Confocal 510 Tutorial

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1 Confocal 510 Tutorial You will have to log on in order to use the microscope. You will be charged according the time you are logged in, so please don t forget to log out after you are done. If you don t have an username and password, go to the microscopy website at microscopy.uc.edu, choose Light Microscopy. Click on New User Application in the left menu bar. Fill out and allow 1-2 days for processing. 1

2 Table of Contents Check list for trained users p 3 Typical Protocol p 6 Scope Parts p 8 Setting Up p 12 Beampath Configuration p 25 Acquisition p 47 Saving and Cleaning up p 61 Z-stack & Z-line Acquisition p 65 DIC p 78 Appendix of Background Information p 105 2

3 check list for trained users: Start Up Turn on nitrogen gas for air table mercury lamp under microscope microscope setup with main switch (between monitors) computer if necessary (depends how it was shut down) Start LSM 510 program, start in expert mode Switch on needed lasers ( acquire, laser ) Put your slide on stage. Use brightfield and/or epifluorescence to focus image and select area of interest. Get your beampath configuration ( acquire. config ) file from either an old data file (load file, choose button reuse ) a provided configuration file (choose config button and select appropriate file) set up your own (see pages how to do that) 3

4 check list for trained users: Acquire Images Pull slider all the way out to allow scanning. acquire scan channels: Choose large pinhole and detector gain to start with. Or use find for a first image. Find brightest image by focusing. Adjust pinhole and detector gain approximately. acquire scan mode: Choose zoom/rotation, scan speed, start with low resolution. Adjust pinhole, detector gain, amplifier offset for each channel. Use the range indicator under palette to help you to use the whole range of intensity values. Acquire scan - mode: choose final resolution, select averaging. Make single scan. Save image if satisfied, adjust further if necessary. 4

5 check list for trained users: Shut Down Turn lasers off. The fan for the argon laser should run for 5 mins. You can close the program, but don t switch the main switch (remote) off. Burn your data onto a CD. Data left on the computer will be deleted after one month. Shut off nitrogen tank at main air valve If you used oil, clean any oil from the objective lenses with lens paper. Turn off the microscope; leave the 10x objective in place. Log out of the computer. Once the computer is shut down, turn off main switch. Turn off the mercury lamp under the microscope Cover the microscope but not the lamp housing 5

6 Sign up for confocal time I Go to calendar.yahoo.com Yahoo ID is confocal510 Password is signup Choose Sign in. Choose a day or a month in the calendar part Choose a time 6

7 Sign up for confocal time II Write your name in the field title Choose the start time and duration Save If you can t make it, please delete your reservation as soon as possible so others can use the confocal. Please sign out after you are done. 7

8 Microscope Parts I Becoming familiar with the different parts of the microscope and what they do will help you to collect a good image quickly. Below you can see the (from left to right) z stage controllers xy stage knobs neutral density filters (NDF) focus course/fine toggle for focus (C/F) light source toggle (LST) transmitted light intensity knob power switch 8

9 Microscope Parts II The eyepieces - adjust for your eyes by turning the dials of each lens. Keep the DIC slider out while you use fluorescent or laser light! If the slider is out, it allows the laser to scan the sample. To protect your eyes, it also blocks the light to the objectives. To view your sample through the eyepieces, the slider has to be in. Slider: In: light to eyepieces Out: light to confocal DIC polarizer 9

10 Microscope Parts III You can rotate the objective turret manually or it can be computer driven. The stage is where your sample sits. With the condenser and/ or the field aperture FA), you can adjust the transmitted light passing through your sample. The Z stage buttons raise and lower the stage to change objectives or to apply water or oil. 10

11 Manual Controls You can rotate the filter cube turret with the two buttons below the focus knob on the left side of the scope. The objective lens turret can be rotated by using the two buttons below the focus knob on the right side. 11

12 System Start Up I Inflate the air table by turning the main valve on the air tank clockwise about 2 turns. You should hear the air flowing. Turn on the mercury bulb below the microscope for epifluorescence. You should see light to the left and at the back of the Axioplan 2 scope. If you see no light, call for help. Please make sure you do this before turning on any other electronic device. 12

13 System Start Up II Turn on the remote control button below the monitors on the table. This will turn on the computer and related equipment. If the computer does not come on, turn it on. Turn on the microscope with the green toggle switch on the right side of the Axioplan 2. You have to turn on the microscope before you start the program. 13

14 System Start Up III Once NT is running on the computer, you will see the control+alt+del sign. Press ctrl, alt and del at the same time. This will get you the Novell login window. Input your user name and password. Click OK. 14

15 LSM Software I Doubleclick the LSM 510 icon. Make sure you switched the microscope on before you start the program. (If not, exit program, switch on microscope, start again). The Zeiss software will startup and you will see the LSM 510 switchboard window. You need to make sure Scan New Images is selected and then click Start Expert Mode. 15

