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Zeiss LSM 510NLO-Meta multiphoton/confocal
• Location: LSN 429 (see map)
• Contact: Barb Carolus, 621-2047
• Administration: Imaging Facility, Div. of Biotechnology, Arizona Research Laboratories.
• Fees for use: See below
Reminder: SWEHSC Investigators (including their staff and students) should contact the Cellular Imaging Core directly for assistance with planning experiments, questions regarding specimen handling, and assistance with analysis or the interpretation of results.
NOTE: All users of this instrument will be required to attend the Laser Radiation Protection course offered by the University of Arizona's Radiation Control Office before they can be trained.
Fees:
Fees for Hourly use: Unassisted use by trained users is $21/hr. Use of the pulsed infrared laser (multiphoton) is $28/hr. Operator assisted use of the instrument is $46/hr.
Fees for Training: Training will consist of three 3 hour sessions over the course of 2-3 weeks. Training will be charged at the hourly rate ($21/hr).
Capabilities:
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The instrument has available the following excitation wavelengths: 458, 477, 488, 514, 543 and 633 nm. In addition, the tunable pulsed infrared multiphoton laser can be used to excite a variety of dyes that are normally excited by wavelengths from the ultraviolet (e.g., DAPI, Hoescht) through the middle of the visible light spectrum (approx 550nm) .
- The instrument can capture grayscale DIC (Differential Interference Contrast, or Nomarski) transmitted light images as well as confocal images from 3 or more fluorescent dyes.
- The maximum image size is 2048 x 2048 pixels (at 12 bits=4096 levels of intensity) .
- "Stacks" of images can be acquired in Z (focus depth) , Lambda (wavelength scan using the META) or T (time series).
- FRET (fluorescence resonance energy transfer) , FRAP (fluorescence recovery after photobleaching) , Ratio imaging (e.g., pH or ion-sensitive dyes) .
- Using the multiphoton laser and a Bioptechs Delta T3 cell culture system we can perform time-lapse imaging of live cells.
Advantages & Disadvantages :
| Advantages |
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Disadvantages |
| Confocal: Crisp digital images can be acquired of fluorescently stained specimens that avoid the out-of-focus haze that is a common problem with widefield fluorescent microscopes. |
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Confocal: The intense illumination from the laser can rapidly photo-bleach (fade) some fluorescent samples. Does not work well with dim fluorescent samples. Photo-damage to living cells limits its use with live samples. |
| Multiphoton: Infrared light penetrates deeper into tissue than visible light wavelengths. The multiphoton effect occurs only in the plane of focus, therefore, out of focus areas do not have problems with photo-bleaching. Typical UV-excitable dyes can be used in live cells without the DNA-damaging effects of UV light. |
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Multiphoton: The pulsed infrared laser must be manually tuned to the desired wavelength. Only certain fluorescent dyes respond to the multiphoton effect. |
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