Integrated Microscopy Facility
Danforth Plant Science Center
1. Microscope Stand: Inverted microscope with a focus maintenance system, DIC transmitted light detector, and X-Y scanning stage for montaging. A galvo stage is included that is a piezo-controlled Z-axis stage control for use when acquiring image stacks with the resonance scanner.
2. Objectives (Plan Apo):
- 10x and 20x objectives for identifying regions within samples for higher magnification imaging and for imaging large fields of view at low magnification.
- 40x oil immersion, 1.3 NA. This objective will enable imaging of large fields of view, without montaging, at the high resolution offered by the 1.3 NA.
- 40x water immersion, 1.1 NA, 680 µm working distance. The working distance of this objective is an amazing 680 µm beyond the cover slip, providing deep imaging in thick samples.
- 63x oil immersion, 1.4 NA, for high resolution imaging.
- 63x water immersion, 1.2 NA. This is the highest resolution water immersion lens and can be used for live cell imaging of tissues where the deep imaging of the 40x water lens is not needed and where the increased resolution produces a benefit.
- 100x oil immersion, 1.44 NA. This is the highest resolution lens in our system.
3. Scan head:
- Acousto-optical beamsplitter (AOBS): This programmable beam splitter provides flexibility for programming the optimal cut-off for the laser line being used. The cut-off is only a few nm, improving throughput of emission signal to the detector and preserving sample viability. The AOBS can be programmed to pass multiple laser lines virtually simultaneously.
- Tandem scanner, 8 kHz. The scan head contains both a conventional and resonance scanner. The conventional scanner can be used for imaging large fields of view in a single scan. The resonance scanner provides high speed scanning (8,000 lines per second; 27 frames per second, 512 x 512 pixels each). In addition to imaging rapid events, rapid scanning reduces photobleaching rates, preserving cell viability, especially important in time lapse imaging.
- Abbe-Konig rotator for optimally rotating the field of view.
There are five detectors, two conventional PMTs and three HyD detectors. Detection bandpasses are custom fitted by using sliding mirrors for each detector that select bandpasses from the emission wavelengths that are dispersed efficiently by a prism. All five can be used when there is a need to detect five different signals. They can also be used for spectral unmixing, using up to five data points, when needed to separate multiple overlapping emission signals.
The HyD detectors are hybrid detectors in that they use a conventional photocathode (and vacuum tube) and an avalanche amplification step (http://www.leica-microsystems.com/science-lab/detectors-for-sensitive-detection-hyd/). The HyD uses a GaAsP photocathode of high sensitivity and the detector design helps prevent damage to the photocathode and also use it for high dynamic range detection—allowing imaging of both bright and dim signals in the same image. These detectors can be set up for single photon counting using the Leica software.
4. Lasers (over 200 lines are available):
- 405 nm diode laser for excitation of short wavelength-absorbing dyes and for photo-activation of fluorescent proteins.
- multi-line 100 mW argon laser, emission lines available for selection by the AOBS: 458, 476, 488, 496, 514 nm.
- White light laser: broad spectrum emission, 470-670 nm, with any single nm selectable, up to eight total per scan.