Core Facilities
USF Health - College of Medicine

3i Olympus Spinning Disk Confocal Microscope

This system consists of an Olympus IX81 inverted microscope equipped with the 3i Yokogawa spinning disk scanner and CCD cameras that allow fast high-resolution image acquisition with revolving laser arrays.  Fast scanning and highly sensitive EMCCD camera on this system minimizes phototoxicity to living cells and bleaching of samples without sacrificing fluorescence detection, which is critical for long term live cell imaging.  This system is equipped with motorized xy stage movement and fast piezo z focusing module that allow users to monitor multiple regions of interest automatically during live imaging.  With 4 lasers (405, 488, 561, and 640 nm), users can acquire high resolution multicolor images in 4D.  This system has a precise high-speed laser scan head for FRAP, FLIP, photoconversion, and photoactivation analyses. The microscope is also equipped with a live cell environmental chamber that maintains the temperature and CO2 level constant. The image analyses including 3D rendering, colocalization, and FRAP can be performed qualitatively and quantitatively with Slidebook 5.0 software.

 

Features

 

·         Olympus IX81 inverted microscope with ZDC2 focus drift compensating mechanism.

·         Yokogawa CSU-X1 A1 spinning disk scanner (5000 rpm), producing between 300 to 2000 frames per second.

·         Two CCD camera choice; EMCCD for fast imaging and HQ CCD for high resolution imaging.

·         Multicolor 2D, 3D, and 4D image acquisition.

·         Quantitative and qualitative analysis including colocalization and stereology. 

·         Long term imaging of live cells at multiple places in glass bottom dish or well plates.

·         Analysis of molecular dynamics with FRAP, FLIP, and photoactivation.

 

 

Specifications

 

·         Objective lenses

o    10x UPLSAPO 0.40 NA, WD 3.1mm.

o    20x UPLSAPO 0.75 NA, WD 0.65mm.

o    40xW3 UAPO Water immersion, 1.15 NA, WD 0.25mm.

o    60xW UPLSAPO Water immersion, 1.20 NA, WD 0.28mm.

o    100xO UPLSAPO Oil immersion, 1.40 NA, WD 0.13mm.

·         Fluorescent filter cubes (excitation/emission) on Olympus microscope

o    DAPI (350 nm/460 nm)

o    FITC/GFP (480 nm/535 nm)

o    TRITC/RFP/Cy3 (540 nm/605nm)

 

·         Excitation and emission filters for image acquisition on Yokogawa scanner

o    Quad excitation dichroic 405/488/568/647 nm

o    445/45 nm single emission

o    525/50 nm single emission

o    617/73 nm single emission

o    692/40 nm single emission

o    440/521/607/700 nm quad emission

·         CCD cameras

o    Photometrics Evolve EMCCD: very high sensitivity with low noise- excellent for high-speed imaging (up to 750 frames per second) and for low-light application.

o    Photometrics CoolSNAP HQ CCD: high resolution imaging with megapixel resolution and small pixels; relatively fast frame capture rate up to 104 frames per second.

Excitation Lasers

Laser source

Excitation wavelength

Dyes & Fluorophores

Emission color

405 cube laser

405 nm

DAPI, Hoechst

Violet

488 cube laser

488 nm

Alexa 488, Oregon Green, FITC, GFP,

EGFP, DiO, Cy2

Green

561 Saphire laser

561 nm

Cy3, TRITC, mCherry,  Alexa 543, Alexa 594

Orange-red

640 cube laser

640 nm

Alexa 633, Alexa 647, Cy5, TO-PRO3, DRAQ5

Far red


User Guide

3i SPD User Guide

Slidebook user manual (Version 5.0.0.1)