Core Facilities
USF Health - College of Medicine


    Olympus FV1000 MPE multiphoton laser scanning microscope

Supports multicolor fluorescent studies for imaging of living, whole mount or thickly sliced specimens. Dynamic biological processes can be imaged hundreds of micrometers within living cells and tissues. Provides support for applications where phototoxicity/photobleaching are a concern such as time course studies of living cells and tissues. Low magnification lens and long working distance stage allow imaging of large samples, embryos, and animals. A range of microprobe objectives are available for minimally invasive in vivo imaging of deep tissues. This microscope also offers conventional confocal laser scanning of samples on slide.

Features

  • Conventional laser scanning fluorescence microscopy of fixed and live samples with visible lasers and DIC imaging.
  • Multiphoton laser scanning with Coherent Chameleon Ultra II IR lasers ranging from 680 to 1080 nm.
  • Independent grating and slit for precise spectral imaging of dyes with similar emission wavelength.
  • Z-series for 3D image.
  • Time series for time-lapse imaging (high-speed imaging up to at 16 frames/second)
  • FRAP (fluorescence recovery after photobleaching), FRET (fluorescence resonance energy transfer) and photoactivation.

Specifications

  • Objective lenses
    • 4x U Plan APO 0.16 N.A.
    • 5x M Plan N 0.10 N.A.
    • 10x U Plan APO 0.40 N.A.  
    • 20x LUC Plan FLN 0.45 N.A.
    • 25x XL Plan N 1.05 N.A. Water immersion
    • 40x LUM Plan Fl/IR 0.8 N.A. Water immersion
    • 40x U Plan FLN 1.30 N.A. Oil immersion
    • 60x LUM Plan Fl/IR 0.9 N.A. Water immersion
    • 60x U Plan APO 1.42 N.A. Oil immersion

  • Fluorescent filters (excitation/emission) for fluorescent viewing. 
    • DAPI (330-385 nm/420 nm; violet DNA dyes such as DAPI and Hoechst)
    • NIBA (470-495 nm/510-550 nm; fluorophores from blue to yellow range, such as CFP, YFP, GFP, Alexa-488, FITC)
    • WIG (530-550 nm/ 575 nm for redish fluorophores, like Cy-3, TRITC, DsRED)
  • Multiphoton Emission Filter set
    • G/R (emission filters; 495-540 nm & 575-630 nm; dichroic 570 nm; suitable for GFP/RFP, FITC/TRITC, Alexa 488/Alexa 594)
    • C/Y (emission filters; 460-500 nm & 520-560 nm; dichroic 505 nm; suitable for CFP/YFP)
    • V/G (emission filters; 420-460 nm & 495-540 nm; dichroic 485 nm; suitable for DAPI, Hoechst/GFP, Alexa 488, FITC)

Excitation Lasers

 

Laser source

Excitation wavelength

Dyes & Fluorophores

Emission color

405 Diode

405 nm

DAPI, Hoechst

Violet

Multi-line Argon

457 nm

CFP

Cyan

488 nm

Alexa 488, Oregon Green, FITC, GFP,

EGFP, DiO, Cy2

Green

514 nm

YFP, EYFP

Yellow

Green HeNe

543 nm

Cy3, TRITC, mCherry,  Alexa 543, Alexa 594

Orange-red

Red HeNe

633 nm

Alexa 633, Alexa 647, Cy5, TO-PRO3,

Far red

 

 

Fluorochrome excitation with multi-photon IR laser.

Emission color

Dye

Excitation (FV1000-tested)

Blue/cyan

Alexa 350

780-800 nm

Hoechst

780-800 nm, 900-1100 nm

DAPI

780-800 nm, 900-1100 nm

CFP

800-900 nm

Green

Oregon Green

800-860 nm

Alexa 488

800-830 nm, (860 nm, 910-920 nm)

EGFP

920-990 nm

Bodipy

900-950 nm

FITC

750-800 nm, (860 nm, 910-920 nm)

DiO

780-830 nm

Yellow / Orange

YFP

890-950 nm

DiA

800-860 nm

Red

DiI

830-920 nm

Rhodamine B

800-860 nm

Alexa 568

780-840 nm

 

Download and view Olympus FV1000 User Guide.

 

For viewing and basic image handling, a free viewer software (FV10-ASW viewer) is available.

 

For more information on FV1000MPE system, click on http://microscope.olympus-global.com/en/ga/product/fv1000mpe/index.cfm

 

Images
 

Multiphoton vs. Single-photon laser scanning.

One of the main advantage of multiphoton laser scanning microscopy comes from the use of infra-red (IR) lasers and signal detection without pinhole.  IR lasers seem to penetrate more deeply than shorter-wavelength visible lasers and, with no need for pinhole, more light signals are collected, consequently increasing the resolution and contrast of image from deep and thick sample.  This enhancement is well demonstrated by the figure below, in which a Drosophila embryo (~200 µm thick) stained with FITC-conjugated anti-a-tubulin antibody was imaged with 488nm visible laser (single photon) or 800nm IR laser (multiphoton) on Olympus FV1000 confocal microscope.

 


 
Multiphoton laser scanning image.
3T3 cells are stained with DAPI and anti-tubulin (green) antibody and imaged with 800 nm to 920 nm laser to visualize both DAPI and green Alexa fluoro 488 antibody. As seen in the figures, DAPI and Alexa 488 exhibit different excitation and emission profiles over various wavelength; DAPI is excited well with 820~840 nm laser, whereas Alexa 488 shows better exciation with 880~920 nm laser. It is noticeable that some DAPI emissions appear to be detected in Alexa 488 (green) channel at shorter wavelength (i.e. 800 nm), about which one needs to be careful during double immunofluorescence imaging.