Articles submitted for the Fall 2004 SWEHSC newsletter (Cellular Imaging Core)

With the image editing power inherent in programs like Adobe™ Photoshop™, scientists need to know the difference between appropriate adjustments to an image and inappropriate manipulations that may constitute scientific fraud. This issue becomes even more important as journal editors are becoming increasingly adept at spotting inappropriate alterations.*

The Cellular Imaging Core has been considering these issues over the last decade and has had a set of guidelines on the WWW since 2001. These guidelines are posted at several campus imaging facilities, they have been used in classrooms for discussion, and they were recently used as a background document by the US department of Health & Human Service's Office of Research Integrity for the "Integrity in Digital Imaging Working Group Meeting" held in January 2005.

The guidelines are available as a web page or as a four page Adobe™ Acrobat™ (PDF) file. See: Digital Imaging: Ethics

* What’s in a picture? The temptation of image manipulation (July 5, 2004) M. Rossner and K. M. Yamada, J. Cell Biology 166 (1):11–15

From the Fall 2004 - SWEHSC Newsletter
USING LIGHT TO MEASURE CONTRACTION FREQUENCY

Douglas W. Cromey, MS - Manager, SWEHSC Cellular Imaging Core

Cultured heart cells contract anywhere between sixty and eighty times per minute. Center Investigator Dr. Qin Chen (Pharmacology) approached the Core to see if we could image and measure the contraction frequency in these cells. Her goal was to observe changes in the frequency of contraction before and after the cells were exposed to toxic chemicals.

There were a number of technical hurdles to be overcome before we could begin this study. To keep the cells alive at a constant temperature of 37°C on the microscope, we used a culture system from Bioptechs. To capture the time-lapse images, we used the University’s Zeiss LSM 510NLO-Meta confocal microscope. By selecting a single cell and scanning the laser illumination very rapidly, we were able to collect images at the rate of about 4 images/second.

To see the contractions, Dr. Chen’s lab loaded the cells with a fluorescent indicator dye called Fluo-4 (Molecular Probes). Fluo-4 fluoresces brightly when in the presence of calcium. Calcium levels increase when cells are contracting and decrease between contractions.

We are still working out the details of this protocol. We have been able to successfully see and measure the intensity changes in the contracting cells. Sample images for one time series are to the left. The images have been colorized with the warmer colors indicating higher calcium levels (higher intensities). The graph shows how the intensity changed over a 13.4 second time span.

Movie of contracting cardiomyocyte (colorized AVI - 11MB).

Scientific details:
Yan Lin, M.S. and Jerome Terrand, Ph.D. from Dr. Chen’s lab cultured the cells and performed the dye loading for this experiment.

The cells are primary cardiomyocytes grown in 10% FBS, low glucose DMEM (Dulbecco's Modified Eagle Medium) for the first 3 days and 0.5%FBS, low glucose DMEM for days 4 and 5. The cells were grown on Bioptechs Delta-T glass bottomed dishes. The Fluo-4 calcium dye was loaded for 30 minutes at a concentration of 8 uM (microMolar) and then washed with low glucose DMEM without phenol red.

The Zeiss LSM 510 confocal used a 488nm excitation wavelength (200mW Argon laser) which was attenuated to 0.1% of full power. The lens used was a Zeiss 40X achroplan "dipping" lens. A 256x256 pixel image was zoomed to 4x and scanned at maximum speed (no averaging) to yield the 4 frames/sec speed (note: we can scan slightly faster, however the image quality degrades).

Caveat: we have seen some indications that even with low laser power, the light and dye combination may be too intense for the cells. We are currently refining the parameters of this experiment.