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«A dummy’s guide to image analysis used in the comet assay Barbara Vilhar University of Ljubljana Biotechnical Faculty Department of Biology Help! ...»

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A dummy’s guide to image analysis

used in the comet assay

Barbara Vilhar

University of Ljubljana

Biotechnical Faculty

Department of Biology

Help! There is a comet in my computer! i

Contents

Do you have a problem?

1 Fluorescence microscopy

2 Detection of emitted light

3 Measurement of the comet parameters

3.1 Grey values

3.2 Segmentation

3.3 Geometric calibration

3.4 Measurement of geometric parameters

3.4.1 And I measured my comet at 400× magnification

3.5 Measurement of light intensity parameters

3.5.1 Integrated light intensity

3.5.2 Percentage of DNA in the tail

3.5.3 Tail extent moment

3.5.4 Comet profile

3.5.5 Olive tail moment

4 Recorded image and displayed image

4.1 Look-up table (LUT)

4.2 Live image and grabbed image

5 Image acquisition

5.1 Histogram of image grey values

5.2 Image contrast and brightness

5.3 Image saturation

5.4 How can I evaluate the quality of my image?

5.5 Shading

5.6 How can I detect shading in my image?

5.7 Why a 10-bit camera beats an 8-bit camera

5.8 Binning

5.9 Which image does my software measure?

5.10 How do I get a good quality image out of my comet equipment?

6 Concluding remarks

–  –  –

Figures Figure 1. The principle of fluorescence and absorption and emission spectra of DNAbound ethidium bromide

Figure 2. The path of light through a microscope during observation of fluorescence of ethidium bromide

Figure 3. Light emitted from a comet on a slide is detected as an image

Figure 4. Information on an image is coded as grey values

Figure 5. Segmentation of the comet image

Figure 6. Geometric calibration of the image analysis system.

Figure 7. Scaling of a printed image.

Printed images should contain a scale bar

Figure 8. Grey values for the head, the tail and the background region of the model comet image

Figure 9. The comet profile for a comet recorded from a microscope slide

Figure 10. The comet profile of the model comet image

Figure 11. The intensity centroids

Figure 12. The grabbed and the displayed image

Figure 13. Types of images with respect to colour

Figure 14. Look-up tables (LUTs)

Figure 15. Effects of image acquisition settings on the information contained in an image of a comet

Figure 16. The histogram of image grey values

Figure 17. Image contrast and brightness

Figure 18. Image saturation

Figure 19. Viewing image histograms and grey values in Photoshop

Figure 20. Examples of indicators of the pixel grey value

Figure 21. Effects of uneven illumination of the field of view

Figure 22. Detection of image shading in Photoshop

Figure 23. Comparison of an 8-bit and a 10-bit camera

Figure 24. The effects of binning

Figure 25. Different versions of user interface for adjusting the camera settings

Figure 26. Examples of contrast and brightness settings of the displayed image

Figure 27. Measurement of the comet parameters: an overview

Help! There is a comet in my computer! 1 Do you have a problem?

Here I will talk about the basic principles of image analysis used in the comet assay. This should help you spot and solve potential problems in your measurements, avoid measurement errors, understand the settings available in software packages and the language used in user manuals, and improve your communication with the software companies when you run into trouble. The rest of this chapter is crap and you should move to the next one or quit reading. Now, this is how all the problems began.

Not so long ago, my good friend Prof. Lite Microscope was elegantly standing on the real desktop in our lab. He lived a happy and respectful life. A fine screwdriver and a drop of oil fixed all his troubles, although he hardly had any. These days, he is choking under the weight of digital cameras, ambushed by monitors, keyboards, graphics tables and printers.

An entanglement of cables ties him to his new master, Mr. P.C. Computer. The life of imprisoned Prof. Microscope has moved to the virtual desktop. Every day he is humiliated by software crashes, electronic mice, digital bugs, digital worms and digital viruses. The only one that ever looks down his shiny objectives is Ms. Camera Digital. His newly acquired, chip-controlled motors are desperately humming. Prof. Microscope suffers, and I suffer with him. I have no idea what the mean Mr. Computer might be doing to the digital results of my real experiments. I just sit in the dark for hours on end and click. Not so long ago, I used to understand my science.

Does this sound familiar to you? Do you actually know how the computer calculates the parameters of your comet assay? Do you understand the instructions and explanations in the handbook that came with your comet software? And is it important to know this at all?

Judge for yourself. I bought expensive software from a well-respected company, ran the computer and started clicking. I noticed straight away a problem with my data – the graphs that I plotted were somewhat, but not completely, in disagreement with previously published results from other labs. However, my results had remarkably small standard errors, so I concluded that the computer was doing a very fine job. I blamed the new method for staining of the slides that I used, and spent several months trying to perfect my staining skills. The strangest thing was that whatever I did, my results were always very similar, and always in disagreement with other labs. In the end I realised that it was not I, it was the computer that spoiled my results. So I sat down, learned the basics of image analysis and analysed the software algorithm step by step. In the end I found out that the software was surely measuring something, but that this something was not at all a meaningful parameter. The bad news was that the software could not be easily fixed – the whole measurement procedure was based on erroneous assumptions and at odds with the laws of physics. The next thing I did was learning how to program the computer for image analysis.





Help! There is a comet in my computer! 2 An annoying problem with computers is that, unless they crash, they will always give you a result. The above story is true, but it is not about the comet assay. Nevertheless, it is about measurement of the amount of DNA in nuclei on a microscope slide (to determine genome size in different organisms). I have to admit here that I have never prepared one single microscope slide for the comet assay. But I recently tried to find out how the comet assay works and I talked to researchers and students from several comet labs. While I got excellent explanations about DNA damage and variations in the procedure for preparation of the slides, the image analysis part of the experiment was more obscure. In most cases, the researchers bought the software and hardware, and then started to click. They had no time to study image analysis. Still, it is not really sensible to treat the only part of the experiment in which parameters are measured as a tightly closed “black box”.

