Transitional Core

  • Located in the Coker Life Sciences building, serving the South Carolina College of Pharmacy and other departments at the University of South Carolina
  • Providing training and services in microscopy, flow cytometry and quantitative PCR

Microscopy and Flow Cytometry Facility

MFCC

Robert Doran, Ph.D.

Robert Doran, Ph.D.
CLS 506 
Microscopy and Flow
Cytometry Core
doranrm@mailbox.sc.edu  
(803) 777-6566

Co-Director

MFCC

Chang-uk Lim, Ph.D.
CLS 506 
Microscopy and Flow Cytometry Core 
limc@sccp.sc.edu
(803) 777-4689 

Co-Director

Flow Cytometry Core Resources

BD LSR II Flow Cytometer with HTS option

Flow Cytometer

The BD LSR II has digital electronics for increased speed, a 3-laser optical bench, and can detect 8 fluorescent parameters plus 2 physical parameters and a time parameter (see picture).

  • Coherent Sapphire solid state 488 nm
  • JDS Uniphase 1344P HeNe 640 nm
  • Coherent Vioflame solid state 405 nm

     

Beckman Coulter FC500 Flow Cytometer

The Flow Cytometry Core has a Beckman Coulter FC500 MCL System with CXP software for both technician-assisted and user-independent data acquisition. The Cytomic FC500 MCL system has a uniphase argon ion 488nm laser and five color capability (FITC, PE, ECD, PC5 or APC and PC7).

The FC 500 System provides automated tube based acquisition for cell based assays.

Utilizing powerful CXP software, investigators can create custom panel reports and perform multiple tube panel based analysis.

The automated QC database allows daily monitoring of instrument QC. 

BD FACS ARIA Cell Sorter

Aria Cell Sorter

The BD FACS Aria is a high-speed cell sorter. New-generation digital electronics allow high speed analysis and sorting (>20,000 cells per second).

The FACS Aria optical system is configured with three excitation lasers and can detect 9 fluorescent parameters simultaneously (plus 2 physical parameters and a time parameter).

Additionally, the FACS Aria offers numerous sorting options which include 4-way sorting, sorting to microtiter plates or glass slides, and customizable settings to optimize sort yield and purity.

The FACS Aria laser excitation wavelengths and partial list fluorochromes that can be utilized on the   instrument are listed in the table.

Microscopy Core Resources

Leica Microsystems DM IRE2

There are two of these systems, one within an environmental chamber for live cell imaging. Each uses the Leica DM IRE2 inverted stand with motorized objective turret, z positioning, and fluorescence filter changing. The objectives on each include 10x/0.25n.a. Ph1, 40x/0.6n.a. Ph2 HCX PL FLUOTAR, 63x/1.4 HCX PL APO and 100x/1.4 HCX PL APO. The microscopes use phase contrast with the 10x and 40x objectives and differential interference contrast with the 63x objective.

100W mercury arc lamps provide excitatory light for fluorescence imaging and there are filter cubes for DAPI, GFP (FITC & Cy2), TRITC (Cy3 and Texas Red) and Cy5 (Alexafluor633). Both DM IRE2 microscopes have CCD cameras for fluorescence imaging, one a Leica DC350 FX and the other a Photometrics Coolsnap HQ. The microscopes and cameras are controlled with personal computers running Leica FW4000 software. The software enables the user to set up image acquisitions incorporating z-series, time series and multiple color channels. The FW4000 software also performs basic analysis and image processing functions.

 Leica Microsystems     

Olympus IX81 Fluorescence Microscope

The Olympus IX81 has an inverted motorized stand controlled by the IX2-UCB control box. Fluorescence illumination is from a Sutter Instruments Lambda LS Xenon lamp with liquid light guide. Fluorescence filter changing is done by the Sutter Instruments Lambda 10-3 filter changer. There are filter cubes for DAPI, FITC, and TRITC. For more rapid filter changing for live cell imaging there is a filter wheel containing emission filters for DAPI, FITC, TRITC and Cy5. Also for live cell imaging there is a Bioptechs Delta T5 culture dish temperature controller. The objectives include a UPlanFL N 10x/0.30 Ph1, a LUCPlan FL N 40x/0.60 Ph2 with correction collar for long working distance, a UApo/340 40x/1.35 oil iris which transmits UV light for 340/380 ratio imaging, a PlanApo N 60x/1.42 oil and a UPlanSApo 100x/1.40.

Images are captured on a Hamamatsu C10600 camera connected to a personal computer running MetaMorph Basic. This software controls microscope and camera settings, saves the settings for future use and provides features for image processing and analysis. For example, 2D or 3D image deconvolution can be performed to remove out of focus light from fluorescent images. The system also includes the MetaFluor software for ratiometric imaging commonly used for measuring calcium fluxes.

Carl Zeiss LSM 700 Confocal Microscope

The LSM700 has a  scan head  with 2 photomultiplier tube  (PMT) detectors on an inverted Axio Observer Z1 stand.  There are four solid state lasers producing lines at 405nm, 635nm, 555nm and 488nm.  A transmitted light PMT collects transmitted light images using differential.  A secondary dicroic beamsplitter facilitates acquiring lambda scans to be used for linear unmixing. 

The entire microscope stage and objective turret are enclosed in an environmental chamber to have stable temperature and CO2 control for live cell imaging. There is a motorized stage for acquiring multiple fields in separate wells of a multiwell plate at each time point in time lapse data acquisition. 

 

3D reconstruction of a nucleolar protein (green) and metabolically labeled RNA (red)

Lasers

  • 405, 488, 555, and 639nm
     

Capabilities

  • Confocal point scanning
  • 3D reconstruction
  • Multi-field time lapse in 24 well plates
  • Ratiometric concentration determination
  • Tile and stitch
  • Linear unmixing
  • Fluorescence recovery after photobleaching (FRAP)
  • Forster resonance energy transfer (FRET)

Leica Microsystems AS MDW

The AS MDW was developed for live cell fluorescence imaging. The system uses the DM IRE2 stand described above. In addition to a mercury lamp it has a Xenon lamp in a monochromator. This latter source is more stable over time and causes less phototoxicity in comparison to the mercury lamp. The AS MDW also has a motorized stage so that multiple fields can be imaged, over time, in the same experiment, increasing the number of cells analyzed. There is a humidified chamber which fits on the stage and can be perfused with custom mixed gases, such as 5% CO2 in air, to maintain the proper pH of bicarbonate-buffered media. The system is equipped with N PLAN L 40x/0.55 CORR, HCX PL APO 63x/1.30 GLYCEROL and HCX PL APO 100x/1.4 OIL objectives. Differential interference contrast optics provide contrast in bright field images. The 63x objective is optimized for 37°C and is on a Piezo mount for 3ms focus changes during z stack acquisition. On the bottom port of the stand is a Photometrics Coolsnap HQ camera. Multiple image planes can be acquired at each time point and the resulting data can be processed by 3D image deconvolution.

    Leica AS MDW

Carl Zeiss Axiovert 200

There are three identically configured Zeiss Axiovert microscopes on air tables.  Each is equipped with 10x, 20x and 32x objectives, phase contrast optics, a Zeiss AxioCam MRc color camera, and a PC interface running Zeiss Axiovision. These are used for routine phase contrast and color imaging of live and stained specimens.