Prioritizing speed

Related Articles Tips for choosing a microscope setup Going Live How it Works: Two-Photon Microscopy Pooling resources Mix and match Deep down view Sticking to the surface User: Peter Robin Hiesinger, a neurogeneticist at UT Southwestern Medical Center in Dallas. Project: Real-time imaging (seconds to minutes) of synaptic transmission at the neuromuscular junction of the developing fly larva; time-lapse imaging (hours to days) of neuronal outgrowth in cultures of dev

Nov 1, 2007
Alla Katsnelson

User: Peter Robin Hiesinger, a neurogeneticist at UT Southwestern Medical Center in Dallas.

Project: Real-time imaging (seconds to minutes) of synaptic transmission at the neuromuscular junction of the developing fly larva; time-lapse imaging (hours to days) of neuronal outgrowth in cultures of developing brain.

Problem: Hiesinger needed a system that could handle both extremely short and extremely long timescales.

Solution: Hiesinger's real-time imaging experiments require the acquisition of as many as 25 images per second. Although a spinning-disk confocal microscope can achieve the speed, it lacks the flexibility to change the pinhole size to restrict out-of-focus illumination and adjust for the sample's level of fluorescence. Instead, Hiesinger chose a Leica resonance-scanning confocal (Leica TCS SP5), essentially a standard laser-scanning confocal microscope, but with a beam that oscillates at a very high resonance frequency (8,000 Hz, as opposed to 400-1,000 Hz). This translates into a resolution of about 15 frames per second for images scanned at a resolution of 512 x 512 pixels. Hiesinger also chose the Leica system because it can be built as a "tandem scanner," which can be booted in either the resonance or the conventional confocal mode for the long-term experiments. "In our case, the solution was obvious," Hiesinger says.

<figcaption>Top: Cultured developing fly brain labeled with photoreceptor-specific GFP (green) and pan-neuronal RFP (red), imaged in conventional confocal mode. Bottom: Neuromuscular junction in fly larva (blue: muscle; green: presynaptic boutons, red: active zones) imaged in resonant confocal mode. Credit: Top: Peter Robin Hiesinger</figcaption>
Top: Cultured developing fly brain labeled with photoreceptor-specific GFP (green) and pan-neuronal RFP (red), imaged in conventional confocal mode. Bottom: Neuromuscular junction in fly larva (blue: muscle; green: presynaptic boutons, red: active zones) imaged in resonant confocal mode. Credit: Top: Peter Robin Hiesinger

Other companies have recently introduced similar modifications on the traditional confocal microsope, notes Maddox, such as Zeiss' line-scanning (5Live) or Nikon's swept-field (LiveScan) models. Both scan with a slit instead of a pinhole for faster imaging.

Cost: ~$340,000