Observation of fast biological processes requires high-speed imaging systems. However, confocal scanning microscopy has an inherent bottle-neck: the serial recording of picture elements. Consequently, camera-based systems and other rapid acquisition approaches are often applied. With the Tandem Scanner Leica offers a high-speed confocal alternative (Figure 1). The Tandem Scanner unites the FOV scanner with a resonant scanning system based on switchable galvanometric mirrors.
It allows for line scan frequencies of up to 12 kHz, which results in about 40 fps at a scan format of 512x512 pixels or 428 fps at 512x16 pixels. This is a significant increase of more than 30% over Leica’s previous implementation with 8 kHz line frequency which is still available. These high scan speeds are useful for a number of biological questions, including rapid imaging of second messengers like Ca2+, kinetic measurements, fluid flow or cell motility (Figure 2). To control such high scan frequencies without compromises in image quality and with high geometric fidelity an intricate real-time control system is needed.
Next to high speed imaging the resonant scanning system reduces the photon dosage delivered to the sample. The mechanism by which this is achieved is also known as anti-triplet relaxation. Live samples thus benefit from low photobleaching and increased cell viability. For details on anti-triplet relaxation see also the corresponding article on Leica Science Lab by Rolf Borlinghaus.
Figure 1 The Tandem Scanner unifies resonant and non-resonant versions of galvanometric mirrors (scan mirrors for x-direction). Switching is performed by sliding the x-galvanometers back and forth. Using the resonant galvanometer frame rates up to 428 fps are possible, while the non-resonant mirror optimizes the field of view and provides adjustable speed in steps of 1 Hz.
Figure 2 HeLa Kyoto with GPI-YFP, H2B-mCherry imaged rapidly using Tandem Scanner 12 kHz, Galvoflow and HyD reveals rapid filopodial movement. Sample courtesy of: Jutta Bulkescher, EMBL, Heidelberg. Tubulin/H2B cell line: Steigemann et al. (2009) Cell 136:473-84; GPI-YFP construct: Keller et al. (2001) Nat. Cell Biol. 3:140-149