Instrumentation Summary                                     updated: 1/28/07

1. Confocal Microscope – Zeiss LSM 510-META

2. Confocal Microscope – Zeiss LSM 510

3. Confocal Microscope – BioRad Radiance 2100

4. Epifluorescence Microscope / Olympus IX71 (inverted) with Andor iXon camera &Till Monochromator

5. Epifluorescence Microscope / Zeiss Axioskop (upright) with Axiocam HR color camera

6. Epifluorescence Microscope / Nikon TE2000 (inverted) with Hamamatsu ORCA camera

7. Image Analysis Workstation

8. Microinjection System (Eppendorf)

1. Confocal Microscope – Zeiss LSM 510-META

• Location: CRF 212
• 4 Lasers for fluorescence excitation

• 405 nm laser diode - Fluorophores include DAPI, Hoechst, Photo-activatable GFP
• Argon (458,488,514 nm lines) - Fluorophores include fluorescein, GFP, CFP, YFP, Alexa 488
• Helium Neon 1 (543 nm) - Fluorophores include rhodamine, Cy3, Alexa 546, Texas Red
• Helium Neon 2 (633 nm) - Fluorophores include Cy5, TOPRO 3, Alexa 633

• Objectives: 10x, 20x, 40xDIC, 63xDIC (all fluorescence objectives)
• META detector for spectrum acquisition (411 - 743 nm range)
• Software for spectral "unmixing", separates closely overlapping fluorophores such as GFP and fluorescein
• META detector can image up to 8 different fluorophores simultaneously
• Two additional (PMT) channels for fluorescence detection
• Transmitted light detector for acquiring simultaneous DIC images
• Multi-track image acquisition reduces channel crosstalk in multiple labeling experiments
• FRAP (fluorescence recovery after photobleaching) and region of interest scanning possible
• Acquires a series of images along the Z axis (Z-stack) and processes the images to produce a 3-D projection image
• Mounted on Inverted (Axiovert 100) microscope
• Live-cell imaging capabilities with
• Medical Systems Inc. stage microincubator (temperature and CO 2 control)
• Coverslip dish holds cells grown on 25 mm diameter glass coverslips; fits into microincubator

2. Confocal Microscope – Zeiss LSM 510

• Location: CRF 216
• 3 Lasers for fluorescence excitation
• Argon (458,488,514 nm lines) - Fluorophores include fluorescein, GFP, CFP, YFP, Alexa 488
• Helium Neon 1 (543 nm) - Fluorophores include rhodamine, Cy3, Alexa 546, Texas Red
• Helium Neon 2 (633 nm) - Fluorophores include Cy5, TOPRO 3, Alexa 633
• Objectives: 4x Achroplan, 10x, 20x, 40x, 63xDIC (all fluorescence objectives)
• 3 PMT channels for simultaneous fluorescence detection
• Transmitted light detector for acquiring simultaneous DIC images
• Multi-track image acquisition reduces channel crosstalk in multiple labeling experiments
• FRAP (fluorescence recovery after photobleaching) and region of interest scanning possible
• Acquires a series of images along the Z axis (Z-stack) either by:
• moving the microscope stage (conventional method – less precise)
• moving the microscope objective with piezoelectric positioner (Piezosystem Jena – more precise). Precise position information is important for successful image deconvolution.
• Z-stack images can be processed to produce a 3-D projection image
• Mounted on an Axioplan 2 upright microscope

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3. Confocal Microscope – BioRad Radiance 2100

The BioRad confocal is well suited for live-cell experiments because it is mounted on an inverted microscope and because its argon laser intensity can be reduced to low levels, thus minimizing photobleaching in experiments in which multiple images are captured over time.

