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Femto3D-RC

/ Spectrometers

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.

The unit achieves significantly better optical resolution and produces less stray light than most filter-based and other spectrometers of its size. For example, STS has 14- bit resolution and has low power consumption of just 0.75 W. Also, STS has triggering functions for instances where precise timing is necessary – for example, synchronizing measurements with an external event such as the pulsing of an excitation lamp for fluorescence. STS takes advantage of recent advances in cMoS detectors that elevate optoelectronic performance and improve system reproducibility.

It uses a 1024-element eLiS-1024 linear image sensor that’s responsive from 200- 1100 nm and has great sensitivity (6.74V/lux-second typical). This new generation of cMoS detectors offers excellent performance with great value.

Scanning modes of the microscope

  • 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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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
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  • RollerCoaster scanning
  • Stripe scanning
  • 4D scanning
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