Comet Labs WN591 Uživatelský manuál

A dummy’s guide to image analysis used in the comet assay Barbara Vilhar University of Ljubljana Biotechnical Faculty Department of Bio

Help! There is a comet in my computer! 7 2 Detection of emitted light Our eyes, supported by our brain, are amazing instruments. We process incred

Help! There is a comet in my computer! 8 Figure 3. Light emitted from a comet on a slide is detected as an image. An image of a real comet on a m

Help! There is a comet in my computer! 9 3 Measurement of the comet parameters Now we recorded an image, and finally we come to image analysis. Ma

Help! There is a comet in my computer! 10 Figure 4. Information on an image is coded as grey values. An image of a comet is composed of separate p

Help! There is a comet in my computer! 11 Figure 5. Segmentation of the comet image. During segmentation, the regions of interest (ROI) for measu

Help! There is a comet in my computer! 12 Figure 6. Geometric calibration of the image analysis system. We record an image of a micrometer ruler u

Help! There is a comet in my computer! 13 3.4.1 And I measured my comet at 400× magnification... While we are talking about lengths, scaling fact

Help! There is a comet in my computer! 14 3.5 Measurement of light intensity parameters Several parameters measured in the comet assay describe t

Help! There is a comet in my computer! 15 intensity from the total light intensity that we recorded. If we do not do this, we will compromise the

Help! There is a comet in my computer! 16 Table 1. Subtraction of the background signal for each pixel column of the comet on the model comet imag

Help! There is a comet in my computer! i Contents Do you have a problem?...

Help! There is a comet in my computer! 17 However, we will now not average the grey values for the whole background region – we will instead calcu

Help! There is a comet in my computer! 18 3.5.4 Comet profile We have now done all the necessary things to tackle a new task – the comet profile o

Help! There is a comet in my computer! 19 Figure 10. The comet profile of the model comet image. Box-segmentation (A): The recorded comet intensit

Help! There is a comet in my computer! 20 Let us look at our model comet image (Figure 11). First, we will determine the position of the intensity

Help! There is a comet in my computer! 21 4 Recorded image and displayed image We assumed at the beginning of our calculation of comet parameters

Help! There is a comet in my computer! 22 Figure 12. The grabbed and the displayed image. Changing the settings of the displayed image that we see

Help! There is a comet in my computer! 23 Images of orange comets on a black background (Figure 13C) are very popular. Notably, most systems for c

Help! There is a comet in my computer! 24 same illusion of continuous motion as we have when watching movies, which are also composed of separate

Help! There is a comet in my computer! 25 Figure 14. Look-up tables (LUTs). A LUT contains instructions for translation of the grey values on the

Help! There is a comet in my computer! 26 5 Image acquisition The first rule of image analysis states that all care should be taken to record imag

Help! There is a comet in my computer! ii Figures Figure 1. The principle of fluorescence and absorption and emission spectra of DNA-bound ethid

Help! There is a comet in my computer! 27 5.1 Histogram of image grey values It is, however, not important at all for our measurement whether the

Help! There is a comet in my computer! 28 The two basic properties of an image, related to the range of grey values that are used to store informa

Help! There is a comet in my computer! 29 Figure 17. Image contrast and brightness. Images of the same comet are shown in A, B and C. The grabbed

Help! There is a comet in my computer! 30 5.3 Image saturation The recorded image is not suitable for measurement of the comet parameters if is ei

Help! There is a comet in my computer! 31 they violate the linear relationship between the amount of DNA on the slide and the recorded grey values

Help! There is a comet in my computer! 32 Some comet software packages feature an option to set the maximum number of allowed saturated pixels. A

Help! There is a comet in my computer! 33 white regions, and those regions of the image that have the grey value lower than the grey value selecte

Help! There is a comet in my computer! 34 Let us now drag the slider from left to right for the image shown in Figure 19. At grey value 0, the who

Help! There is a comet in my computer! 35 5.5 Shading Our measurement was based on the assumption that a linear relationship exists between the am

Help! There is a comet in my computer! 36 Uneven illumination of the field of view cannot be completely eliminated even in an optimally aligned mi

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 as

Help! There is a comet in my computer! 37 on the right side. In the image with severe shading (Figure 22D: 1-5), the background is clearly much br

Help! There is a comet in my computer! 38 5.7 Why a 10-bit camera beats an 8-bit camera In the comet assay, a camera serves as a detector of ligh

Help! There is a comet in my computer! 39 Figure 23. Comparison of an 8-bit and a 10-bit camera. While an 8-bit image has 256 available grey valu

Help! There is a comet in my computer! 40 Having a higher pixel depth brings another advantage. When we start recording images for a comet experim

Help! There is a comet in my computer! 41 the signal at a high enough resolution to allow for reliable measurement of the comet profile. The optim

Help! There is a comet in my computer! 42 after we mark the comet borders, we return the image back to original prior to measurement. How can we f

Help! There is a comet in my computer! 43 Figure 25. Different versions of user interface for adjusting the camera settings. The main settings ar

Help! There is a comet in my computer! 44 Figure 26. Examples of contrast and brightness settings of the displayed image. For adjustment of the c

Help! There is a comet in my computer! 45 6 Concluding remarks I said “linear relationship” so many times that I would really like to avoid it in

Help! There is a comet in my computer! 46 Notes I learned about image analysis and the comet world from many people, and I feel indebted to them a

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 ab

Help! There is a comet in my computer! 3 1 Fluorescence microscopy We are running a comet experiment. We prepared the slides and ran electrophores

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

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

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.