Cell Imaging...Disk Scanning Confocal Back to Cell Imaging Home
Contact info:
Chris Rodesch
crodesch@cores.utah.edu
Phone: 587-7964
Fax: 585-6364		  
  • Dual Nipkow disk scanning confocal head (see below for details) coupled to either a cooled CCD camera or a video rate, intensified CCD camera
  • Provides high scan speed imaging (30 frames per second (fps) at full resolution and brightness or reduced resolution or brightness imaging at 60 fps)
  • Enables imaging with drastically reduced incident illumination energy
  • Ideal for analysis of live samples that either require rapid frame rates or are photosensitive
  • Krypton/Argon laser providing excitation at 488 and 568 nm; switching between lines at rates up to 30 Hz
  • Go to examples (Netscape users scroll down to examples)

    *** Example files on this page require the QuickTime plug-in, which can be downloaded here***

 

 

 

 

 

 

Disk-Scanning Microscopy System...Some Details:

The unique operational features of the disk scanning confocal system begin with the expansion, recollimation, and masking (1,2,3 in diagram) of a laser light source so that a 7 x 10 mm rectangular area is uniformly illuminated. This field of laser light is then passed through a pair of tandemly rotating Niplow disks (4, 5 in diagram). The first disk contains an array of 20,000 radially arranged microlenses that serve to collect and focus the illumination through circular pinhole apertures (diameter = 50 um) present in the second disk. These apertures function in a manner similar to the confocal apertures in confocal laser scanning microscope (CSLM) systems, excluding stray light and out-of-focus fluorescence emission from the final images. At any instant, 1,000 microlenses focus illumination through 20,000 pinhole apertures. A dichroic mirror placed between the two Nipkow disks directs the fluorescence emission to the detector, selectable between an ocular port or camera port.
Drawing adapted from: Ichihara et al., 1996 Bioimages 4(2)57-62 and H. Ishida et al., Tokai Univ.

Scan Speed (= 360 Hz; imaging speeds of up to 30 or 60 fps):
A 30 degree rotation of the disks produces a full field-of-view scan across the sample, resulting in 120 full scans per single disk rotation. The disks spin at a rate of 30 rotations per second, producing 360 full field-of-view scans per second. As noted above, full resolution and brightness imaging can be accomplished at 30 frames per second (fps), or reduced resolution or brightness acquisition can be performed at 60 fps. Frame averaging procedures can be employed as a method for reducing noise levels in experiments requiring high frame rates, and images are quantifiable when full resolution imaging is performed and processor calibration procedures have been properly implemented. The video sequence to the right provides an example of active cells imaged at 30 fps.		 Scan Speed and Illumination Intensity Demonstration:
Three video clips are contained in the sequence to the left. The first two present the same cells constantly imaged at 30 fps over a 13 minute interval using a 16 frame averaging procedure for noise reduction. Note that little change in cellular activity occurs after this extended sampling period. The third clip demonstrates the effect of reducing the frame number in the averaging procedure. HeLa cells were loaded with a fluorescent dextran conjugate overnight, then chased for approximately 1 hour.
     
Reduced Illumination Intensity (= reduction in specimen photodamage):
Perhaps the greatest limitation imposed by the use of CSLM is its propensity for inducing phototoxic and photobleaching effects due to its requirement for point-scanned laser illumination. The disk scanning confocal system, employing an intensified CCD camera, enables a significant incident illumination energy level reduction relative to levels typically used for CSLM. Frame integration imaging schemes enable further incident energy level reduction. These levels permit extended imaging of live samples at relatively high frame rates. The video sequence above demonstrates the effect of reduced illumination intensity on live cells. The sample was continuously imaged for 13 minutes with no apparent change in cellular activity. The video sequence to the right demonstrates the use of frame integration. These cells were imaged using laser excitation attenuated approximately 90% relative to the sequence above.

(Back to Top)

Frame Integration Demonstration:
The video sequence to the left demonstrates the use of frame integration for further illumination intensity reduction. Cells were imaged using laser illumination attenuated approximately 90% relative to the intensity used for the sequence above. The sequence was acquired using approximately 1 second integration intervals. Playback rate is approximately 10x real time.

Stained cells in this sequence and the sequence above courtesy Mr. Michael Vaughn and Dr. Jerry Kaplan, Department of Pathology.

Experimental Example:
Human mammary epithelial cells were labeled for one hour with 1 ug/ml of a fluorescently labeled Fab fragment derived from an antibody raised to the HER2 protein. Cells were then imaged without removal of the antibody solution. The Fab fragment is localized and trafficked within endosomes. Playback rate is approximately 10x real time.

Video sequences and protocol courtesy Dr. H. Steven Wiley, Pacific Northwest National Laboratory.		  

(Back to Top) (Back to Cell Imaging Home)