Confocal Microscopy & Advanced Imaging

What's New - Advantages - Equipment - Objective Lenses - Laser List - Long Term Goals - Other Websites - Protocols

What's New

To better serve the needs of the Stokes Institute and CHOP investigators the Pathology Core Laboratory hired Dr. Edward Williamson operate this facility. Dr. Williamson is experienced in confocal microscopy. As the new Confocal Specialist, Dr. Edward Williamson is providing research support and training to facilitate the use of various advanced microscopy in their research. Dr. Williamson's experience is proving to be an invaluable asset to labs needing collaborative support in organizing larger research initiatives. Dr. Williamson availability working as a consultant with individual investigators on existing projects helps to bring new or novel techniques that use our equipment and technology.

Microscopy is a labor-intensive pursuit, and it is helpful to have someone streamline experimental design and analysis. Confocal and associated analysis software is extremely complex and we are trying to incorporate particular tools requested by investigators to take advantage of confocal microscopy. Therefore, several changes in the newly reorganized confocal facility have been made.

First, users who plan to make extensive use of confocal technology are being trained to use the microscope independently, and at a discounted rate. After these users have demonstrated competence, they are given independent use & extended access to the instrument.

Second, Dr. Williamson is actively working to assist users embarking on projects involving advanced imaging technologies, e.g. spectral imaging, live cell imaging, FRET, FRAP and electron microscopy.

Third, the confocal core is closely integrating operations with the pathology core enabling users to move smoothly from histochemical analysis to higher resolution studies using the confocal microscope.

Fourth, the core has generated a user reagent database, and has established several informal user meetings to facilitate the exchange of information and collaborations among Stokes Investigators.

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Advantages of Confocal Microscopy

The biggest advantage to confocal microscopy is the ability to collect images exclusively from a single plane in the specimen. In "normal", or widefield microscopy, focusing on a specimen allows light from the image, or plane of focus,to reach a dectector e.g the eye, of camera. However, light from above and below the plane-of-focus also reaches the dectector, and this results in a blurred imaged that is difficult to interpret. Confocal microscopy blocks this out of focus light and eliminates blurred specimens, because the image is only collected from the in-focus plane resulting in clearer images allowing better interpretation of data. After the in-focus light at the image plane reaches a detector (PMT; photo multiplier tube, or charge coupled device), it is collected, and stored electronically in either a tiff or jpeg format for subsequent analysis. An optical section of the sample can be moved thru the Z-axis to create a series,or stack, of images perpendicular to the x-y plane. This stack can then be used to reconstruct three-dimensional images using the appropriate software.

The advantages to confocal microscopy can be summarized below

  1. An increase in effective resolution by about 10-15% over conventional microscopes.
  2. Reduces blurring of the image due to light scattering outside the focal plane. The confocal can view conventional specimens to give especially sharp images that mayotherwise be interpretable.
  3. An improved signal-to-noise which gives greater sensitivity of image detection.
  4. The area of the laser scan results in increased optical magnification, not empty electronic magification.
  5. Z-axis scanning can produce 3D views of the sample.

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Microscope

We have a Leica SP-2 AOBS Laser Scanning Confocal Microscope that uses laser light to excite fluorophores at very specific wavelengths, and can also choose the specific window, or spectrum of light, that is collected from samples that are bound to antibody-conjugated fluorophores. This spectral capability replaces the excitation filter & emission filters found on most confocals with a AOTF & AOBS devices that select for a very narrow wavelengths. This is a unique capability, and is not found on most confocal microscopes making this an especially useful instrument.

Objective Lenses

Note: Larger N.A. lences produce better resolution, and should be used in most circumstances.

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Laser/Laser Lines

LaserLaser LineComments
UV Laser405nm(used for DAPI, Hoechst, ECFP, Alexa 405, Cascade blue)
Ar/ArKr458nm & 476nm(used for FITC, CFP, GFP, YFP)
Ar/ArKr488nm & 496nm(used for FITC, GFP, Alexa 488, Cy2, YOYO-1, DiOC)
Ar/ArKr514nm(used for YFP, TOTO-1, Oregon Green 514, Rhod-123, Bodipy-FL)
GreenHeNe543nm(used for TRITC, Alexa 546, CY3, Propidium Iodide, DsRed, Alexa 555)
Orange HeNe594nm(used for Allexa 494, Texas Red, and similar fluors)
HeNe633nm(used for far Red fluors, e.g. CY5, Alexa 633, Alexa 647, APC, TOTO-3, and DRAQ-5)

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Long Term Goals

While our first priority is to meet the existing demand for confocal microscopy, we are also expanding services in new areas that were not previously well supported at CHOP. The first of these is live cell imaging, and we are currently upgrading our equipment to accommodate users who would like to work with live cells. While we already have significant expertise in live cell imaging, we are also expanding the services that we can offer to include FRET/FRAP imaging.

Other Websites

Operating The LeicaTCS-NT Confocal Microscope

Spectral viewers

Companies

Software

Protocols

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