Here are some arguments from the comet world. A colleague called me on the phone one day saying that she noticed abnormal values for the comet parameters in recent experiments. None of the researchers that spent days and months with the comet machine knew what went wrong. I only saw this machine once for an hour or so. Nevertheless, I was able to diagnose the problem and also propose the solution after only a few minutes of phone conversation. How did I do it? Well, I previously borrowed the software user manual, and because I know the principles of image analysis, I also understood how the black box works.

On another occasion, I had the idea to write a macro program for the comet assay. So I borrowed a CD with comet images from another colleague, who was then a comet beginner using another comet software package than the one mentioned above. When I viewed the images on my computer, I realised that there was something wrong with image contrast. In addition, quite a few spots on the image were overexposed, which means completely white. With such images, you do measure something with your computer, but your results are either imprecise or wrong. Optimising the settings of the camera solved the problem.

Now let us open the “black box”.

–  –  –

1 Fluorescence microscopy We are running a comet experiment. We prepared the slides and ran electrophoresis. Now we have to measure the amount of DNA that migrated out of nuclei. The most important thing to remember is this: Measurement is based on a known relationship (e.g. linear, logarithmic) between the amount of DNA in the sample and the parameter used for detection of DNA. In all steps of the measurement procedure, we have to take care not to compromise this relationship.

The first thing we need to do is stain DNA to make it detectable. Staining has to be quantitative: the amount of stain that binds to DNA must be stoichiometrically proportional to the amount of DNA present in the sample. In other words, the relationship between the amount of DNA and the amount of the staining substance bound to DNA must be linear.

The quantitative DNA dye usually used in the comet assay is ethidium bromide. During incubation in the staining solution, ethidium bromide binds to DNA on the comet slide.

Ethidium bromide is an intercalator with little or no sequence preference and binds to DNA at stoichiometry of one molecule per 4-5 base pairs of DNA. Hence, the relationship between the amount of DNA and the amount of ethidium bromide bound to DNA is linear.

Ethidium bromide is a molecule with special properties - a fluorophore. Fluorophores are molecules that absorb the energy of light (photons) of specific wavelengths. This process is called excitation. Fluorophores then release the absorbed energy in the form of light energy (photons). This process is called emission. Because some energy is “lost” during the excitation-emission cycle, the energy of emitted photons is lower than the energy of absorbed photons. In other words, the wavelength of emitted light is longer than the wavelength of absorbed light. Fluorescence is thus emission of light or other electromagnetic radiation at longer wavelengths by matter as a result of absorption of shorter wavelengths. Emission lasts only as long as the stimulating irradiation is present.

Each fluorophore absorbs and emits light of specific wavelengths. For example, DNAbound ethidium bromide absorbs green light and emits orange light (Figure 1A). Once ethidium bromide is bound to DNA, its fluorescence increases about 10-fold (compared to free molecules).

To be able to see the comets under a microscope, we need to illuminate the ethidium bromide-stained slide with intense green light. The source of high intensity light is usually a mercury lamp. Because a mercury lamp emits light of different wavelengths (including UV), we need to place an excitation filter between the lamp and the slide. This filter only passes green light and blocks all other wavelengths (Figures 1, 2A).

Help! There is a comet in my computer! 4

–  –  –

Figure 1. The principle of fluorescence and absorption and emission spectra of DNA-bound ethidium bromide.

A: DNA-bound ethidium bromide (EB) absorbs green light (high energy) and emits orange light (low energy). B: DNA-bound ethidium bromide absorbs green light (peak at ~530 nm) and emits orange light (peak at ~610 nm). The excitation filter on the microscope passes green light and blocks all other wavelengths. The emission filter passes wavelengths longer than the excitation light wavelength (orange and red). Note that ethidium bromide has another absorption peak in the UV range of the light spectrum (not shown) – this peak is exploited for visualisation of DNA on agarose gels with a UV transilluminator. See Figure 2 for the position of filters in the microscope light path.

Help! There is a comet in my computer! 5 When a slide is illuminated with green light, DNA-bound ethidium bromide on the slide emits orange light in all directions (Figures 2B, 2C). During observation of an object through eyepieces of a microscope, we see only light that is collected by the objective. We now have two problems: the emitted light is very weak and some of the strong excitation green light is reflected from the slide. To prevent excitation light disturbing our observation of fluorescence, we need to place an emission filter between the object and the eyepieces. This filter only passes light with longer wavelengths than the excitation light. The emission filter for ethidium bromide passes orange light and blocks green light (Figures 1, 2B, 2C). The second problem is easier to solve: because the intensity of the emitted orange light is very weak compared to the intensity of daylight, we have to observe the slide in a dark room.

Both the excitation and the emission filter are usually mounted together in a cubical filter holder, which is typically located in a filter wheel just above the objective revolver. Each fluorophore requires different combinations of excitation and emission filters to match its specific absorption and emission properties.

Let us return to our task to measure the amount of DNA on the slide. We already know that the relationship between the amount of DNA and the amount of ethidium bromide bound to DNA is linear. The good news is that also the relationship between the amount of DNA-bound ethidium bromide and the intensity of emitted light (the number of emitted photons) is linear. We can thus conclude that the relationship between the amount of DNA and the intensity of emitted orange light is linear. So all we need to do now is to somehow measure the amount of emitted light, and we will know how much DNA there is on the slide. This is where we need to start talking about images.

Help! There is a comet in my computer! 6

–  –  –

Figure 2. The path of light through a microscope during observation of fluorescence of ethidium bromide.



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