FV-MPE Multiphoton Microscope


Location

RL B.716

Capabilities

  • Multiphoton imaging of thick in vivo and in vitro samples
  • Simultaneous photostimulation or dual-line imaging
  • Long, working-distance objectives optimized for multiphoton
  • High-speed extended IR (1300 nm) multiphoton imaging (30 FPS at 512 x 512, 438 FPS at 512 x 32)
  • 4 non-descanned detectors (2 GaAsP and 2 multi-alkali PMTs)

Cost

Internal user $30/hour
Internal service fee $69/hour

Detailed Specifications

Microscope

  • Olympus IX83 fully motorized upright microscope
  • Twin IR laser coupling optics with full laser enclosure for independent control of two IR lasers
  • Motorized mirror assembly permits user-selectable stimulation + imaging or simultaneous dual-laser line excitation
  • AOM laser attenuation (0%-100%, 0.1% increments) provides user selectable laser intensity adjustment and laser beam blanking to minimize photo-damage
  • Fully-automated IR beam expander optimizes beam diameter to maximize image resolution with any selected objective
  • Deep Focus Mode adjusts beam diameter in accordance with laser scattering sample conditions to facilitate deeper imaging
  • Quadralign 4-axis auto-alignment optics provides both 2 horizontal axes (X & Y) and 2 angular axes (Olympus-exclusive) of automatic laser realignment for precise multicolor imaging and precise co-alignment with two-laser systems
  • Prior z-deck linear-encoded motorized x-y stage with z-height adjustment
  • Possibility for upgrade to carry out in vivo electrophysiology

Tandem Scanners

  • Olympus SIM scanner for simultaneous imaging and photostimulation
  • Olympus-patented SIM scanner provides additional galvanometer scanning unit for region-of-interest stimulation simultaneous to imaging for optogenetic stimulation, uncaging, etc.
  • Wide choice of stimulation modes enable users to select point stimulation, rastered regions-of- interest, or Tornado (spiral) scans for high-efficiency stimulation

Left: Crosstalk occurred after simultaneous excitation o GFP and DSRED with a single wavelength IR pulsed laser (950nm). Right: Two fluorescence proteins are clearly separated after individual excitation of GFP and DS-RED with a dual wavelength IR pulsed laser (950nm/1040 nm).

Light Sources

  • Main scanner laser–Spectra Physics INSIGHT DS+ -OL pulsed IR LASER, tunable from 680 nm to 1300 nm, 120 fs pulse width at specimen plane
  • Stimulation laser–Olympus-specific SPECTRA PHYSICS MAI TAI HP DEEP SEE-OL pulsed IR LASER, tunable from 690 nm to 1040 nm, 100 fs pulse width at specimen plane
  • Olympus HGLGPLS widefield fluorescence illumination light source 130 W

Conventional Fluorescence Filters

  • U-FBVW; blue-violet excitation, 400-440/455/460 LP
  • U-FBNA; blue excitation, 470-495/505/510-550 BP
  • U-FGW; green, 530-550/570/575 LP

Both maximum intensity projection from 23 slices

Deep Focus Mode improves image brightness

Left: Normal image; Right: Deep Focus Mode; Image Image data courtesy of: Urs Zielger and Jose Maria Mateos, Center for Microscopy and Image Analysis, University Zurich. Mouse line L15 kindly provided by Pico Caroni FMI, Basel

Objectives

  • XLPLN10XSVMP, Olympus ultra-long working distance 10X MPE-optimized multi-immersion objective 60NA, WD 8 mm, correct collar for refractive indices 1.33-1.52
  • XLPLN25XWMP2, Olympus ultra 25x MPE water-immersion objective 1.05 NA, 2 mm WD
  • 5x NA 0.1, WD 20 mm dry (MPLN5X-1-7) and 40x, NA 0.80, WD 3.30 mm water-immersion (LUMPLFLN40XW(F)) objectives on swing nosepiece

Optimized for best live-tissue and intravital multiphoton imaging. Widefield design maximizes fluorescence light capture. Correction collar provides correction for spherical aberration across a wide variety of tissue refractive indices 400nm-1600nm transmission coatings. Chromatically corrected for 700nm to 1300nm IR lasers.

Dichroic(s)

  • AOBS serves as tunable dichroic for all lasers

Third harmonic generation imaging of porcine adipose tissue; Unlabeled porcine adipose tissue was irradiated with femtosecond laser at 1250nm, second harmonic is detected from collagen fibers at 625 nm and third harmonic from lipid interlaces at 416 nm.

 

Detectors

  • Includes 2 channels of GaAsP PMTs for maximum sensitivity and 2 channels of multi-alkali PMTs for maximum spectral range
  • Transmitted light detector for Brightfield/DIC Image Acquisition

Olympus GaAsP detectors provide approximately twice the quantum efficiency of standard multi-alkali PMTs. All non-descanned detectors are positioned as close as possible to the specimen and signaling optics have been enlarged to facilitate maximum detection efficiency of scattered fluorescence.

Computer and Software

  • HP Windows 764-bit Workstation and 30” Monitor, 2TB HD, 128 & 256 GB SSDs, 32GB DDR3 RAM
  • FVMPE-RS system software including sequence manager, stage control, multipoint mapping, and data analysis
  • Full-feature acquisition software provides complete control of system components including laser wavelength and intensity attenuation, laser coupling optics, scanners, & PMTs
  • Stage Control Software provides XY motorized stage control, map image acquisition for easy target locating, tiling acquisition, and image stitching
  • Multipoint Mapping Software provides high-speed multipoint stimulation, multipoint acquisition, and optogenetic mapping
  • Sequence Manager allows programming of complex imaging tasks with microsecond precision within imaging tasks and millisecond precision between imaging tasks for up to two weeks
  • Embedded 64-bit 3D rendering tool
  • Olympus cellSens Dimension provided for post-acquisition data analysis

Tutorials

iBiology tutorial by Kurt Thorn on multiphoton microscopy

 Software manual

Module 1: Basic introduction and lightpath

Module 2: Dye, detector, and laser selection

Module 3: Detectors

Module 4: Objectives and image acquisition