16 LSM Software II When the LSM software opens, you will want to create a new database. To do this, click File then New. Create on the D (DATA) drive of the computer. The file name should be your name and the date. Click Create. When you click Create, a new database will open and you should minimize the window for now. To open an existing database, click the Open button. Find your database and click on it and then click Open. 16

17 Igniting the Lasers I Next, click the Acquire button on the control strip. Then click the Laser button. You only need to turn on the lasers you will be using Click each HeNe laser you want to use to the On position and click the argon laser to the Standby position. The status will read Warming up. 17

18 Igniting the Lasers II Once the argon laser is warm, the status will read Ready and you can click it On. Slowly change the Output to read 60% this should make the Tube Current %. This adjustment will insure stability of the laser. Close the laser control window. 18

19 Microscope Software Controls I Once you have the lasers on, you will need to find your sample with the microscope. Most microscope operations can be controlled manually or by the Zeiss software. To use the software controls, click the Acquire button on the top menu and then Micro on the bottom menu. Focus and XY stage have manual controls only. From this menu, you can change the filter cube positions, objectives lenses, and transmitted light settings. If you see only the top part of the window click on more to show the bottom part. Click on the icon to open the window for the control of the option you want. 19

20 Microscope Software Controls II Select one of these three filters for your fluorophore. This filter affects only what you see through the objectives; it won t affect the laser. Filter Mercury bulb light for brightfield (halogen bulb) You can click the shutter of the mercury bulb open and close by clicking on reflected light If you want to use the manual controls for the halogen lamp, you have to unselect remote and close the window. 20

21 Viewing Specimen With Brightfield I Place your slide on the stage and secure it with the slide clamp on the stage. Make sure you push the slide all the way to the back to secure it. Turn on the transmitted light by clicking the transmitted light box on the microscope window in the software. Adjust the intensity either using the intensity knob on the microscope or using the control on the computer screen. 21

22 Viewing Specimen With Brightfield II To view your specimen with the ocular lenses, you must push the slider knob all the way in to VIS. You will see the light come on the stage. To adjust the intensity of the transmitted light you can also use the neutral density filter wheels on the right side of the scope. The numbers on the wheels are a representation of the amount of reflected light passing through the filter. (i.e.. a 6% filter will produce a darker view than a 50% filter because it allows less light through). Focus your sample with a low magnification objective and check your Kohler illumination (See DIC section for more info on Kohler illumination). Then, switch to a higher magnification. Find the area you wish to image. 22

23 Viewing Specimen With Fluorescence To view fluorescence, you need select the proper filter cube for viewing (FITC, Rhodamine or DAPI) using the either the reflector menu in the Axioplan control box or the manual control under the left focus knob. After you have selected the proper cube, push the reflected/transmitted light toggle button and you should see the reflected/fluorescent light come on. 23

24 Confocal Image Acquisition After you have a focused image on the microscope, you can acquire a scanned image. Pull the slider all the way out to the LSM position. This will allow the laser to collect data, and it will also close the light path to the eye pieces so you cannot accidentally look into a laser. You may want to check if the lasers that you will be using are on by clicking on the Laser button under the Acquire menu. 24

25 Beampath Configuration (BC) Overview There are three different detector channels available for a signal plus one for a brightfield/dic image (ChD). The detector (photomultiplier) measures only light intensity, not wavelength. By choosing the appropriate filters and beam splitters you can make each detector wavelength specific. Beam splitters and mirrors direct light to the different detectors. The following pages tell you how to configure the beampath for single (single track) or multiple (multi track) dyes. You will find an explanation of these building blocks in the appendix of this manual. You can construct your own beam configuration, use predefined configurations, or reuse configurations you have used before. 25

26 BC: The REUSE button Don t forget that you can load a configuration from a previously acquired image. If you select Reuse, the current acquisition settings (including the configuration) will be set to the parameters of the saved image. 26

27 Beampath Configuration (BC) Single Track Click the Acquire button in the top row and then the Config button in the second row. If you are doing only one dye, use single track mode. You don t have to design your configuration new every time. There are stored configurations you can use. To access those, click on Config button. To view preset configurations such as FITC, click on the downward arrow. After selecting one preset configuration CHOOSE Apply. Then, select Close. 27

28 BC-Single Track-FITC only-excitation Let s examine how this configuration would be built if it were not already saved. Step 1 Select the appropriate laser line Select Excitation. Turn on the appropriate laser. Select the appropriate % transmittance. Why does one usually keep the 488 line around 10-20% while the other lines are often run at 100%? The 488 line is much stronger than the other lines. Why is there no excitation filter? The laser is so specific for wavelength that no excitation filter is needed. 28

