2/sat 25 ◦C [127]) is used respectively. The measurements are usually performed at a temperature of 22◦C. The detection volume ω0 is then calculated with:
ω0 =p
4DτD (3.1)
Usually the measured diameter is 300±10 nm. For FCS measurements, data are collected between 10 and 60 s for each curve, depending on the expected diffusion coefficient. The data are analyzed either using the software Quickfit 3.0 [128] or using a self-written fitting routine with Python code (Python Software Founda-tion, https://www.python.org/). Measurements, where strong fluorescence peaks, caused by aggregates, affect the correlation curves are excluded from the analysis.
If bleaching occurs, the trace of fluorescence intensities over time is fitted using a double exponential [129] and the data are re-correlated to overcome the decrease in intensity. A Levenberg-Marquardt non linear least-square algorithm is used to fit the data.
FCS measurements in living neurons
The sample containing living neurons in Tyrode’s solution is mounted on the sam-ple stage of the microscope. Measurements are performed on mature hippocam-pal cultured neurons expressing EGFP-synapsin, mEGFP, membrane EGFP and mEGFP-SNAP25. Data are acquired from different positions along axons for each protein analyzed. Each position is measured at least 20 times, with acqui-sition times between 10 s to 30 s for each round of acquiacqui-sition. All of the data are then fitted with a Levenberg-Marquardt non linear least-square routine using a self-written Python code. Data, in which large fluorescence peaks are observed or with pronounced photobleaching, are excluded from the analysis. The cell culture, transfection and manipulation was carried out by our collaborator Sofiia Reshetniak from the University Medical Center of Göttingen in the Institute for Neuro- and Sensory Physiology.
PCH experiments
The data for each PCH are collected for a time duration between 300 s and 800 s.
To decrease the computational time, the data are acquired in slots of 10 s each and then combined together, excluding the runs where aggregates were present. The acquired photon arrival times are binned with a binning time between 2 and20µs, stored and used to build the experimental PCH curves. A histogram of the counts per bin,k, is created and normalized. The experimental PCH, p(k)is fitted with the theoretical model described in [8] using a Levenberg-Marquardt non linear least-square algorithm. All the scripts used during the PCH calculation and analysis are custom written with Python code. The OPE correction parameter F for our experiment is fixed to 0.6 and the dead-time is measured to be 86 ± 19 ns (45 ns is the value provided the manufacturer).