Two photon microscope combining galvanometer scanning and piezoelectric objective positioner.
Real-time 3D microscopy at Femtonics does not just mean the ability to measure 3D image stacks, but also to collect signals from different depths of the sample fast enough to resolve its functionality, following events all through the three-dimensional space of the sample with a high signal-to-noise ratio. We provide a fast high resolution 3D two-photon laser scanning method (Roller Coaster Scanning) capable of imaging long dendritic segments resolving individual spines and inputs with a temporal resolution of a few milliseconds.
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| SCANNING |
|---|
| Measurement mode |
| Two-photon imaging mode with four non descanned detectors |
| Transmitted IR scanning mode |
| Transmitted gradient contrast mode (or DIC mode) viewed through CCD camera. |
| 3D scanning |
| RollerCoaster scanning (3D - up to 150 Hz) |
| Tilted frame scanning (3D - up to 8 Hz) |
| Stripe scanning (3D - up to 8 Hz) |
| Fast XYZT scanning (3D - up to 8 Hz) |
| Multiple Line Scan mode of scanning (2D - up to 1 kHz) |
| Folded Frame Scanning (2D - up to 100 Hz) |
| XY(t), X(t), XYZ |
| Roller Coaster Z- Scanning range |
| 25 µm @ 150 Hz (optimal for calcium imaging) |
| 90 µm @ 40 Hz |
| 100 µm or 300 µm @ 10 Hz (selectable) |
| SOFTWARE |
| 3D seal-time scan control and analysis software |
| • Extends functionalities of the MES software |
| - Flexibility of protocol design |
| - ΔF/F, ΔG/R calcination |
| - Real-time display and analysis of multichannel measurements |
| - Integrated parallel data acquisition and analysis of electrical recordings Automated calibration software |
| - Zoom, automatic scan adjustment, time series acquisition |
| • 3D data acquisition and real-time 3D control of the measurement |
| • 3D spatial normalization and data projection |
| MICROSCOPE |
| Wide field-of-view (e.g. 650 µm x 650 µm with 20x obj.; full optical resolution) |
| Spatial resolution characteristics of 2D two-photon imaging is preserved (down to 350 nm) |
| Pixel dwell time: 0.5 µs - 10 ms |
| High quality mode-locked lasers from SpectraPhysics, Coherent or Toptica |
| Olympus BX61 based, Objectives from various vendors |
| Non-descanned whole field PMTs optimized for scattered photons |
| Measurement workstation PC, Dual display, 16 GB memory, 64 bit Windows 7 |
| 3D DENDRITIC IMAGING |
| A ~ 25 fold increase in access rate to imaging 40-50 µm long continuous dendritic segments |
| Imaging long dendritic segments (up to 250-300 µm) |
| Resolution of individual spines |
| Simultaneous measurement of 15-50 inputs |
| Drift compensation for high S/N |
- RollerCoaster scanning (3D trajectory scanning)
- Tilted frame scanning (3D arbitrary frame scanning)
- Stripe scanning (fast line – Z scanning)4D scanning
- All 2D scanning modes possible with galvanometer based scanners:
- Frame scanning,
- Folded/Multi-frame scanning,
- Line scanning,
- Point scanning,
- XYZ stack
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