Multiphoton/Confocal Microscope

Reminder: SWEHSC Investigators (including their staff and students) should contact the Cellular Imaging Shared Service directly for assistance with planning experiments, questions regarding specimen handling, and assistance with analysis or the interpretation of results.

Training:
All users should contact Doug Cromey for training. All users of this instrument are required to attend the Laser Radiation Protection course offered by the University of Arizona's Radiation Control Office before they can complete their training.

Guests (other lab members) who wish to be present when the multiphoton laser is in operation are also required to attend the laser course.

Capabilities of the Zeiss LSM 510Meta-NLO
Confocal microscopy improves on routine fluorescence imaging by removing light from the out-of-focus planes in samples. The result is improved resolution and greatly improved contrast in the images.

• The confocal has available the following excitation wavelengths: 458, 477, 488, 514, 543 and 633 nm. In addition, the tunable pulsed infrared multiphoton laser (~700-1000nm) 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 confocal currently has the following objective lenses: 10x Plan Neo-fluar (dry, NA=0.30), 20x Plan Apochromat (dry, NA=0.75), 25x Plan-NeoFluar IMM (oil/water/glycerine, NA=0.80), 40x Plan-NeoFluar (oil, NA=1.30), 40x IR-AcroPlan (LWD immersion lens for cell culture media, NA=0.8), 63x C-ApoChromat (water, NA=1.2), 100x Plan-ApoChromat (oil, NA=1.40).
• The confocal can capture grayscale DIC (Differential Interference Contrast, or Nomarski) transmitted light images as well as confocal epi-fluorescence 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.
• Multiphoton has been used for generating second-harmonic images, as well as improved penetration in thick samples.
• Using the motorized XY stage, multiple images can be captured to create large montages or to capture data from specific points in a sample in a time course experiment.
• The META (spectral detector) can be used to create customized filter sets, for "un-mixing" overlapping fluorescence emissions, and more.

Fees

Service	Fee
Routine use by previously trained users	$32 per hour
Confocal use with the pulsed infrared laser (multiphoton)	$40 per hour
Operator assisted use	$78 per hour
Training	$46 per hour

These fees are current as of Oct 1, 2008.

Advantages & Disadvantages

Confocal
Advantages - 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.
Disadvantages - 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
Advantages - 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.
Disadvantages - The pulsed infrared laser must be manually tuned to the desired wavelength. Only certain fluorescent dyes respond to the multiphoton effect.