• Location: CRF 212
• 3 Lasers for fluorescence excitation
• Argon (458,476,488,514 nm lines) - Fluorophores include fluorescein, GFP, CFP, YFP, Alexa 488
• Green HeNe (543 nm) - Fluorophores include rhodamine, Cy3, Alexa 546, Texas Red
• Red Diode (633 nm) - Fluorophores include Cy5, TOPRO 3, Alexa 633
• Objectives: 20x, 40x, 60x (all fluorescence and DIC)
• 3 PMT channels for simultaneous fluorescence detection
• Transmitted light detector for acquiring simultaneous DIC images
• Lambda strobing feature reduces channel crosstalk in multiple labeling experiments
• FRAP (fluorescence recovery after photobleaching) and region of interest scanning possible
• Acquires a series of images along the Z axis (Z-stack) and processes the images to produce a 3-D projection image
• Mounted on Nikon TE2000 inverted microscope
• Live-cell imaging capabilities
• 20/20 Technology stage microincubator (temperature and CO 2 control)
• Cells grown on 25 mm glass coverslips fit microincubator chamber

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4. Epifluorescence Microscope / Olympus IX71 (inverted) with Andor iXon camera & Till Monochromator

• Location: CRF 218
• Fluorescence excitation - Hg lamp
• Alternate excitation: Xenon lamp with Till system monochromator (15 nm slitwidth)
• Objectives: 20x (phase), 60x (PlanApo-water), 100x (PlanApo-oil)
• Low-light level Andor iXon camera
• Image splitter – Cairn Optosplit II
• Software: Andor iQ – controls image acquisition and excitation wavelength (monochromator)
• Filter sets:
• FITC
• Rhodamine
• Quantum dot - filterset excites and allows viewing of multiple quantum dots simultaneously (500 nm LP emission)

(additional discrimination of Qdots available using narrowband filters in Image Splitter)

• Live-cell experiments/Single-particle tracking
• Ratio imaging – Ca 2+ and pH probes (fura-2, BCECF)
• Slide and objective heater

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5. Epifluorescence Microscope / Zeiss Axioskop (upright) with Axiocam HR color camera

• Location: CRF 220
• Objectives: 10x Phase, 20x Phase, 40x, 63x, 100x (all fluorescence objectives)
• Filter sets:
• DAPI
• FITC
• Rhodamine
• DAPI/FITC/RITC triple cube
• Camera: Zeiss Axiocam HR (color, digital)
• SlideBook software (Intelligent Imaging Innovations) controls image acquisition and analysis (see #7)
• 35mm film camera (Zeiss, automated) also available

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6. Epifluorescence Microscope / Nikon TE2000 (inverted) with Hamamatsu ORCA camera

The Nikon microscope is the platform for the BioRad confocal, but is also used for epifluorescence viewing of cells in culture, or other applications requiring an inverted microscope.

• Location: CRF 212
• Objectives: 20x, 40x, 60x (all fluorescence and DIC)
• Filter sets:
• DAPI
• FITC
• Rhodamine
• CFP
• YFP
• Camera: Hamamatsu Orca 100 (monochrome, digital)
• Software: Wasabi (Hamamatsu) controls basic image acquisition

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7. Image Analysis Workstation

• SlideBook Image Analysis software (Intelligent Imaging Innovations)
• Location: CRF 216
• Can directly import images in multiple formats:
• Zeiss LSM confocal
• BioRad confocal
• tif images
• Can import single plane, Z series and time series images
• Can create multi-channel images from separate images (including BioRad confocal and Wasabi images (Hamamatsu camera))
• Scaling information retained
• Colocalization analysis (Pearson’s coefficient; colocalization display view available)
• 3 dimensional rendering of Z series images (volume and surface views)
• Fluorescence intensity quantitation
• Deconvolution (no neighbor, nearest neighbor and constrained iterative methods)
• Object counting (in two and three dimensions)
• Morphometry on regions of interest: area/perimeter/center of area (for single plane images) or volume/surface area/center of volume (for Z series images)

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8. Microinjection System (Eppendorf)

Although not currently in use, a microinjection system is available and can be mounted on a Zeiss IM35 inverted microscope. Microinjection is a powerful tool but learning to do it successfully requires persistence.

• Location: CRF 218
• Eppendorf Transjector 5246 system
• Mounted on Zeiss IM35 inverted microscope
• DKI Vertical Pipette Puller for making micro-injection pipettes
• Micro-injected living cells can be subsequently imaged with the Zeiss Meta System Confocal, the BioRad confocal or the Olympus Inverted microscope.

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