Overview
The Olympus FVMPE-RS Multiphoton system is an upright microscope utilising dual pulsed two-photon far-red lasers for deep tissue imaging. The system also includes a resonant scan head allowing for extremely rapid imaging for tracking cell migration in living tissue.
The Olympus system is capable of imaging a wide range of endogenous and antibody labelled samples and generating second harmonics of unstained structures in whole tissue. The system also has special objective lenses designed to image cleared tissue to image up to 8 mm into organs.
The system is compatible with most samples but is ideally suited for preclinical models and large tissue imaging, giving scientists a chance to image deeper and over prolonged periods of time.
Video: Captured on the Olympus FVMPE-RS, green and magenta ‘tendrils’ show the network of blood vessels that are essential for the eye to form. Credit: Stephen Mieruszynski and Leigh Coultas
Hardware
Objective magnification | 5x | 10x | 20x | 25x | 25x |
Immersion media | Air | Multi | Water | Water | Clarity |
XY-resolution (µm) | 4.97 | 2.49 | 1.24 | 0.99 | 0.99 |
Working distance (mm) | 20 | 8 | 2 | 2 | 8 |
NA | 0.10 | 0.60 | 1.00 | 1.05 | 1.00 |
Light sources
- Olympus U-HGLGPS halogen light source + generic DAPI/GFP/RFP filters
- Mai-Tai eHP DeepSee multi-photon laser
- Insight DS+ multi-photon laser
Detection sources
- 2 x PMT detectors
- 2 x GaAsP detectors
Technological Specifications (capabilities)
Available modalities
- Z-stack
- Time-series
- Tile scan
- Multi positions
Additional hardware
- Resonant scan head
- SIM scanner
Unique features
- The system has a structured illumination microscopy (SIM) scanner, allowing for simultaneous imaging with one laser while using the other laser for photo-conversion or cell-ablation of small areas.
- The MATL (Multi-Area Time Lapse) feature allows for highly customisable experiments of multiple areas.
- The deep focus mode allows users to choose between depth of imaging and optimal Z-resolution.
Limitations
- Lower resolution in Z-axis than traditional confocal
- High laser powers can be detrimental to sample integrity
- Broad excitation profiles with single laser increase possible channel cross-talk
- Excitation profiles not characterised for all fluorophores
Resources
Nikon: Fundamentals and applications of multiphoton microscopy
Acknowledgement
This microscope was purchased with the generous support of the Australian Cancer Research Foundation (ACRF).
Video: A dissected retina image has been carefully removed to preserve its 3D structure, then imaged using a multiphoton microscope to allow the full structure to be acquired. Previously, imaging a retina would require the sample to be flattened. Credit: Leigh Coultas, Stephen Mieruszynski, Lachlan Whitehead