29 BC-Single Track-FITC only-detection Now we re going to skip to the end of the light path to turn on the appropriate channel detector. tickbox Channel 1 Channel 2 Channel 3 There are three different detector channels available for a epifluorescent signal plus one for a brightfield/dic image (ChD). Check the tickbox to activate the appropriate detector. Click on the Ch1 button to select a color. Select the color and then select Close. Why do you need to select a rectangular color square for the detector? The photomultiplier tube (PMT)/detector is not a color detector so you need to pseudocolor your image. DIC Channel 29

30 BC-Single Track-FITC only-detection Now we re going to pick the appropriate emission filter. The detector (photomultiplier tube) measures only light intensity, not wavelength. To make your detector (channel) color/wavelength-specific, you need to put an emission filter in front of it. To do this, click on the emission filter button to reveal a drop down list of available emission filters. Is LP505 the best option for FITC (emission 519 nm)? Yes, if your specimen has very little emission other than for FITC. We ll talk about other options in a few slides. Emission filter button Drop down list of available filters LP XXX longpass filter. Will let all light of a wavelength longer then xxx (here 530nm) through and block all light of a shorter wavelength. 30

31 BC-Single Track-FITC only-detection How do you decide which detector to use? Not all filters are available for all channels, so your filter choices will determine which channel you use. What might make channel 1 better than channel 2 other than the emission filter selection? The path to Channel 1 has fewer optical elements than for channel 2 or 3 and so less light is lost there. Ch1 Emission filters Ch2 Emission filters Ch3 Emission filters 31

32 BC-Single Track-FITC only-between Now we re going to direct the excitation light toward the sample and the emitted light toward the detection channels by selecting primary beamsplitters. Excitation and Detection Primary beamsplitter button The Primary beam splitter is next in the path after the laser. The primary beam splitter, HFT xxx(/yyy/ ), deflects the indicated laser lines but lets all other wavelength pass through. It is used to deflect the laser onto the specimen and allows the emitted fluorescent light to pass through. Select the primary beamsplitter button to see the available beamsplitters. For FITC, you will use HFT 488. What would HFT 458/543 be used for? It would reflect the 458 and 543 nm lines toward the sample and let all other wavelengths (including light emitted from the specimen) pass toward the detector. 32

33 BC-Single Track-FITC only-between Now we re going to direct the emitted light to the various detection channels by selecting secondary beamsplitters or mirrors. Excitation and Detection The next position in the path determines whether the emission is sent to Ch1 or to Ch2 and 3. There are four types of options: 1) A secondary beam splitter behaves like a long-pass filter for the light longer than the cutoff wavelength and like a mirror for the shorter wavelengths. This will send the shorter wavelengths toward where? Ch2 & 3 2) None nothing in the path, the beam will travel on straight 3) Plate lets beam travel straight like None but has elements which insure correct beam guidance, will be set by the program when necessary. 4) Mirror deflects light of all wavelengths. Use to guide the beam to Ch2 & 3. 33

34 BC-Single Track-FITC only-between Excitation and Detection So what about the remaining buttons in the paths? Set by the software The button that is just before the Ch1 emission filter button is always set by the software. What does the button just prior to Ch2 and Ch3 emission filters do? This button allows one to deflect all the emission to Ch2 with a mirror, send all emission to Ch3 with a plate or split the emission with a secondary beamsplitter. 34

35 BC-Compare FITC configurations Here are the two prestored configurations for FITC. How do you decide which configuration to use? If you are only staining with FITC, then use the FITC(cy2) setting. It uses channel 1 with the long pass filter to gather the most emission. If your sample fluorescences at a longer wavelength because of autofluorescence or perhaps because it is labeled with rhodamine, then you use FITC(narrow band) setting. It uses the bandpass filter. How do you decide which bandpass emission filter to use for sending FITC emission to Ch2? The options are shown to the right. Use the wider one (BP ) for more sensitivity. If there is bleed through of the rhodamine emission into the FITC channel, then you will need to try the more narrow filter (BP ). 35

36 BC-Single Track versus Multitrack Why do we never want to see someone with this configuration? Because if you are using more than one dye, you should be using the multitrack configuration in order to minimize bleedthrough of one dye into another dye s channel. 36

37 BC-How to build a multitrack configuration Easy way to get two tracks: Load a single track configuration for the dyes (e.g.,fitc/rhodamine) in track 1 and in track 2. In track 1, have only laser and detector for FITC switched on. In track 2, have only laser and detector for rhodamine switched on. The beamsplitter must be the same between tracks because there is not enough time to move them. The only thing that there is enough time to change is which laser is blocked. Let s practice by setting up a configuration for doing a rhodamine and FITC-labeled specimen. 37

38 BC Multitrack Click the Config button under the Acquire menu. Because you are doing more than one dye, select Multitrack. There are no presaved multitrack configs in a new account. After you have built and saved multitrack configs, you can access them by clicking on config button. To view preset configurations, click on the downward arrow. After selecting one preset configuration CHOOSE Apply. Then, select Close. 38

