Imaging of Synaptic Vesicles

For imaging of immobilized SVs, a home-built total-internal reflection fluorescence microscope (TIRFM) was used, based on an Axiovert 200 microscope and a back-illuminated EM-CCD camera. This setup had a multi-line argon gas laser which provides multiple laser lines in the range between 488-514 nm wavelength at a 225 mW maximum power, and a diode laser which provides a 641 nm wavelength at 100 mW maximum power. A 488/10 nm filter was placed in the argon laser path to select the 488 nm laser line. As depicted in Figure ‎2-3, both laser lines were guided to a two-meter optical fiber through two solenoid shutters, six high surface quality mirrors (M1-M6) as well as a dichroic beamsplitter (Table ‎2-2). The laser beam from the fiber was deviated by a right angle prism by 90° and directed to a filterset cube on the reflector turret of the microscope through two achromatic doublet lenses. These lenses, with focal length of 75 and 65 mm, focused the lasers at the back-focal-plane of a PLAN-FLUAR 100x 1.45 NA objective. In addition, a micromanipulator was coupled to the fiber-prism holder to control the horizontal and vertical movement of the prism. A total-internal reflection angle was achieved by de-centering the laser beam using this micromanipulator.

Moreover, a dual line beamsplitter (ZT488/640rpc) was placed in the filter cube to be able to illuminate the sample with both lasers and UV light through the objective. No excitation filter was used in the filter cube, and either a dual band 538/685 filter or a 515/30 nm bandpass filter was used as the emission filter. Images were acquired using Andor IQ2 software (Andor Technology) which offers tight synchronization of the EM-CCD camera with external events such as opening of the laser shutters via TTL triggers.

Moreover, this software allows for programing different imaging protocols, in which

Materials and Methods |35 exposure time, imaging frequency, sequence of external events, etc. can be set according to the purpose of the experiment.

Figure ‎2-3 Laser alignment in TIRF setup.

The multi-line argon laser and diode laser were guided to an optical fiber via a dichroic beamsplitter (DBS) (LM01-503-25, AHF analysentechnik) and six 25.4 mm-diameter broadband dielectric mirrors (M1-M6). A 488/10 nm filter was placed in the argon laser path to select for the 488 nm line of the laser.

In order to test the quality of TIRF setup and compare it with epifluorescence, 0.2 µm yellow-green fluorescent beads were immobilized on PLL-coated coverslips, and a green fluorescent dye (Pyranine, Exc450 nm/Em511 nm) was added to the bath solution.

Beads were illuminated with the 488 nm line of argon laser and their emission was collected in both TIRF and epifluorescence mode. As shown in Figure ‎2-4, TIRF excitation could effectively eliminate background fluorescence and in turn improve the signal-to-noise ratio (SNR) compared to epifluorescence.

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Figure ‎2-4 Widefield versus TIRF illumination.

Representative fluorescence images of immobilized yellow-green fluorescent beads, illuminated with the 488 nm line of the argon laser in widefield (A) and TIRF (B) mode, in the presence of a green fluorescent dye (Pyranine) in the bath solution. C) Normalized intensity cross-section through one single bead in image A (red line) and B (green line). As shown, the background signal was reduced in TIRF mode. Calculating SNR by dividing the background-subtracted peak intensity by the standard deviation of the fluorescence values in the background resulted in an SNR of 61.6 in widefield, and 142.5 in TIRF, indicating a ~2.3 fold improvement of SNR in TIRF compared to widefield illumination.

2.6.3.2 Coupling a UV Flash Lamp to TIRF Setup

In order to provide a mercury lamp-based illumination source for the TIRF setup, a slider had been designed to direct the light from the lamp to the microscope through one of its side-openings. It consisted of an optical fiber holder, two mirrors (M1 and M2) and two lenses (80 and 75 mm focal length) (Figure ‎2-5). When the slider was coupled to the microscope, M2 blocked the laser path and only light from the mercury lamp could illuminate the sample. In order to equip the setup with an uncaging system, the mercury lamp was replaced with a Xenon-flash lamp. Moreover, to perform UV-uncaging simultaneously with illumination of the sample with lasers, M2 was replaced with a 425 nm longpass beamsplitter which allowed for more than 95 % transmission of both laser lines while 99% of UV light was reflected.

Materials and Methods |37 Figure ‎2-5 Coupling UV light to the setup through a side opening of the microscope.

The optical fiber connected to the flash lamp was coupled to a slider. The slider consisted of two lenses (focal lengths of 80 and 75 mm) and two mirrors (M1 and M2). M2 was replaced with a beamsplitter (F48-425, AHF analysentechnik) for transmission of both lasers and reflection of UV light. By placing the slider in the microscope through one of its side openings, uncaging could be performed during imaging with lasers.

The flash lamp was controlled by Andor IQ2 software via TTL triggers. As a control experiment to test whether the UV light was aligned properly, 0.2 µm yellow-green fluorescent beads were immobilized on PLL-coated coverslip and 40 µM DM-Nitrophen-calcium (provided by Dr. Kun-Han, Dep. Membrane biophysics, MPIbpc, Göttingen, Germany) as well as 2.5 µM of the calcium indicator, Fluo-4, were added to the bath solution. The sample was illuminated in epifluorescence mode with the 488 nm line of the argon laser and a UV flash was triggered during imaging. An increase in Fluo-4 intensity was observed upon triggering the flash lamp, indicating that the UV light could efficiently uncage DM-Nitrophen-Ca²⁺ and release free calcium (Figure ‎2-6).

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Figure ‎2-6 Quality check of UV light alignments.

Representative fluorescence response of the Ca²⁺ indicator, Fluo-4, in the solution to uncaging of DM Nitrophen-Ca²⁺ by two subsequent UV flashes. Release of Ca²⁺ by photolysis resulted in a transient increase of Fluo-4 fluorescence. Diffusion of Ca²⁺ out of the field of view diminished the fluorescence intensity.

2.6.3.3 Solution Exchange System

In order to perform fast solution exchange, the setup was equipped with a six channel perfusion valve control system (Table ‎2-2). The valve controller, which could be triggered manually or externally through Andor IQ2 software, provided synchronized opening/closing of multiple valves. To be able to exchange the whole bath solution in a short time, a custom-designed imaging chamber was constructed by the workshop of the Max-Planck Institute for Biophysical Chemistry (Göttingen, Germany). The chamber was designed to encompass a low bath volume (<100 µl) and equipped with three inlets and one outlet. Moreover, a peristaltic pump was used for fast removal of solution. With this solution exchange system, more than 80% of the bath solution was exchanged in 200 msec.