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Show it all – right away!
Digital multichannel fluorescence microscopy in developmental biology
Egg formation in the fruit fly is a complex process. In the Department for Zoology/Developmental Biology at the University of Kassel, Germany, we are especially interested in investigating errors that can take place in the maturing egg chamber. The various imaging methods available with a microscope, such as different fluorescences or Nomarski optics, make only a part of the cell structures visible. However, these image contents can be merged digitally via a CCD color camera and the analySIS image analysis software by Soft Imaging System, creating images that "show it all – right away".
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The fruit fly (Drosophila melanogaster) is one of the most popular research objects in genetics and developmental biology. The reason for this is that the fruit fly has just eight chromosomes, is easy to breed in large numbers and new generations can be produced every two weeks. Over time, scientists have been able to elucidate many important questions regarding cell development and heredity mechanisms, using crossbreeding experiments, artificially induced mutations and gene analyses. Almost 100 years ago fruit flies were discovered with white instead of red eyes, and these mutants were bred. Today, their tumor genes are being closely examined in order to gain new understanding which can be used in the battle against cancer in humans. One primary focus of our research in the Department of Zoology/Developmental Biology at the University of Kassel is oogenesis: egg formation in the fruit fly. It is an extremely complex process in which the interaction between somatic cells and gametes controls the development and differentiation of the various types of cells.
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From stem cell to egg cell
One exciting process is how gametes are formed. A stem cell divides asymmetrically into two daughter cells – one, a gamete and the other a new stem cell. The gamete then divides four times which leads to a group of 16 connected gametes. One of these develops into the oocyte, the precursor to the egg cell. Its 15 sister cells become nurse cells and later provide the oocyte with proteins, organelles and RNA molecules via ring channels. The group of gametes is separated via the ingression of somatic cells and is encapsulated to form what is referred to as a follicle. Such a follicle can be clearly seen in figure 1. The somatic cells that have ingressed and that now form the casing of the maturing egg cell, are called follicle cells. In the further course of development, one can observe an increase in size, morphological changes of the individual cell types as well as migration of the follicle cells.
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 Figure 1:
Where is the oocyte, the precursor to the actual egg cell? Usually it is located at the spot marked with an "O" on the front end of the cell cluster. However, anyone investigating developmental defects during egg formation requires more precise information. The image with the Nomarski optics (A) and the Hoechst 33258 fluorescence acquisition (B) make the follicle cells (F) and the nurse cells (N) visible. This is where digital image processing can step in and help out.
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Making errors visible
An improper separation can lead to abnormal differentiation of the gametes. It can also cause the formation of aberrant follicles with the wrong number of gametes. We use fluorescence microscopy to investigate this kind of defective development. Multichannel fluorescence microscopy is possible thanks to digital image acquisition and image processing. We were able to demonstrate defects during oogenesis following abnormal separation in fruit flies with a mutation in what is known as the egghead (egh) gene. To identify and analyze aberrant follicles, we use fluorescent markers to label the cells involved. A combination of Hoechst 33258 with rhodamine-coupled phalloidin makes it possible to classify the individual cell types precisely. Hoechst 33258 stains DNA and thus the cell nuclei. Phalloidin binds to actin and thus stains oocytes, which characteristically contain a prominent actin cytoskeleton.
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Why use multichannel fluorescence?
We incubated fixed organs with 2 µg/ml Hoechst 33258 and 2.5 µg/ml rhodamine-coupled phalloidin for two hours. Then these were washed and prepared for microscopy. Soft Imaging System's ColorView II digital camera, flanged onto the microscope, acquires the fluorescence images and transfers them to the PC. The camera is controlled by the analySIS image analysis software, also produced by Soft Imaging System. This software enables acquisition, processing and visualization of multichannel fluorescence images. This means that both the blue Hoechst 33258 fluorescence and the red rhodamine phalloidin fluorescence can be acquired simultaneously. If necessary, the images can be adjusted before they are superimposed to create one composite image. The blue Hoechst fluorescence is converted to green for better color contrast. Figure 2 shows examples: two pairs of images (A and B; D and E) are combined into one multichannel fluorescence image respectively (C and F).
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Images that show it all
On the left side of figure 2, several wildtype follicles can be seen (A, B, C); on the right is an aberrant follicle from a combination of two egh alleles, thus having a mutation in the egghead locus (D, E, F). The upper images show actin stained by rhodamine-phalloidin (A, D), the middle images show DNA with Hoechst 33258 (B, E) and the lower images are the respective composite images (C, F). The large, polyploid cell nuclei of the nurse cells are readily visible in B and E. The small cell nuclei of the oocytes, on the other hand, are not. This is where the labeling of actin in the cytoskeleton of the oocytes can help (A, D). By this means, a total of four oocytes become clearly visible in the aberrant follicle (see arrows in D). Three of these are redundant and positioned incorrectly. In the Hoechst 33258 acquisition (E), the two lateral oocytes remain completely invisible. It is only in the digitally generated multichannel fluorescence image that all cells and cell types can be reliably identified. In addition, the analySIS software can be used to automatically determine and document the number of gametes in a follicle by detecting the Hoechst 33258 stained cell nuclei. This means that developmental defects in the generation of the egg chamber as a whole can be made visible and quantified.
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 Figure 2:
Using digital multichannel fluorescence to obtain a composite image. Under the fluorescence microscope, the actin emits a red color, stained with rhodaminyl phalloidin. This makes the cytoskeleton of the cells visible. The cytoskeletons of the oocytes are especially visible because they contain high levels of actin (A, D). The DNA stained with Hoechst 33258 has a blue color. By this means, the large, polyploid cell nuclei of the nurse cells become visible (B, E). The digital composite image then combines the information of the individual component images and all cell types are easily recognizable (C, F). For better color contrast, the blue was converted to green. In the Hoechst 33258 image by itself (B, E), the oocytes (arrows in A, D) could not be located.
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The total digital approach
This example of the kind of work we do every day demonstrates how digital image processing can be made useful for microscopical investigations – beyond the familiar advantages of doing everything digitally. Digital image acquisition, which is more convenient, less expensive and more environmentally friendly than conventional photography, makes it possible to obtain images where you can literally see more. Another possibility is to superimpose a normal brightfield image or a Nomarski interference contrast image over a fluorescence image. In addition, a digital image database makes archiving and retrieving images, related data and analyses a lot easier.
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Author
Dr. Martin Hollmann | FB 18 – Zoology/Developmental biology (Prof. Dr. Mireille A. Schäfer)| University of Kassel | Heinrich-Plett-Str. 40 | D-34132 Kassel | Germany
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