Full Color Multiple Label Confocal Imaging

by Pete DeVries (pdevries@facstaff.wisc.edu)

The confocal microscope allows researchers to view flourescently labeled proteins at a single focal plane. This instrument is designed to remove out-of-focus fluorescence when viewing flourescently labeled 3-dimensional specimens.A laser scans the specimen and only the light from the plane of focus is recorded by the photomultiplier tube.

This work was done in conjunction with Steve Paddock, Jim Langeland and Professor Sean Carroll of the Howard Hughes Medical Institute at the UW - Madison Laboratory of Molecular Biology.

BioRad Confocal Principles:
Courtesy of Charles Thomas of the UW-Madison Integrated Microscopy Resource

QuickTime movie illustrating the ray paths in a laser-scanning confocal microscope. Special mirrors allow light of certain frequencies to pass through while reflecting other light.
Confocal Light Path (QuickTime 2.0 - 200K)

QuickTime movie illustrating the ray paths for an in-focus object. Most of the light from the object pass through the confocal aperture and so are unattenuated.
Ray Paths for In-focus signal (QuickTime 2.0 - 27K)

Quicktime movie illustrating the ray paths for an out-of-focus object illustrating how most of the light from such an object is blocked by the confocal aperture.
Ray Paths for Out-of-focus interference (QuickTime 2.0 - 27K)

The Acquired Images: (Excitation Spectra)

Each image is acquired by collecting the light emitted by a specific flourochrome when it is excited by laser light. Below are three different images of one specimen. Each image shows the location of one of the three different flourochromes used to label proteins in the sample.

Rhodamine-Label (568nm)
First Image JPEG

Flourescein-Label (488nm)
First Image JPEG

Cyanine-5-Label (647nm)
First Image JPEG

Psuedocoloring: Assign Each Image to One of Three Color Channels
Earlier researchers had created 8-bit red-green images by merging two 8-bit grayscale images into one 8-bit color image. This used only 4-bits of intensity information from each image, resulting in a color image with only 16 shades of red and 16 shades of green.It would be better to merge files by assigning each 8-bit image to one of the red, green or blue color channels of a 24-bit color image. I wrote a suite of PC DOS programs for merging BioRad Confocal files. The resulting images retained the full 256 gradations of each original image, while allowing researchers to view the location of up to three labeled proteins in one color image. Steve Paddock and Jim Langeland in Professor Sean Carroll's lab worked out the microscopy and sample preparation. They were able to successfully triple-label and image developing fruit fly embryos.

Rhodamine-Label assigned to the Red Channel
Rhodamine Image JPEG

Flourescein-Label assigned to the Green Channel
Flourescein Image JPEG

Cyanine-5-Label assigned to the Blue Channel
Cyanine-5 Image JPEG

Instructions: Using the RGBmerge to combine three BioRad Confocal Files

Three files which have equal dimensions and are in register can be merged using the RGBmerge program. One file will be assigned to the red channel of an RGB file, another file will be assigned to the green channel and the third file will be assigned to the blue channel.

To create the merged file, at the C: prompt type rgbmerge followed by the red file name, the green file name, the blue file name and the name you want for the merged file. At the end of the line press Enter. Below is an example of how this might look.

C:\ rgbmerge hbkrr05.pic hbkrf05.pic hbkrb05.pic hbk05.rgb

The program will display the file dimensions, and these should be written down.

The program will begin the merging process. A new file will be created. In this example it will be named hbk05.rgb. Copy this file to an IBM-formatted floppy disk.

Reading Merged or Colorized BioRad Images into PhotoShop

Use PC Exchange to read the IBM floppy disk on the Mac.

Check that the Mac is set to Millions of colors.

Open PhotoShop, Select Open As... from the File menu.

Select the Raw file format, and double-click on the file you wish to open.

Enter the X and Y dimensions of the file, and enter 3 in the channels box, Interlace off

Click on OK to open your file.

The file's image will appear in color.

First Image JPEG

Articles on Multiple Label Confocal Imaging

Paddock S.W., Langeland J. A., DeVries P. J. and Carroll S. B. (1992) Three colour immunofluorescence imaging of Drosophila embryos by laser scanning confocal microscopy. BioTechniques 14 1993 pp. 42-48

Paddock, S.W., Hazen E.J. and DeVries P.J. (1997) Methods and applications of three colour confocal imaging.BioTechniques. January 1997 (22: 120 - 126.)

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