Advanced Imaging Facility

The imaging facility currently has 6 major instruments, a benchtop system under development, a macroimager, two dissecting microscopes, 4 major software packages for image analysis or acquisition, and associated supporting hardware.

Leica SP5 AOBS Point Scanning Confocal Microscope

The SP5 is a state of the art point scanning confocal microscope. It produces the best out of focus rejection of all the instruments in the facility (most "confocal": highest contrast, best axial resolution) and has the most flexible array of excitation and emmission bands. However, it is not the most sensitive instrument for small numbers of chromophores and live cell imaging. The SP5 has tools for performing photobleaching and photoactivation experiments.

Description of Leica SP5:

• Excitation lines: 405, 442, 457, 476, 488, 514, 520, 561 and 633 nm. 
• Emission bands: continuously customizable over three detector channels with AOBS system
• Channels: three confocal, 1 transmitted light
• Objectives: 63X Oil n.a.=1.4, 63x Glycerin n.a.=1.3, 63x water n.a.=1.2, 40x oil n.a.=1.25, 20x multi-immersion n.a.=0.7
• Sign-up: Agendo Calendar  https://agendo.cirklo.org/carnegiescience/weekview.php?resource=1
• Requirements for use: Need to take training and be approved by a designated trainer. Contact Heather Cartwright.
• Room: 408

Spinning Disk Confocal Microscope 1

The spinning disk instruments have less out of focus rejection than does the SP5, but they have superior sensitivity and photobleaching characteristics for extended imaging of dim subjects in living cells. Both systems feature high speed imaging acquisition and piezo-driven stage focusing, multiple laser lines for excitation, high speed excitation selection, shuttering and attenuation by AOTF, and detection by back-thinned, high QE EMCCD cameras.

Description of system 1:

• Available excitation lines: 443, 488, 514, 568 nm
• Excitation/emmission pairs: 443/cyan plus 514/yellow, 488/green plus 568/red (see scope manual for exact specs)
• Stand: Leica DMI6000 with Adaptive Focus Control, motorized focus, objective turret and dichroic carousel.
• Objectives: 100x Oil na=1.4, 63x Oil na=1.4, 63x glycerin na=1.3, 63x water na=1.2, 20x Multi-immersion na=0.7
• Camera: Photometrics QuantEM EMCCD. 512x512, QE greater than 90%, on chip electron multiplication.
• Auxilary focusing: MadCity Labs piezo focusing stage
• Photobleaching: Micropoint FRAP apparatus
• Multichannel imaging: Dualview image splitter for GFP/RFP or CFP/YFP.
• Software: SlideBook.
• Sign-up: Google Calendars
• Requirements for use: Need to take training and be approved by a designated trainer. Contact Heather Cartwright.

Spinning Disk Confocal Microscope 2/FLIM Imaging System

The spinning disk instruments have less out of focus rejection than does the SP5, but they have superior sensitivity and photobleaching characteristics for extended imaging of dim subjects in living cells. Both systems feature high speed imaging acquisition and piezo-driven stage focusing, multiple laser lines for excitation, high speed excitation selection, shuttering and attenuation by Crystal Technology programmable AOTF, and detection by back-thinned, high QE EMCCD cameras.

In addition to being a spinning disk confocal microscope, System 2 is also a Fluorescence Lifetime Imaging Microscopy (FLIM) imaging system. Fluorescent energy has four characteristics can be exploited to get information from a specimen: intensity, wavelength, polarization and lifetime of decay. FLIM exploits the latter, which describes the probability over time of an excited electron falling back to the ground state and generating an emission photon. The decay time is sensitive to the environment of the chromophore and the means by which the chromophore is excited. For example, lifetime shows a large shift when a chromophore is excited by Forstner Resonance Energy Transfer (FRET) as compared to direct absoption of an excitation photon. This property makes FLIM valuable for detecting FRET and it is less sensitive to variables that can affect fluorescence intensity, which is the basis for conventional FRET measurement.

System 2 also has a 405 nm excitation line for near-UV excitation of chromophores such as DAPI, a camera with improved signal to noise characteristics, and a fully motorized stage for repeated visitation of multiple sample locations and for automated image tiling.

Description of system 2: 

• Available excitation lines: 405, 443, 491, 515, 561 nm.
• Excitation/emmission pairs: 443/cyan plus 514/yellow, 488/green plus 568/red, 405/green/red (see scope manual for exact specs).
• Stand: Leica DMI6000 with Adaptive Focus Control, motorized focus, objective turret, dichroic carousel, field stop, condenser.
• Objectives: 100x Oil na=1.4, 63x Oil na=1.4, 63x glycerin na=1.3, 63x water na=1.2, 20x Multi-immersion na=0.7.
• Camera: Photometrics Evolve EMCCD.
• Auxilary focusing: MadCity Labs piezo focusing stage.
• FLIM: Pockel's cell to modulate excitation energy, Lambert gated image intensifier to modulate emission.
• Software: SlideBook.
• Location: Room 401
• Sign-up: Google Calendars
• Requirements for use: Need to take training and be approved by a designated trainer. Contact Heather Cartwright.

Ratiometric macroimaging systems

Roper HQ camera, Leica MZ6 stand, Sutter filter wheel for whole seedling or colony screening.

Ratiometric epifluorescence imaging system 1

Ideal for analysis of FRET sensors in mammalian cells (HEK293T cells, HepG2 cells), in arabidopsis roots or leaf slices.

Custom assembled system: Sutter light source Lambda DG4, millisecond switching of excitation wavelengths, equipped with Chroma ET/sputtered excitation filters, liquid light guide, Inverted fluorescence microscope Leica DMIRE, objectives: Vacucell perfusion chamber for mammalian cells, World precision perfusion chamber for plants, Automate computerized perfusion control system, Sutter emission filter wheel, Roper Dualview image splitter, Chroma ET/sputtered emission filters, Roper QuantEM CCD camera. Fast computer with Slidebook 5.1 software with FRET module. 

Ratiometric epifluorescence imaging system 2

Ideal for screens of RNAi collections, chemical libraries, analysis of single yeast cells, set up for sensor design on MITOMI (Quake).

Custom assembled system: Till Photonics spectral light source, tunable, liquid light guide, Inverted fluorescence microscope Leica DMIRE, objectives, motorized Märzhäuser XY stage, 96 well plate holder: World precision perfusion chamber for plants, microfluidic perfusion system Cellasic, Automate computerized perfusion control system, Sutter emission filter wheel, Roper Dualview image splitter, Chroma ET/sputtered emission filters, Roper QuantEM CCD camera. Fast computer with Slidebook 5.1 software with FRET module.

FEI Quanta 200 Environmental SEM

The Quanta 200 is a very flexible instrument that allows wet and living specimens to be imaged without special preparation. It features a cooled stage and 5 detectors: backscatter, ET, Large Field Detector, Gaseous Secondary Electron Detector 1000 microns and Gaseous Secondary Electron Detector 500 microns.

• Sign-up: Goodle Calendars
• Requirements for use: Need to take training and be approved by a designated trainer. Contact Clayton Coker or Kathy Barton.
• Instrument Managers: Clayton Coker and Kathy Barton.
• Room 410