Gottfried Schatz Research Center | Molecular Biology and Biochemistry

1. Array confocal laser scanning microscopy (ACLSM) with Patch Clamp

We have received the ACLSM by a governmental infrastructure program back in 2003. Together with Visitron Systems and VisiTech we designed a customized instrument that perfectly matches our needs. Initially, it was designed to have a beam splitter using two RS CoolSnap HQ for detection. Notably the implementation of a filter wheel in front of each camera allows us to use multichannel acquisition just like any other conventional laser scanning microscope. Right from the beginning, the device was equipped with a patch clamp system that was not harmed at all by the nipkow disc. However, since the vertically installed camera introduced some vibration that did not allow for patch clamp experiments while using this device we have recently rebuilt the system and are now using one camera for confocal and one for conventional (simultaneous) imaging. Currently we are setting up the ACLSM almost weekly on our demands and experiments planned. The ACLSM is based on the principle of a scanning disc that has multiple holes introduced by Paul Nipkow (for more details see NipkowDiskMicrosope). There are some nice stories about how and when Paul Nipkow invented this idea that can be considered as the first TV system on Christmas Eve 1883. Now, more than 100 years after this great invention, the principle of the Nipkow disc has been upgraded by the introduction of a second disc that has lenses, thus boosting light transmission to approx. 50%. The general principle of the Nipkow Scanning head (see also Yokogawa disc) is that a laser beam is collected through a microlense disc via a pinhole array (see image below) and a lense (microscope objective) onto the sample. These two discs turn and, thus, allow for scanning the whole frame very fast. Emission light passes the pinhole array and gets reflected on a dichroic within the two discs directly onto a camera chip. Somehow, this device can be used like a conventional Imaging device and is, in our opinion the device of choice for working with living cells. We have equipped our ACLSM with a 405 nm laser diode and we recommend to do so if you are going to buy such device. It makes the ACLSM even more flexible and one can measure dyes like fura-2 (no ratiometric) or sapphire. It also allows FRET measurement with the CFP/YFP- and sapphire/DsRed-based FRET pairs that have been introduced so far. In our experience, the great advantages of an ACLSM compared to conventional CLSMs are: • strongly reduced cquisition time • less phototoxicity • easier to handle with far less image analysis time • emission spectra separation is also available (see MES) • due to its modular setup, laser alignment, filter exchange or any kind of rebuilding is simple • it is very open to extensions like filter wheels, beam splitters, or simultaneous imaging • it is very open to many of the great Imaging programs (e.g. MetaMorph®) • it is cheaper and much more flexible The drawbacks compared with conventional CLSM are: • excitation needs more powerful lasers (> 20 mW) • resolution is lower, while working in living cells, it might be identical • laser alignments are more often necessary • no classical bleaching experiments can be performed (e.g. FRAP, FLIM) • it is great for single cells but has limitations in tissue slices or model systems (e.g. matrigel)

2 & 3. Fluorescence Imaging Devices with Patch Clamp

Together with the ACLSM, these devices are our most frequently used instruments. Basically, we use hem to measure fura-2, Rhod-2, cameleons, pericams, picchu, apoK1er and all other dyes/proteins/sensors we are using in our studies. Late in the year 1998, I was granted by the FWF and the government of Austria to build two imaging system for conventional fura-2 imaging that also allows high resolution microscopy and patch clamp. Based on inverted Nikon microscopes and a 150 XBO lamp, these devices are equipped with filterwheels and the very nice and solid Quantix camera. 3D-scanning is available using a z-stage driver and image deconvolution is done by MicroTome/VolumeScan from VayTeck. For conventional imaging we first used a customized imaging program (thanks to Andy Haer) from IonOptix that was perfect for Ca2+ measurements. Finally, we moved to MetaFluor® and this configuration is about the setup we are still using day by day. To allow nice FRET measurements, the systems are equipped with Optical Insight's Dual-View™ that fits great to the MetaFluor®.

4. Microfluoremetry & Patch Clamp

This device is actually not much in use anymore, but it clearly deserves to be listed here. It has been our first device for ratiometric Ca2+ measurements in single cells. Notably, it also allowed for simultaneous patch clamp experiments. This device was established by Prof. Michael Sturek at the Dalton Cardiovascular Research Center when I (wfg) asked him to join his laboratory as a Postdoc. Among the many, many things Mike taught me in science, the building of such a device, collecting all the parts around the world was very important too. Already in the USA, I applied for a research grant at the FWF and requested funding of such a device. Back in Austria, and after the FWF granted my project, Mike came over and helped me to install this system in summer 1994. We put it together piece by piece and it has been running heroically for 10 years until Imaging systems became so much attractive that nobody wanted to work with it anymore. This was the way I was introduced to fluorescence microscopy and patch clamp and I still consider it to be the best one to do it by your own hand. Thanks Mike! This is a schema how this device was built. Basically it used an XBO lamp and a filter-wheel (360 or 340/380 nm) to excite the cells mounted in a chamber that was installed into an inverted microscope. Emission light had to pass an aperture, which allowed for selecting the area of interest, and was subsequently reflected by a dirchroic and intensity was measured using photon counting by a PMT. To allow pipette positioning the cells were also illuminated by a red light (>600 nm) that passed the dichroic and allowed visualization of the cell and the pipette during fura-2 experiments. This device was also equipped by a patch clamp system that utilizes a conventional bessel filter and amplifiers. Both signals (current as well as fura-2) were handled by the miraculous OP-400 (build by M. Martensen) that allowed for having everything on one screen.

5. CLSM Zeiss LSM 410

Although we are very happy with our ACLSM and the Imaging devices, we have an old, actually very old, Zeiss LSM410 still in use. As indicated above, FRAP and FLIM experiments are not possible with our systems at the moment. Thus if we want to perform these experiments we are using the 410.

6. Fluorescence Imaging device incl. beam splitter for FRET measurements

We just put some equipment together and built up a nice working Imaging system using a SPOT camera, 150 XBO lamp (Opti Quip) and a beam splitter (W-View, Hamamatsu) that is suitable for single wavelength imaging or FRET measurements.

 

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