39 BC Multitrack To setup a multitrack, you first need to decide whether to switch tracks after each Frame or each Line. Tissue sections seem to have brighter images if one switches between frames while cells do fine when switching between lines; Zeiss acknowledges this but can t explain it. Switching between lines is easier to use. Start by switching between lines and try switching between tracks only if you have trouble. Select the appropriate button. 39

40 BC Multitrack An easy way to get multiple tracks is to load the multiple dye config for the single tracks into both track 1 and track 2. Then, modify each track to be specific for one dye. We ll do fitc and rhodmine as an example. Start by selecting the Store/Apply Single track button. Select the down arrow and then click on the desired multiple dye config (here: FITC/RHODAMINE). Select apply and then close to put this config. In the first track. 40

41 Now, the first track should have the fitc/rhodamine config. BC Multitrack Click Add Track to get a second track. Click Store/Apply Single track to apply another copy of the FITC/RHODAMINE config into the 2 nd track. Select the down arrow and then click on the desired multiple dye config (here: FITC/RHODAMINE). Select Apply and then Close to put this config. In the 2nd track. 41

42 BC Multitrack Both tracks are setup to do both dyes. We need to get rid of one dye in each track. You can slide the right edge of the Name column to the right to reveal the entire name of each track. Click twice slowly on the name of the first track and erase the FITC/ part of the name. Click twice slowly on the name of the second track and erase the /RHODAMINE part of the name. 42

43 BC Multitrack Now that we ve changed the names, let s change the configs to match. Select the Rhodamine track by clicking on it. Deselect the Ch2 detector box. Select Excitation. Deselect the 488nm laser. 43

44 BC Multitrack Now let s change the FITC configs to match its name. Select the FITC track by clicking on it. Deselect the Ch2 detector box. Select Excitation. Deselect the 543nm laser. 44

45 BC Multitrack Now, check the emission filters on each track to make sure that they are what you want. (e.g., You may want a wider band pass filter for the FITC channel) To save this multitrack config, select Config on the configuration control dialog box. Type a new name in the space next to Configurations. Click Store AND ONLY THEN click Close. If you Close before Storing, the config won t t be saved. Type new name here 45

46 Beampath Config: Notes for Do-it- Yourself Configs You can build each track from scratch. The beamsplitter must be the same between tracks because there is not enough time to move them. The only thing that there is enough time to change is which laser is blocked. Modifying a track that was loaded using the Store/Apply Single Track button can save you time. When you are done, trace each path to make sure that you have things set up correctly. Not all emission filters are available at all channels. You will probably have to put your longest wavelength dye on CH1. Ch1 is the path with the least glass/most sensitivity of the three fluorescent channels. 46

47 Image Acquisition Set Up I Click the Scan button under the Acquire menu and the scan control window will open. Make sure the window indicates Mode and that Frame scan is clicked. Click the Find button and the computer will approximate levels and generate a starting image. 47

48 Image Acquisition Set Up II The Mode window allows you to adjust digital resolution scan speed scan direction, pixel depth,, scan averaging, zoom/rotation When you first start learning the confocal, you should try out these settings to see how they affect your image. 48

49 Image Acquisition Set Up III resolution file size print size at 300 dpi 512x kb 1.7 (4.3cm) 1024x MB 3.4 (8.7cm) 2048x MB 6.8 (17..3cm) Choose the number of pixels for your image. Use at least 1024x1024 for a final image. You can also choose your own value. The value for x must be a multiple of 4, but any value between 1 and 2048 pixel is acceptable for y is the maximum value for x as well as y. Use 8 bit image (256 gray values). Use unidirectional scan for a single image. Bidirectional scan can reduce the scanning time (but not photobleaching) e.g. while scanning z-stack. The alignment is not always correct, so check if works for your image before scanning a stack. Ask for help if you have problems with it. Use Line for Mode. For averaging Method, use mean. Use only for final images. Its use will increase photobleaching, and it will take longer to scan. Average 2-4 images. Averaging more than 4 images will only give a very 49 slight improvement. Scan speed: With a slower scan speed, more photons will be measured by the detector (lower noise, better signal), but your sample will also bleach more. The recommended start value is 7.

50 Image Acquisition Set Up IV You can zoom into the image. This is a true zoom (the photodetectors scan a smaller area but keep number of pixels). However, if you use the zoom to magnify the image beyond the resolving power of the microscope, you will produce empty magnification no spatial detail is added and your sample will bleached more than necessary because it takes longer to scan. The maximum resolution of the light microscope is about 0.2 um. Sampling theory (Nyquist theorem) says you should sample at least twice your maximum frequency. Further increase in sampling will not increase the spatial information much, but will contribute to bleaching your sample. So the pixel size of your image should not be much below 0.1um. You can check the pixel size if you click on info on the right menu bar next to your scanned image. This will give you the pixel size for this image in microns. In addition to zoom, you can also rotate your scanned image area or choose an area not in the center. It is recommended to use the center as much as possible, as the quality of all lenses 50 is best in the center and image aberrations are smallest there.

51 Image Acquisition Set Up V Click on the Channels button of the Scan Control window. This window gives you control over pinhole diameter (thickness of optical slice) brightness and contrast of your image (gain and offset) laser intensity. By clicking the Ch1 or Ch2 buttons, you can choose which laser line you wish to adjust. Always adjust both lines before making your final scan. Pinhole: the pinhole diameter determines the thickness of the optical section you will look at. By increasing the pinhole diameter/optical section, you are also including more of the out of focus regions. Try to use 1 Airy Unit to start with. 51

52 Image Acquisition Set Up VI Detector gain: controls the sensitivity of the detector and will increase or decrease the brightness of your image. So if you have a dim sample you can increase the detector gain and boost the intensity of the light detected in your sample better than opening the pinhole so always try this first. This is a gain setting and will amplify your signal including noise if set too high. Ampl. Offset: will adjust the black level, or the background so it affects the image contrast of your sample. If the black level is too high, you may be blocking out some of your signal so use sparingly in most cases. Ampl. Gain: this will boost your signal by increasing the overall intensity of the collection. Because of this you also will increase the noise in your collected image very bad. Always try to keep the gain as low as possible. 52

53 Image Acquisition Set Up VII Excitation: controls the intensity of the excitation lasers. The HeNe lasers can be boosted to 100% without any problems. The argon laser is not usually used over 30%. If the laser is too high, your sample will bleach. If your signal is too dim, try adjusting the detector gain or try using a broad band filter during collection if increasing the laser is bleaching the sample. 53

54 Why is the Crop button evil? When you use the crop button to zoom in on a sample, you often oversample. Here the value for a single pixel in the XY dimension is 10 nm. Pretty small considering the theoretical resolution is only 200 nm. And oh yeah, the far red dye (pseudocolored blue) faded. 54

55 Warning Once you have achieved an image that you are happy with, save it. There is no overwrite protection in the software so if you have an image you like and you do not open a new scan window, the software will scan over your last image and it will be lost after once or twice you will remember to save as you go. If you have a double-labeled sample, scan it using the multi-tracking option to prevent bleedthrough. We can help you set this up. If you plan on doing any quantitative confocal measurements, please see us first. 55

56 Scanning Your Image If you used the Find button to acquire an initial image, you will need to use the described controls on the scan window to optimize your image. Your goal is to produce an image which represents the way your sample looks not the way you want it to look. This takes practice. First click Fast XY and using the fine focus on the microscope, focus up and down through your sample. Find the brightest area of your sample then click Stop. start image brightest image previous image with adjusted pinhole/gain 56

57 Single and Split Views If you have more than one channel, it will be much easier to adjust each if you view them in the split screen configuration. Click on split xy in the menu bar of the image. The rangefinder palette, which is describe on the next page, will not work for two channels shown in a single screen. Switch between your channels in the channels setting menu. red channe l green channel overlay of both 57

58 Optimizing your image Click Palette on the image window tool bar and the Color Palette window will open. Choose Range Indicator and close the window. Now, you will have an image where red represents the white saturated (overexposed) pixels and the blue represents the black saturated (underexposed) pixels. Click Fast XY on the scan window and change Pinhole, Detector Gain and Ampl. Offset to adjust your image to have just a few red and a few blue pixels. Make sure you do this for each channel...red, green and transmitted light if you are using it. Click Stop. Choose Palette and then No Palette to see your image. 58

59 Adjusting for Your Final Image Once you are satisfied with the range of saturation in all channels, you can create your final image. Go back to the scan control window by clicking the mode button. Set the resolution to at least 1024x1024. You can reduce noise by averaging your scan. Usually you will do a line average with a mean calculation and a average number of 2. After making the changes you want click single. Repeat the above steps as needed to obtain the image you want. Remember that you may only have a couple of tries at this before your sample fades so be careful and keep track of how your scan time changes as you change parameters. 59

60 Adjusting for Your Final Image Here is the difference in the initial image, the final image and a view of the individual channels. 60

61 Saving Your Image When you are satisfied with your image, you need to save it to your database. Press Save As in the image window and the Save window will open. Choose your database, or create a new database by clicking the new MDB button. Name the file and include any other information in the Description or Notes sections. 61

62 Image Viewing in Your Lab If you would like to view your images on your own computer, you must download the Zeiss image browser from this website: The downloads are at the bottom of the page. There are instructions for using this browser later in this tutorial. The Zeiss software does not have a Macintosh version. Macintosh users can view images (but not databases) using ImageJ with an additional plugin for viewing lsm images. 62

63 Shutting Down the System I Click the Laser icon and turn off the lasers. The HeNe lasers do not require cooling. After the Argon laser is turned off, you will need to wait about 5 minutes for the fan to cool the laser. When the laser is cooled, you will hear the fan go off. During the 5 minutes, you can switch the computer off, but not the main power switch (remote control). If somebody else is signed up for the confocal within the next hour, leave the argon laser on standby and leave the mercury lamp on. Log off the computer, but leave the computer and the main system on. 63

64 Shutting Down the System II While you are waiting for the Argon laser to cool, you can exit the Zeiss LSM software by clicking the X in the right corner of the LSM 510 menu bar. A message will come up reminding you not to power down the system until the laser is cool. Click OK. After exiting the Zeiss software, burn your data to a CD. Data left on the computer will be deleted after one month. Choose the LOGOUT option. This will log you out of the system. If you do not logout, the system will continue to charge time to your account. Next, go to Start and Shut Down to shutdown the computer once the computer is safe to be shut down, turn off the remote control only after the argon laser has cooled down (ca. 5 mins). Turn off the microscope and leave the 10x objective in place. Make sure you have cleaned off any oil with lens paper and glass cleaner dry wipe, wet wipe, dry wipe. Cover the scope but avoid covering the lamp housing. Turn off the Hg lamp under the air table. Shut off the nitrogen tank. You are finished thank you for your careful cooperation. 64

65 Z Stack Acquisition Select the Z Stack button on the Scan control Dialog box. Select the Mark First/Last tab. This tab will allow you to tell the software the starting and stopping point for you stack (more on that on the next page). Then, you will define the interval. If you turn the right focus knob clockwise, the stage will raise and you will be looking deeper into your sample. The focus motor will be more accurate if it is working against a load. So, make the first slice the one with the stage as low as needed to see the most shallow 65 part of your sample.

66 Z Stack Acquisition Use Fast xy to view your sample while adjusting the right focus knob counterclockwise. When you reach the most shallow part of your sample, stop scanning. Then, click the Mark First button. Then, scan while turning the right focus knob clockwise until you get to the deepest part of your sample. Stop scanning and click the Mark Last button. The Z stack size is shown. 66

67 Z Stack Acquisition In order to set the interval, click on the Z slice button. Missed info Proper sampling The diagram will indicates the result of the current 1 um interval. Since the optical sections are only 0.8 um thick, this interval would miss info. If you click the Optimal Interval button, the interval will be set a about half the optical section thickness. Why do you suppose that is considered optimal? By sampling each part of the tissue twice, you are following the Nyquist theorem recommendations to avoid omission and oversampling. Select Close on the Optical Slice 67 dialog box

68 Z Stack Acquisition Now, check Num Slices and make sure that it is not too high. Click on Mode and check the Scan Time for each slice. This takes into account averaging and scan speed. The product of this scan time and the number of slices will be how long you have for coffee. If the total time is acceptable, click Start. 68

69 Z Stack Acquisition Options for stacks that are too large Reselect the first and last slice to have a shorter stack. Use region of interest to capture the most important part of your image. Use the dual direction scan to decrease the scan time. Selecting a Region of Interest (ROI) Temporarily, deselect Z stack. You will not lose your settings. Use Fast XY to scan while adjusting focus until you get to the widest slice through desired area. Then, select Single to capture a image at final resolution. Why not define a ROI on a Fast XY image? If you try to select a ROI on a Fast XY scan, the ROI will not be in the correct 69 position when you do the stack, unless your final resolution is 512x512.

70 Z Stack Acquisition Select Acquire on the first row of the main menu. Then select Edit ROI on the second row. Select the square tool. Make sure that the box is clicked next to Fit Frame Size to Bounding Do NOT select Close for the Edit ROI box Draw the region on your image. 70

71 Z Stack Acquisition You may want to scan with Fast XY while adjusting the focus to make sure that the ROI is a good size for all the slices in your stack. You will be asked if you want to scan the whole image area. Select Yes. If you select Mode, you will see that the Scan Time has decreased. Now, reselect the Z stack button. Select Start to acquire the stack. What danger exists if you do not save the stack right after acquisition? You will write over the top of the stack if you start a new scan without opening 71 a new window.

72 Z Stack Acquisition Another way to save time is to do bidirectional scanning. Select the Mode button. Select the reversing arrow button. You might want to select Auto to let the software adjust the scan. Bad If the scan is misaligned, you will get bad data. Here, the red and green images are shifted with respect to one another. I avoid using bidirectional scanning because I do not know how to tell if it is misaligned. If may be possible to get more guidance from Zeiss. 72

73 Z Line Acquisition Another way to save time is to do a line scan in Z. This will yield a stack that is one pixel thick, has the same Z depth, and is as wide as your line is long. After setting up as if you were going to take a traditional stack, select the Line button. Then, select the Line Sel button. 73

74 Z Line Acquisition When you select the, Line Sel button, an image will be freshly scanned to allow you to draw the line. Draw a new line where you would like the Z line scan to occur. If you select Mode, you can see that the Scan Time is quite short. Newly drawn line Then select Start. 74

75 Drawn line Z Line Acquisition The resulting linescan is a series of images, each a Z single pixel wide, at each depth along the drawn line. It is as if you sliced the sample along the drawn line and viewed the cut edge. Save the image to your database. Lateral (XY) dimension versus Z Lateral Dimension 75

76 Z Line Acquisition If you then select Line Sel to do the next line or if you go back to Frame scanning, you should understand that the zoom, rotation, and offset have been changed. I usually, hit Mode & Reset, and then reenter the zoom that I was using. If you fail to fix this you could end up over or undersampling the X and Y dimensions. Remember, every objective has a combination of zoom and frame size that yields the best resolution. 76

77 Z Line Acquisition Depressing the Fast Z Line and then Start allows you to get a Z scan in less time than without depressing the Fast Z line button. I cannot find this function in the Help manual and would not use it for a final image but it might help you adjust gain and other acquisition parameters before a final line scan. 77

78 Trace the DIC pathway when the ocular lenses are used 78

79 Koehler (or Köhler) Illumination Koehler illumination occurs when the condenser and field iris are positioned to optimally illuminate the specimen. Koehler illumination needs to be done for brightfield, darkfield, phase contrast, and DIC. First step: Choose the appropriate objective and focus on the specimen. If the next steps are done without the specimen being focused, then Koehler illumination will not be achieved. Koehler illumination must be rechecked if a different 79 objective is used.

80 Filters Light intensity can also be adjusted by dialing to select the neutral density (ND) filters on the right side of the base of the scope. The greater the value on the dial, the more light that will be passed. Set both dials to 100% for DIC but be careful not to hurt your eyes when you look through the oculars. The Green Interference Filter (GIF) enhances contrast for phase contrast microscopy. The CB filter is used with samples that have been stained with brightfield dyes. 80

81 Do not hurt your eyes With the nd filters pass 100%, you may need to lower the intensity of the halogen bulb. 81

82 The Condenser The condenser is located under the stage. It focuses the light onto the specimen. Its numerical aperture factors into the determination of resolution. Make sure the top lens is in place. The top lens lever should be all the way forward. The top lens can get oil on it and yield a poor DIC image. Top lens lever 82

83 Koehler Illumination- You must rotate the dial on the multipurpose condenser to III in order to set the condenser for DIC with any of the objectives that we have. III will let you do DIC with objectives that have an NA of ~0.7 or higher. Selecting III places the appropriate polarizer and a prism under 83 the sample.

84 Koehler Illumination Adjust the condenser to its highest position using the condenser focus knob on either side of the microscope. 84

85 Koehler Illumination - Close Field Iris To adjust the field iris, use the dial that is on the lower right of the scope. Close the field iris. If the condenser is not too badly aligned, you should see the edges of the leaves of the iris. Open Iris Closed Iris 85

86 Focus the field iris Adjust the condenser so that the field iris is in sharp focus using the condenser focus knob on either side of the microscope. Unfocused Iris Focused Iris 86

87 Koehler Illumination - Center the Condenser The field iris may be off center when view through the ocular lenses. Centering is easier if you open the iris so that its edge almost reaches the edge of the field of view. Center the image of the iris with the condenser centering screws that are located on either side, and just under, the condenser (Only the left screw is shown). When you are done centering, open the iris until it is just outside the field of view. 87

88 The condenser iris controls a balance between resolution and contrast Adjust the slider on the condenser to change the size of the condenser iris. The size of the condenser iris affects resolution and contrast. View the image through the ocular lenses while changing the slider to choose the best balance between resolution and contrast for your specimen. increase resolution increase contrast 88

89 Optimizing the DIC optics Once you set up Kohler illumination on the objective that you are going to use, it will be set up for confocal The first step to change the brightfield pathway into a DIC pathway for the ocular lenses is to push the polarizer into the path Polarizer is out of the light path Polarizer is in the light path 89

90 Toggling between Hg and Halogen lamps. I find it easiest to use the toggle button on the scope to go between HG and Hal. Lamps. To enable this function, select the Micro button on the main menu bar. Select the Transmitted Light button. The Remote button will be pushed in. Click on it so that it is not pushed in. The toggle switch will now work. 90

91 Optimizing the DIC optics The optimal position for the sliding prism may be different for ocular and confocal uses. So, set it up using the oculars but be prepared to adjust when viewed on the confocal. The slider prism is located just above the objective. Rotate the knob to affect the amount of 3D effect you want in your image. The prism after removal from the scope. Let us do this if it is needed. 91

92 Adjusting the DIC with the Prism Rotate the prism knob to adjust the degree to which objects appear raised, depressed, or even with respect to the background. You will want to adjust the slider (prism) while viewing the image on the confocal because the effect will be different from what is viewed through the oculars. Too little of the 3D effect Cells seem raised Cells seem depressed) 92

93 Optimizing the DIC optics Turning the prism knob will affect the intensity of the image and you will have to compensate for that by adjusting the intensity knob (if looking through the oculars) or the gain, etc (if looking with the confocal) Too bright-range indicator Too bright-no palette 93

94 Optimizing the DIC optics I often want to readjust the condenser iris (contrast) after changing the prism. Changing the condenser iris will affect the intensity of the image and you will have to compensate for that by adjusting the intensity knob (if looking through the oculars) or the gain, etc (if looking with the confocal) I often have the iris nearly closed (to the right) to have maximal contrast. increase resolution increase contrast 94

95 Trace the DIC pathway when the confocal is used Where are the PMTs for the fluorescent channels? In the box on top of the scope PMT for DIC 95

96 The polarizer can greatly diminish fluorescence Remove the polarizer if you use fluorescent light with the ocular lenses. Remove the polarizer when you plan to view the specimen with the confocal. Polarizer OUT Polarizer IN Stuff looks unstained 96

97 Remove the polarizer when you use the confocal Polarizer is out of the light path What does this tell you about the laser light if one can remove the polarizer and still get a DIC image on the confocal? The laser is already polarized. 97

98 DIC - configuration To add DIC to the configuration, click the box next to channel ChD. I have not heard of a rationale for adding DIC to one track or another. I add the DIC to the track of the most photostable dye so that the additional scans required to set up the DIC are less likely to cause fading. You can add DIC to a single track, too. I often turn off the DIC channel when doing Z stacks to save time and memory. As a separate image, I acquire one DIC and fluorescent images near the middle of the stack. 98

99 Adjust the Channel settings for DIC 99

100 Channel settings for DIC There is no pinhole aperture for DIC. Too much contrast for most uses if some pixels are near zero (blue) The DIC image will have an optical section thickness of some unknown amount and is not necessarily the same thickness as for the fluorescent channels. I adjust the gain until something in the images nearly maxes out. I do not change the amplifier offset so that there is some pixels near zero because this makes the image have too much contrast. Better contrast 100

101 Adjusting the DIC The uneven illumination is an inherent problem with DIC. If you are planning to crop the image, you can put the part you care about the most in the center of the image. You may want to slide the condenser aperture to the left to decrease the contrast so the uneven illumination is less noticeable. 101

102 Adjusting the DIC There are some dust spots that we have not been able to get rid of. If they are bothering you, you will need to put the area of interest in a part of the field that will allow you to crop out the dust. We are also working with Zeiss to get rid of the dust. 102

103 A slower scan speed may reduce lines in DIC image Scan Speed μs/pixel Scan Speed μs/pixel Scan Speed μs/pixel 103

104 Removing DIC from the overlay The DIC images makes it hard to see colocalization in the overlay. You can remove the DIC component from the overlay by clicking on the channel button. Then, click on the ChD button and select the OFF button in the resulting dialog box. Dialog box for channel colors 104

105 Appendix Things that may be good to know 105

106 The Confocal Principle monochromatic point light source - defined laser lines as excitation light source pinhole excludes out of focus light (decrease of blur) optical sectioning (non-invasive) bleaching only in scanned region (though all of your sample of that area will get bleached, not only what is in focus) non-confocal confocal 106 images from Zeiss

107 Absorption and Emission Light exiting a fluorophore is always of a shorter wavelength than the light emitted by the fluorophore. 107

108 Absorption and Emission examples of exitation (dashed) and emission (continuous line) spectra of three different fluorescent dyes with their appropriate laser lines. Spectra from the fluorescent spectra viewer from Molecular Probes ( 108

109 Common Fluorochrome Families Excitation Emission echnical/f-cy3-5.asp The DAPI-like dyes Ex~345nm; Em ~455: AMCA, Hoechst (Can t do on our confocal) The Fitc-like dyes Ex~488; Em~520: Alexa 488, Oregon Green, YOYO-1 (DNA), Cy2, FITC, egfp (Use the 488 line on our confocal) The Rhodamine-like dyes Ex:~ ; Em~590: Alexa 546, Cy3, Alexa 568, Rhodamine, TRITC, POPO-3 (DNA), Rhodamine X (EM 576), Propidium Iodide (DNA em 617) (Use the 543 nm line on our confocal) The Texas Red-like dyes Ex~596; