Main Body

Advanced Light Microscopy and Image Analysis Core

Equipment

Confocal microscope Leica TCS SP5

Equipped with well-proven, high efficiency photo-multipliers six channels simultaneously detection (5 fluorescent and 1 bright field) and an acousto-optical beam splitter (AOBS). The ABOS has significantly higher transmission and is much faster than more traditional dichroic mirrors. This system collects more photons than any other technique - the result is bright, noise-free images and extremely low photodamage at high speed. A few of its features are highlighted below:

1. True confocal point-scanning - real optical sectioning
2. High dynamic sensors
3. Low dark current PMTs
4. Free tunable emission bands
5. Any dye adaptable
6. Spectral un-mixing- Dye separation: sufficient for 5 channels simultaneously
7. Spot-bleaching FRAP
8. 8 different laser lines available: 458, 476, 488, 496, 514, 543, 594, 633 nm.
9. Objective lenses 10X to 63X.

Currently, we have separated 5 channels in a very challenging sample. See the image gallery for details and images. To see images and data from our Leica system click on poster link. (PDF file size: 2.6M)

What is multi-photon excitation?

The process of a fluorophore being excited by the near-simultaneous absorption of two longer (twice or three times the normal) wavelength photons. Since this can only be achieved at the point of focus, due to needed energy densities, all surrounding fluorophore molecules remain in their un-excited state.

Multi-photon excitation has several key advantages when compared to single-photon excitation:

• Reduction in phototoxic effects to the specimen!!! Long term (think days) live cell imaging, GFP, RFP etc.
• Much deeper imaging-about 400µm as compared to a single photon which is around 100 µm. In some cases imaging depths of several millimeters can be achieved.
• Even broader excitation range, including UV! The tuneability of a multi-photon laser allows nearly complete coverage of the excitation spectrum.
• More efficient and accurate fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) due to the pulsed laser.

See mitosis of vertebrate cell and neurons in image gallery for examples.

This intelliPATH is a flexible immunohistochemistry (IHC) staining system with walk-away capabilities. Its unique technology enables small volumes of reagent (as little as 100 µL per slide) to be uniformly applied over the tissue sections on a slide. The intelliPATH offers continuous batch processing, allowing independent start and finish times for each batch of 10 slides as well as different protocols on each of the 30 slides. This system will be used to automate slide, cell and section preparation which are currently stained manually and can take days per run.

Wide Field Imaging Workstation:

Nikon TE2000

• Fluorescence, Phase, DIC & Bright field imaging modes
• 1, 2, 4,10, 20, 40, 60, 100X Objective lens
• Filter wheels for Dapi, FITC(GFP), TRITC (Texas Red), CY-5
• Temperature controlled (RT → 50ºC) cell chamber for imaging over extended time periods
• Cooled CCD camera (Roper HQ)
• Z-stepper drive
• FRAP (fluorescence recovery after photo bleaching) capabilities
• Software for acquisition, post-processing, feature extraction, alignment, overlays, tracking and much more

Olympus X-70

• Fluorescence, Phase, & Bright field imaging modes
• 2, 4, 10, 20, 40, 60, 100X Objective lens
• Filter cubes for Dapi, FITC(GFP), TRITC (Texas Red)
• Temperature controlled (RT → 50ºC) cell chamber for imaging over extended time periods
• Cooled CCD camera (Apogee Alta)
• Z-stepper drive
• Software for acquisition, post-processing, feature extraction, alignment, overlays, tracking and much more

Optical Projection Tomography (OPT) Workstation:

There are many techniques for 3D imaging such as confocal microscopy. Including wide-field florescence microscopy, CAT scan, MRI, atomic force microscopy. OPT has the advantage allowing large objects (1 cm3) to be imaged at higher resolution than CAT or MRI. OPT allows the specimen to be imaged both with transmitted as well and florescent illumination. With image algorithms, some borrowed from CAT and MRI imaging this technology has a lot of room to evolve and improve both in terms of delectability and specimen type and size.

Specimens (either mounted in glass tubes or native) are imaged in a rational manner and then projected into a 3D volume. See gallery for some really cool movies.

• Fluorescence, Phase, & Bright field imaging modes
• 2, 4, 10 Objective lens
• Filter cubes for Dapi, FITC(GFP), TRITC (Texas Red)

Upright Fluorescent Scope:

• Fluorescence, Phase, & Bright field imaging modes
• 10, 20, 40, 60X Objective lens
• Temperature controlled (RT → 50ºC) cell chamber for imaging over extended time periods
• 3-shot Color Cooled CCD camera

The software controlling the camera is easy to use, flexible and highly intuitive, and a DVD burner is available on this system. This system also has the capacity to perform calibrated measurements from simple distances to complex area/perimeter determinations.

Upright Polarized light Scope:

• Polarized light, fluorescence, Phase, & Bright field imaging modes
• 2.5, 5, 10, 20, 40, 60X Objective lens
• 3-shot Color Cooled CCD camera

Polarized light microscopy can produce both qualitative and quantitative measurement from birefringent specimens (decomposition of a ray of light into two rays when passing through certain samples). These specimens can be alive, fixed or inanimate. The colors in the image are a result of the interference between light waves passing as they pass through the specimen.

The software controlling the camera is easy to use, flexible and highly intuitive, and a DVD burner is available on this system. This system also has the capacity to perform calibrated measurements from simple distances to complex area/perimeter determinations.

Stereo Fluorescent Scope:

• Fluorescence and bright field imaging modes
• 20-200X Zoom
• Various illumination modes including transmitted and reflected
• 3-shot Color Cooled CCD camera

Dual-head Stereo Scope:

• bright field imaging modes
• 20-100X Zoom

This is an idea-teaching microscope allowing two individuals to view the same field-of-view simultaneously.

Microtome (vibrating-blade)

Leica VT1000 Instrument features

• Linear sectioning speed adjustment
• Linear sectioning frequency adjustment
• Sectioning thickness totalizer
• 5 Different amplitude settings
• Freely programmable sectioning window
• Temperature from 4º - 27º C
• Easy mounting and removing of knife holder and buffer tray.
• Double-walled buffer tray providing excellent contrast
• Cold light source and fiber-optical lighting system

Miscellaneous Equipment:

• Cell viewing chambers and thermal regulation:
  Our laminar flow perfusion chambers allow cells cultured on coverslips to be followed at the highest possible resolution while maintaining the ability to rapidly change the bathing media at any desired time, i.e. for fixation or stimulation (Rieder and Cole, 1998). Our Rose chambers allow for long term (up to 3 day) LM studies. Cells can be maintained on any of the microscopes (listed above) with chamber heaters designed and built in house (Rieder and Cole, 1998), which can maintain temperatures between 0^o-50^oC for long (> 3 day) periods.
• Laminar flow hoods and Incubators:
  For maintaining and growing various cell lines used in LM studies.

Things we are planning on implementing:

Protocol pages on the website

• Fixations staining for various samples and molecules
• Operation procedures for instrumentation
• FAQ's