A confocal microscope uses a bright point of light (laser) which is projected as a spot image into the plane of focus of the sample. The sample fluoresces as a spot, which is focused onto a pinhole located in front of the detector. The pinhole rejects the out-of-focus light from the sample. The bright illumination spot, the fluorescent spot, and the pinhole are all in conjugate focal planes, hence the name "confocal". The confocal principle was patented in 1957, but laser scanning confocal microscopes did not become commercially available until 1987.
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. The result is greatly improved contrast and a slight increase in resolution in the images.
Disadvantages - The intense illumination from the laser can rapidly photo-bleach (fade) some fluorescent samples. Confocal microscopy does not work well with dim fluorescent samples. Live cell imaging requires some care to avoid causing photo-toxicity.
Widefield fluorescence and confocal images of 20um thick intestine. (Source)
All interested users should contact Doug Cromey for training. Training typically requires three sessions of 2 to 2.5 hours each. Two people from the same lab can be trained together (larger groups are highly discouraged, since the training is very hands-on).
All confocal users are required to complete the Imaging Laser Protection Course offered by the University of Arizona's Office of Research Laboratory & Safety Services (RLSS) before they can finish their training.
|Service||Leica Confocal - Fees (as of 2017)|
|Training||$39/hr (a training fee is being considered)|
|Routine use (after training)||$39/hr|
|Operator-assisted use||$51/hr operator time + $39/hr instrument time = $90/hr|
Please note: Due to Cancer Center policies, individuals from labs that are not affiliated with the UACC will not be given after-hours access to the UACC buildings or room 0960. Labs that need this type of access should inquire about the requirements for affiliation.
The confocal is a Leica SP5-II resonant scanner confocal microscope (Leica Microsystems, Buffalo Grove, IL). It has the following features:
- Excitation wavelengths of 405, 458, 476, 488, 514, 543, and 633nm
- Objective lenses: 5x/0.15NA PL Fluotar, 10x0.4 PL Apo, 20x/0.7NA Plan Apo, 20x/0.7NA Plan Apo multi-IMM (oil, water, glycerin), 40x/1.25NA PL Apo (oil), 63x/1.4 NA PL Apo (oil)
- Inverted microscope stand with environmental control chamber for live cell imaging (multi-well plates, 35-70mm dishes with coverslip thickness bottom).
- Capable of capturing transmitted light images (DIC optics are currently not available)
- Maximum image size available is 8192 x 8192 pixels.
- "Stacks" of images can be acquired in Z (focus depth), Lambda (wavelength scan) or T (time series).
- FRET (fluorescence resonance energy transfer), FRAP (fluorescence recovery after photobleaching), Ratio imaging (e.g., pH or ion-sensitive dyes) .
- The instrument includes a tandem scan head with resonant scanner that allows 8kHz scanning speed. The resonant scanner can provide imaging speeds of up to 28 512 x 512 images/second.
- Using the motorized XY stage, multiple images can be captured to create large multi-image montages or to capture data from specific points in a sample in a time course experiment.
- The instrument's prism and slit technology allows tremendous flexibility in designing custom filter settings for any experiment.
Trained users can schedule time on the instrument using the Online scheduler. To ensure equal access, please limit yourself to no more than 8 hours per week (Monday-Friday, 8am-5pm). After-hours use is currently unrestricted.
|Confocal Facility Manager||Doug Cromey, MS, Associate Scientific Investigator, Cellular & Molecular Medicine
|Shared Resource Director||Agnes Witkiewicz, MD, Professor, Department of Pathology
- The Improving Image Quality tutorial explains how to adjust the confocal to get the best images on this instrument.
- The On-off instruction sheet explains the startup and shut down procedures for the confocal.
- The #1.5 Coverslips handout discusses the importance of using the correct thickness to ensure the best image quality.