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Equipment - BioRad 1024 |
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Our main imaging instrument is a Nikon TMD 200 inverted microscope outfitted with a BioRad 1024 confocal head. |
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Koehler-hole mount. The scanning head is mounted through a hole in the base of the microscope stand, immediated below the objective. This configuration optimizes the sensitivity of the microscope by allowing light to pass directly to the scan head from the objective, avoiding losses in efficiency caused by mirrors or prisms in conventional inverted microscope configurations. |
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Water immersion objectives. For high resolution images of live cells we use a 1.2 n.a. water immersion objective (Nikon). Water immserion objectives allow for deeper focusing into living specimens without the same degree of rapidly increasing spherical abberation that occurs with oil immersion objectives. Oil immersion objectives are optimized for obtaining images of specimens mounted just at the coverslip surface. However, as oil immsersion objectives are focused into thick living specimens, which are mostly aqueous, the mismatch of refactive index between the specimen and the immersion oil results in geometrically increasing loss of resolution and light gathering ability due to sperical abberation. Water immserion objectives address this problem by using water as the immersion medium, creating a closer match between the refractive index of the living specimen and the refractive index of the mounting medium. In deep focal planes, which are commonly viewed in living plant cells, these objectives have greater resolution and collect more light than oil immersion objectives of higher numercial aperture. A good discussion of these issues can be found in the book "Confocal Microscopy" edited by Pawley.
Another advantage of water immersion objectives in confocal sectioning is that water is a much less viscous medium than oil and therfore exerts less drag on the coverslip when the objective is moved during focusing. The result is that the coverslip is less likely to flex and displace the specimen during aquisition of a confocal z series. Because the specimen is less likely to move during repeated refocusing, reconstructions from confocal sections are often much sharper than those aquired with an oil immersion objective. |
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Other microscope info:
Detector channels:
2 prismatic photomultiplier tubes (PMT's) in the confocal head
1 PMT mounted on the microscope stand for collecting transmitted light
Filters:
GFP fluorescence
Multi-lable configuration (most used configuration)
500/530/650 triple band pass dichroic mirror
522/20 bandpass emmision filter
Single lable configuration
488 laser line excitation
80/20 neutral mirror
522/20 bandpass filter
Propidium iodide
Multi-lable configuration (most used configuration)
568 laser line excitation
500/530/650 triple band pass dichroic mirror
585 long pass emmision filter
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