April 8th 2014

Maria Helene Kalkvik Cell Biology BI2012

N o r w e g i a n U n i v e r s i t y o f S c i e n c e a n d T e c h n o l o g y

Live cell imaging and advanced image analysis of the Golgi apparatus in plants

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Abstract This experiment was conducted in order to obtain a better understanding of laser scanning confocal microscopy, and also study the movement and stucture of the Golgi apparatus and endoplasmatic reticulum in plant cells. The chosen organells were labled with fluorescent proteins in order to observe them in the microscope. Arabidopsis thaliana was the chosen organism for the experiment. A Leica SP5 microscope was used along with computer software. Image processing softwares Amira and ImageJ were used to analyse the images of the specimen. Overview images of both the ER and Golgi apparatus was captures, and measurements of the Golgi apparatus’ size were contucted. Point spread function of fluorescent beads was determined in order to perform deconvolution.

Abbreviations GFP: Green fluorescent protein PSF: Point spread function FWHM: Full with half maximum ROI: Region of interest ER: Endoplasmatic reticulum LUT: Look up table NA: Numerical aperture

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Table of contents

ABSTRACT ....................................................................................................................................... 2

ABBREVIATIONS ........................................................................................................................... 2

1 INTRODUCTION ........................................................................................................................ 4 1.1 CONFOCAL MICROSCOPY ................................................................................................................................ 4 1.2 FLUORESCENSE ................................................................................................................................................. 5

1.2.1 Green Fluorescent Protein .............................................................................................................................. 5 1.3 POINT SPREAD FUNCTION .............................................................................................................................. 6 1.4 DECONVOLUTION ............................................................................................................................................ 7 1.5 RESOLUTION ..................................................................................................................................................... 9 DYNAMIC RANGE .................................................................................................................................................. 11

1.6 BIOLOGICAL SAMPLES .......................................................................................................... 11 1.6.1 ARABIDOPSIS THALIANA ............................................................................................................................ 11 1.6.2 THE PLANT CELL ......................................................................................................................................... 12 1.6.3 ENDOPLASMATIC RETICULUM .................................................................................................................. 12 1.6.4 THE GOLGI APPARATUS ............................................................................................................................. 12 1.6.5 ACTIN ............................................................................................................................................................. 12

2 MATERIALS AND METHODS .................................................................................................. 13 2.1 SPECIMEN PREPARATION .............................................................................................................................. 13 2.2 BEAD PREPARATION ...................................................................................................................................... 13 2.3 MICROSCOPE ................................................................................................................................................... 13 2.4 IMAGE PROCESSING AND SOFTWARE ......................................................................................................... 13 2.5 OPTIMAL RESOLUTION .................................................................................................................................. 14 2.6 PSF ANALYSIS .................................................................................................................................................. 15 2.7 DECONVOLUTION .......................................................................................................................................... 15

3 RESULTS ...................................................................................................................................... 15 3.1 IMAGE ANALYSIS OF THE GOLGI APPARATUS ........................................................................................... 15 3.2 IMAGE ANALYSIS OF THE ENDOPLASMATIC RETICULUM ........................................................................ 16 3.3 DECONVOLUTION OF THE GOLGI Z-STACK ............................................................................................. 17 3.4 SIZE ANALYSIS OF THE GOLGI Z-STACKS .................................................................................................. 17

4 DISCUSSION ............................................................................................................................... 18 4.1 IMAGE ANALYSIS OF THE GOLGI APPARATUS AND ER ........................................................................... 18 4.2 OPTIMAL RESOLUTION .................................................................................................................................. 18 4.3 PSF ANALYSIS .................................................................................................................................................. 18 4.4 DECONVOLUTION OF THE GOLGI Z-STACK .............................................................................................. 19 4.5 SIZE ANALYSIS OF THE GOLGI STACKS ...................................................................................................... 19

5 LITTERATURE ........................................................................................................................... 21 5.1 ILLUSTRATIONS ............................................................................................................................................... 22

7 APPENDIX ................................................................................................................................... 23 A CALCULATIONS ................................................................................................................................................. 23

A.1 Resolution limit, pixel size and number of pixels needed ................................................................................. 23 B METROLO J ANALYSIS ...................................................................................................................................... 24

B.1 PSF analysis of fluorescent beads .................................................................................................................... 24 B.2 Size analysis of the Golgi stacks ..................................................................................................................... 24

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1 Introduction

1.1 Confocal microscopy Laser scanning confocal microscopy has become an important tool for studying and observing biological cells. Through this technique one can obtain knowledge on how the organells in the cells move through the cytosol, and gain insight in cellular functions. It also gives the opportunity to perform optical cross sections of transparent sampels, withouth physically slicing them into sections. The ability to remove glare from out of focus layers has been an important improvent for studies involving biological imaging. [1] In a laser scanning confocal microscope (LSCM) a laser is used as a light source. The light is then filtered by an acousto-optical tunable filter (AOTF), which allows for regulation of both the wavelength of emitted light from the laser as well as exitation intensity. These microscopes makes it possible for examination of fluorescence emisson from 400 to 750 nanometers. [2] After the light is filtered by an AOTF, the light is reflected by a dicromatic mirror, which only reflects certain wavelengths and let others pass through. The Leica SP5 uses an acousto-optical beam splitter (AOBS) insted of a dicromatic mirror. Here, the crystalline materials only reflect certain wavelengths of light by the interaction of acoustic waves. This is done by manipulates the refractive index of the crystal. [3] The specimen is scanned by the laser, while two high speed oscillating mirrors direct the beam in a certain pattern across a chosen area of the specimen. One mirror controls the light along the x-axis, and the other along the y-axis. When a region of interest is found (ROI), the light moves along the x-axis from a starting point, and then returns to the starting point to scan in the y-dimension. [4] Before the emission light reaches the detectors, it passes through a pinhole aperture. The pinhole’s function is to reduce light disturbance, like blur, from planes above and below the plane of focus. Only a small part of the light from other planes in the specimen will pass through the pinhole (Figure 1). [4]

Figure 1: Illustration of the pin hole’s function in a confocal microscope 2

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1.2 Fluorescense Fluorescense is the ability certain compounds have to emit visible light. When the compund is exited by a photon, electrons move to a higher state of energy. This state is highly unstable which results in the electrons moving back to their original state, and when they do, energy is released. Energy in this case is in the form of light. Different compounds have different electron configuration and orientation of chemical bonds. This leads to emission of different wavelengts as the exited state for the electron may be in an higher or lower state of energy, depending on the compund. Since light can be viewd as a form of energy, differences in energy because of electron configuration, the electrons exited state and its original state, leads to emission of different light. The wavelenth of light required to exite a compound is designated as 𝜆!"# , where the unit is nanometers (nm). Some of the energy is lost as it forms heat and vibration, not only photons (light). Therefore the photons emitted from the compund has less energy, and thus a longer wavelength than the light used to exite the compound. This difference between the exitation wavelength and emission wavelength is called Stokes shift. The emission wavelength is given by 𝜆!", and is also in nanometers.

1.2.1 Green Fluorescent Protein Green fluorescense protein (GFP) was isolated from the jellyfish Aequorea victoria. Research shoes that the molecule is able to fluoresce when it is expressed by itself in any organism, thus making it a well suited marker for biological studies and cell imaging [5]. GFP consists of eleven 𝛽-barrels and one 𝛼-helix, surrounding the cromophore in the core (Figure 2). The cromophore is the part of the molecule that makes fluorescense possible. It has been modified in order to create a more stable and effective version of the fluorescent protein, compared to the wild type found in A. victoria. One of the modified versions are called enhanced yellow fluorescent protein (EYFP). It has a 𝜆!"# of 514 nm and a 𝜆!" of 527 nm. [6]

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Figure 2: A model of Green Fluorescent Protein (GFP). The cromophore is not depicted in the model. The model was created in the software Amira, by using the protein data bank, 1EMA. When studies involving fluorescent proteins are contucted, the protein is fused to proteins who binds to, or target the chosen organelle. When studying ER, the protein is fused to a signal peptide called Arabidopsis thaliana wall associated kinase 2 (AtWAK2 ). The fusion happens at the N-terminus of the peptide, and the ER recognises the signal sequence His-Asp-Glu-Leu at the C-terminus. When studying the Golgi apparatus the fluorescent protein is fused to a cytoplasmatic and transmembrane domain of a protein calles soybean 𝛼-1,2-mannosidase I.[6]

1.3 Point spread function A point spread function is a result of diffraction of light in a specimen. When light travels through the microscope it is defracted as a result of interactions with materials it passes through, lenses. The consequence of light diffraction is that a given point in the specimen will seem larger than it actually is. It can be said that the PSF is a measurement of how many neighboring pixels are affected when one pixel is fully enlightened. The given point will often be surrounded by alternating dark and light rings, as a result of light diffraction. This patterns is refered to as an Airy pattern, and was first discovered by George Biddel Airy. [7] The central disc is called an Airy disc. This pattern in three dimentions is what creates what is called a point spread function, since it describes how light spread out from a single point. [2]

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Figure 3: The corrolation between an airy pattern and a point spread function. The graph (red) shows the PSF as light intensity, with the airy pattern in the background. 3

1.4 Deconvolution Blur is created as a result of the diffraction of light when it interacts with lenses in the microscope. As blur is an undesirable factor when working with image analysis, it can be corrected for by using the PSF. Every point, or pixel, in the image is essensially a point spread function with a corresponding airy pattern. The out of focus blur is a result of the alternating dark and light rings of the airy pattern. The goal is to remove these rings, and obtain a single point in focus. In order to do so, one must apply the point spread function to every point of the object. [8] Deconvolution is a mathematical process based on algoriths, that determines the most likely estimation to reassigh out of focus blur back to its point source. To determine the point spread function, beads with a known size can be used to calculate it. A 3D image of the beads can be analysed with image processing software (Figure 4).

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Figure 4: A 3D visualization of beads in the image processing software Amira. As the PSF shows spreading of light from the point source, it can be used to reverse the blurring effect of convolution. The PSF can be applied to chosen images by using image processing software, and thereby reducing the blur in the image. This creates a more realistic depiction of the specimen.

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Figure 5: The raw image (left) and the deconvolved image (right). The grana in the chloroplasts was almost impossible to observe before deconvolution.

1.5 Resolution Several factors affect resolution of an image taken with a confocal microscope. The numerical aperture (NA) of the objective plays a big role in the resolution of an image. NA is defined by [10]:

𝑁𝐴 = sin 𝛼 𝑥  𝜂 (1)

Where 𝛼 is defined as half the angle of the cone of ligth the objective captures from the focal point, and 𝜂 is the refractive index of the immersion medium used [10]. As a result, when the objective size increases or the refractive index increases, so does the NA. A high NA value gives a higher resolution. The Rayleigh criterion is an optical unit that describes the minimal distance between two point sources at which they are distinguishable from each other. It is given by the formula [9]:

𝐷!! =!,!"!!"#

!" (2)

Where 𝐷!" is the Rayleigh criterion, 𝜆!"# is the wavelength of the excitation light, and NA is the numerical aperture of the objective. In the axial dimension, the Rayleigh criterian can be calculated using the following equation [9]:

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𝐷! =!!!"#!(!")!

(3)

Where 𝜂 is the refractive index of the immersion medium. The most common immersion mediums are water, air, oil and glass. Their refractive indices are presented (Table 1). Table 1: Refractive indices of the immersion mediums water, air and oil/glass [12]

Immersion medium Refractive index Air 1.00

Water 1.33 Oil/Glass 1.52

To illustrate how the NA affects PSF, one can generate different PSFs with varying NA values (Figure 6).

Figure 6: How NA affects PSF. The NA varies from 1.0 (left), 1.2 (middle) and 1.4 (left). The 𝜆!"# was set to 514 for all PSFs. The model was created using the Amira software. The sampling rate is also an influencial factor to resolution. The Nyquist theorem states that it should be two samples per resolvable element to obtain an accurate image.[9] The size of the pixel determines the sampling rate, as one pixel only can have one light intensity value. Therefore the the sampling rate directly correlates to

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the pixel size. The following equation must be used to calculate pixel size in lateral dimentions, in order to fulfill the Nyquist theorem:

𝑃𝑠!" =  !!"!

(4) Where 𝑃𝑠!" is the pixel size. A voxel is relevant when the axial dimension is under study. While a pixel only has two dimensions, a voxel has three, making it a volume element rather than just an area. The voxel size is determined by the pixel size, in the x- and y-dimensions, and also the distance between scans in the z-dimension. The Nyquist theorem can also be applied here. The depth of a voxel should not exceed the value given by the following equation:

𝑃𝑠! =!!!

(5)

Where 𝑃𝑠! is the voxel depth. The number of pixels needed in an image can be calculated using the following equation, by taking the Nyquist theorem in account:

#𝑝𝑥 =   !!!"

(6)

Where the number of pixels needed in both the x and y dimensions are denoted #𝑝𝑥, and 𝑠 represents the size of the ROI.

Dynamic range To ensure good image quality, the microscope and the software needs to be set up and adjusted correctly. The bit depth is an important aspect to consider. A high bit depth will be more likely to produce an accurate depiction of the object under study. A low bit depth results in a larger variance in light intensity [12] The software needs to be adjusted so that the image is shown within the detectors dynamix range. A LUT is usually used, as it gives different colours to different light intensities.[12] One should make sure that no pixels are completely saturated, in order to achieve a better understanding of the pixels intensity relative to each other.

1.6 Biological samples

1.6.1 Arabidopsis thaliana Arabidopsis thaliana is one of the most commonly used species in plant studies. It is a plant with simple growth requirements and a relativly short life cycle of eight weeks, which makes concucting studies on the plant quite easy. Also, A. Thaliana’s

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genome has been fully mapped, and it is therefore often used in genetic experiments. The plant is also susceptible to genetic transformations, only by spraying the plant with a bacterium which holds the gene of interest.

1.6.2 The plant cell The plant cells contain an endomembrane system that incorporates all membrane bound organelles in the cell. These organelles are the endoplasmatic reticulum and golgi apparatus, which will be discussed in the report, and also the nuclear envelope, the cell membrane, vacuoles, lysosomes and transport vesicles. The vaculoles controlles turgor pressure in the cell, as well as it works as a storage space for water and inorganic ions among other things. As a result, the plant vacuole is quite large, which only makes it possible for the other organelles to occupy the small cytoplasmic space between the central vacuole and the cell membrane.[13]

1.6.3 Endoplasmatic reticulum The endoplasmati reticulum (ER) is an organelle consisting of both the rough and smooth ER, which have specialized functions in the cell. It is a continuous network of tubules and sacs. The rough ER plays a prominent role in protein synthesis, as well as protein modification and marking. It is called the rough ER because of ribosomes sitting on its membrane, giving it an uneven surface. The smooth ER is active in lipid synthesis, detoxification and calcium storage, among other things. Overal the ER has a wide range of functions including biosyntesis, metabolism and storage. Like several other organelles the ER is connected to actin filaments which makes movement of the ER possible. [13,14]

1.6.4 The Golgi apparatus The Golgi apparatus consists of several sacks, each enclosed by a membrane. Its functions include modification of protein marking, transport and secretion of proteins and other molecules. Vesicles from the ER fuse together with the Golgi apparatus, where the cargo’s final destination is decided by the Golgi. Either it is sent to the area in the cell where it is required or secreted through exocytosis. The reciving side of the Golgi is called the cis-golgi network, and is the side closest to the ER. The opposite side is the trans-golgi network, where the proteins or molecules are secreted in a vesicle formed by the Golgi membrane. [13,14]

1.6.5 Actin Actin is a part of the cells cytoskeleton. It is necessary for the cell to have a cytoskeleton to obtain its structure, and also for transporting organelles or vesicles containing cargo molecules. Actin contributes to shaping the cell surface, as well as cell movement. Actin makes movement of the Golgi and ER possible. The transport process is mediated by the acto-myosin system, where actin filaments

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represent the road or track the organelles move on, and myosin functions as a motor protein, moving the organelles in a direction. [14] Actin filaments are created by polymerization of actin monomers. Latrunculin B forms complexes with actin monomers, resulting in inhibition of the polymerization process. If the cell is exposed to Latrunculin B, movement of the organelles will be inhibited. [15]

2 Materials and methods

2.1 Specimen preparation An Arabidopsis thaliana plant was marked with fluorochrome YFP or GFP, in order to visualize the golgi apparatus or the endoplasmatic reticulum. A small leaf was removed from a young A. Thaliana plant. The leaf was placed on an object slide with a drop of MQ-water. Any dirt og particles, as well as air bubbles was removed from the object slide, and the specimen was sealed using wax.

2.2 Bead preparation A solution of TetraSpeckTM microspheres was vortexted for two minutes. Three different samples were created by diluting the solution to the factors 1:100, 1:1000 and 1:10 000. The purpose of diluting the solution was to avoid cluttering of the beads, in order for visualization of a single bead to be possible.

2.3 Microscope The type of microscope used was a Leica SP5, along with the Leica Application Suite Advanced Fluorescence (LAS AF) software. An argon laser was used, with an intensity of 30 %. The chosen wavelength of 514 nm light was set to 15 %. The bit depth used was 12 bits, and the pinholde aperture was set to 1 airy unit (AU). One photo-multiplier tube (PMT) detector was set to detec light with wavelengths in the range between 520 and 570 nm. The overview images and the RGB images of the ER and Golgi were taken with a 10x/0.40 air-immersion objective.

2.4 Image processing and software The images of the specimens containing golgi and ER-marked cells were scanned. The images were then analysed and applied a LUT of chosen colour in the computer software ImageJ. The gain and offset values of the images was adjusted to make the structures of interest clear. Scalebars and LUT colour bar, indicating light intensity was added to the overview images. Different ROIs were selected for the movies, resulting in the RGB images, of the ER and Golgi apparatus. The acquisition mode of the microscope software was set to xyt, with a frame rate of 1 frame per second.

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Frame number 1, 6 and 11 were chosen in both movies to illustrate movement of the organelles in the RGB images. The RGB images were processed in the software ImageJ. The first frame is red, the second green and the third blue. Merging the pictures of the three frames create an illustratin of organelle movement in the cells. Since the colours red, green and blue create white when they overlap, white areas represent those areas where no movement was detected. The organelles that moved will have either a red, blue or green colour, or a mixture of two of the colours.

2.5 Optimal resolution The z-stacks of the Golgi apparatus and the fluorescent beads was visualized using a 63x/1.20 water immersion objective. A PMT detector was used for the z-stacks of the golgi apparatuses. For the imaging of the beads a hybrid detector (HYD) in photon counting mode was used. Acquisition mode was set to xyz. Latrunculin B was used on the specimens containing the Golgi apparatuses to inhibit movement. To be sure that there would be no movement in the specimen, one should wait one hour to let the inhibitor work. After one hour the specimen was scanned for ROI. The Rayleigh criterion was applied. The Dxy and Dy was found, and the Nyquist theorem was applied with oversamling by a factor of three. The optimal pixel size was set by adjusting the zoom factor and the number of pixels in the image (Equation 6). The LUT was adjusted so that the pixels of interest were neither fully saturated or at zero intensity. When performing optical cross sections in the z-direction, the start point was set to a plane above the chosen Golgi apparatus, where there was zero light intensity. The same was done with the end point; it was set to a plane well below the Golgi apparatus where the structure was not visible and there was zero light intensity. This is done to ensure that the whole golgi stack was depicted in the z-region. If the regions above or below the Golgi was too large, one can crop the image using an image processing software. The distance between the scans in the z-dimensions was set so that it corresponded with the optimal voxel depth calculated. The stack was visualized using image processing software. The settings listed above was also used for the fluorescent beads, except for the zoom factor, which was set to a higher value. This resulted in greater oversampling in this stack, compared to the stack of the Golgi apparatus. A ROI was chosen based on the requirement of the visualization of three beads to be possible in the chosen aera. The beads also had to be distinguishable from one another. Here, the start and end point was also chosen were there was zero intensity above and below the chosen beads.

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2.6 PSF analysis The z-stacks of the fluorescent beads were analysed in the software Amira. The three chosen beads were marked, and a BeadExtract module was attached to the z-stack module. The BeadExtract module estimates the PSF size based on the average of the three marked beads. The PSF was then visualized using a Projection View module. The PSF was opened in ImageJ, where the MetroloJ plugin was used to calculate size at FWHM of the PSF.

2.7 Deconvolution The computer software Amira was used for the analysis of the z-stacks of the golgi apparatuses. The PSF module from the beads’ PSF analysis were resampled in order to have an sampling rate equal to the z-stack of the Golgi apparatuses. This was done by connecting the two modules to a resample module (Figure 7). A Deconvolution module was connected to the Golgi z-stack module and the resampled PSF module. The correct NA and 𝜆!"# values, as well as refraction index, was entered in the deconvolution module. Deconvolution by maximum likelihood estimation was startet, and 20 iterations were run.

Figure 7: Module window in Amira. The modules containing data is marked green, the operation modules are coloured red and the yellow boxes show visualisation modules. The operation modules process the raw data in order to produce resampled and deconvolved images.

3 Results

3.1 Image analysis of the golgi apparatus An image of a cell marked with EYFP of the golgi apparatus is presented as well as a RGB-image to illustrate movement of the organelles (Figure 8). The RGB-images were created by filming the movement of the organelles and choosing three different frames, numer 1, 6 and 11, where number one is red, 6 is green and 11 is blue. The three frames were overlayed, creating the images shown below. Frame

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number 1, 6 and 11 were used both for the RGB-image of the Golgi apparatuses and the ER.

Figure 8. (To the left) An overview of the Golgi Apparatus marked with EYFP in plant cells of A. Thaliana. The image was taken using a 10x/ 0,4 air immersion objective. The colour scale of the LUT to the right indicates the light intensity raging from zero, black, to fullt saturated pixels, shown in white. (To the right). A RGB-image to illustrate movement of the golgi apparatuses in the cell.

3.2 Image analysis of the endoplasmatic reticulum The images presented are an overview image of cells with an EYFP-marked ER, next to a RGB-image illustrating the movement of the organelle within the cell (Figure 6). The procedure for creating the RGB-image was the same as the one for the Golgi apparatus.

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Figure 9: (To the left) An overview of the ER marked with EYFP in plant cells of A. Thaliana. The image was taken using a 10x/ 0,4 air immersion objective. The colour scale of the LUT to the right indicates the light intensity raging from zero, black, to fullt saturated pixels, shown in white. (To the right). A RGB-image to illustrate movement of the ER in the cell.

3.3 Deconvolution of the Golgi z-stack The software Amira was used to obtain a deconvolved image of the golgi stacks. This was done to remove blur from the image, in order to observe the golgi stacks in 3D with better resolution.

Figure 10. The golgi z-stacks before deconvolution (left) and after (right). The images were created in the Amira image processing software.

3.4 Size analysis of the Golgi z-stacks The deconvolved z-stacks of the golgi apparatus were analysed so that the size measurements of a single stack could be contucted. The software ImageJ was used with the MetroloJ plugin. This software interprets the raw data and produces a gaussian line fit based on the data. The line fit makes it possible to measure FWHM. Three different golgi stacks were analysed, and the average size was calculated (Table 2) Table 2: FWHM size of the three Golgi stacks, and average size based on these data. The asterisk denotes a possibly inaccurate value, as the fittet curve did not incorporate all the data points.

Dimension Golgi 1 [nm] Golgi 2 [nm] Golgi 3[nm] Average size [nm] x 260 447 266 324,33 y 302 502 250 351,33 z 595 617 809* 673,67

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4 Discussion

4.1 Image analysis of the Golgi apparatus and ER The images clearly illustrated the cells structure and organization of the organelles under study. The cell membrane is clearly visible, making it possible to distinguish between to different cells. This is probably a consequence of the vacuole pushing the organelles towards the membrane. Individual organelles can be observed in the cell, and their shape and size can be used for further analysis. In the overview image of the Golgi apparatuses, the stomata is clearly visible. This shows that the cell is one of the cells at the top layers of the leaf. The Golgi apparatuses are clustered together in the stomata guard cells. The overview image also shows that the Golgi apparatus is quite abuntant in cells. Golgi is a very important organelle in the cell because of its role in sorting of proteins, among other things. The overview image of the ER shows a clear network structure. Its long tubular structures creates a large surface area, suggesting that a large surface area is an important tool to secure effiency of the processes occuring in the organelle. The ER seems to be tightly packed near the cell membrane, as the light intensity here is quite high. The underlying network is shown in blue. The RGB images is able illustrate movement of the organelles in the cells quite well. In both the RGB images of the ER and the Golgi apparatuses it shows significant movement. Also, it shows areas where there was not detected any movement, coloured white. It is possible to observe certain areas that are particularly active in both the ER- and the Golgi image. These active areas can be seen as multicoloured strands in the cell. In other areas the movement of the organelles seem more random than directional.

4.2 Optimal resolution The calculated values for the resolution limit, pixel and voxel size were enterted in the Leica softwar for optimal sampling rate. The software automaticly rounded of the numbers, creating a possible source of error. Because of this, the calculations were performed with oversampling.

4.3 PSF analysis The PSF from the bead extract could be seen as a dot surrounded by alternating light and dark rings. This fits well with the theoretical knowlegde of the PSF. This suggests that the z-stacks of the beads were accurate.

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A 3D model of the PSF was generated using computer software, making it possible to view the intensity of the PSF in the z-dimension. Through this model the airy pattern was clearly visible (Figure 8).

Figure 11: Three different cross sections in the z-dimension of the PSF, showing different light intensities through as a pillar in the chosen plane. The airy patterns, or rings, are clearly visible in the PSF. The PSF was created by an NA value of 1.4 and the 𝜆!"# value was created by Amira. The values used to create this model was not from the results.

4.4 Deconvolution of the golgi z-stack The deconvolved image was clearly an improvement from the raw image, as the organelles are easier to see and most of the blur was removed from the image. Deconvolution has shown to be a useful tool when it comes to live cell imaging. However, the process did not work perfectly. This could be due to a deviation between the PSF of the golgi z-stacks and the PSF from the beads. Nontheless, the abberation seems to not have been very significant, as the deconvolved image does not show any obvious flaws and is not missing any crucial information. It seems as though the utmost of blur has been removed, and thus creating a better depiction of the specimen.

4.5 Size analysis of the Golgi stacks The FWHM size for three different golgi stacks were calculated using the MetroloJ plugin. Gaussian line curves where fitted to the light intensity values of the z-stacks. In the analysis of Golgi 3 the gaussin fit did not include all the data points in the z dimension, resulting in a possibly inaccurate R2-value. The R2 value is a measure on how well the fitted line corresponds with the data points. A perfect line will have a R2 value of 1.

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There were differences between the three chosen Golgi apparatuses in size dimensions, suggesting that the organelle is not symetrical in shape. This may be due to the fact that the Golgi apparatus consists of several stacks, or cisternae, which can have different shapes and sizes. The data suggests that all three Golgi apparatuses are largest in the z-dimension. The minimum resolution was larger in the z-dimension, resulting in inaccurate values as the smallest unit will become larger. The data collected from the experiment regarding size of the Golgi apparatus is not representable. For one thing, only one cell type was analysed, and the sample size was also quite low. To achieve a more representable measurement of the Golgi more cell types should be analysed, to remove any inaccuracies.

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14. J. Hardin et al. Becker´s world of the cell. (8. Utg, 2012). San Fransisco: Pearson.

15. A. Kurenda, P. M, Pieczywek, A. Admiak, A. Zdunek. Effect of Cytochalasin B, Lantrunculin B, Colchicine, Cycloheximid, Dimethyl sulfoxide and Ion Channel Inhibitors on Biospeckle Activity in Apple Tissue. Department of Microstructure and Mechanichs of Biomaterials, Institute of Agrophysics, Polish academy of Sciences. Published by: Springer. (2013-12-01).

5.1 Illustrations 1. Cover photo: Plant seed. URL: http://wodumedia.com/wp-content/uploads/Plant-seed-from-freshwater-pond-near-Moscow-Russia.-Photographed-with-fluorescence-10x-objective.--960x1531.jpg [Access date: 01.04.2014] 2. Illustration of the pin hole’s function in a confocal microscope URL: http://www.photonic-lattice.com/en/technology/polarization-longitudinal-slit-technology/ [Access date: 27.03.2014] 3. The corrolation between an airy pattern and a point spread function. The graph (red) shows the PSF as light intensity, with the airy pattern in the background. URL: http://www.asinen.org/ [Access date: 27.03.2014]

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7 Appendix

A Calculations

A.1 Resolution limit, pixel size and number of pixels needed Calculations of the resolution limit was performed using equations (2) and (3). The objective used was a 63x/1.2 water immersion objective, which gives an NA value of 1.2 and 𝜂 = 1.33. The excitation wavelength used was 514 nm. Lateral resolution limit:

𝐷!" =  0.61  𝑥  514  𝑛𝑚

1.2 = 261,3  𝑛𝑚

Axial resolution limit:  

𝐷! =  2(514  𝑛𝑚  𝑥  1.33)

(1.2)! = 951,6  𝑛𝑚

Optimal pixel- and voxel size was calculated using equations (4) and (5): Lateral pixel size:

𝑃𝑠!" =  261,3  𝑛𝑚

3 = 87,1  𝑛𝑚

Axial voxel depth:  

𝑃𝑠! =  951,6  𝑛𝑚

3 = 317,2  𝑛𝑚

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B Metrolo J analysis

B.1 PSF analysis of fluorescent beads The gaussian line fit graphs for the PSF analysis of the fluorescent beads are shown (Figure B1).

(a) (b) (c) Figure B1: Gaussian line fit in the x (a), y (b) and z (c) dimensions. The R2 value is presented (Table B1). Table B1. R

2 values Gaussian curve R2

a 0.99 b 0.99 c 0.98

B.2 Size analysis of the Golgi stacks Gaussian line fits for Golgi apparatus number 1,2 and 3 are presented (Figure B2, B3, B4)

(a) (b) (c) Figure B2: Gaussian line fits in the x (a), y (b) and z (c) dimensions, of Golgi apparatus 1. The R2 value is presented (Table B2). Table B2. R

2 values Gaussian curve R2

a 0.99 b 0.97 c 0.99

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(a) (b) (c) Figure B3. Gaussian line fits in the x (a), y (b) and z (c) dimensions, of Golgi apparatus 2. R2 values are presented (Tabel B3) Table B3. R

2 values Gaussian curve R2

a 0.98 b 0.98 c 0.99

(a) (b) (c) Figure B4. Gaussian line fits in the x (a), y (b) and z (c) dimensions, of Golgi apparatus 3. R2 values are presented (Tabel B4) Table B4. R

2 values Gaussian curve R2

a 0.99 b 0.99 c 0.99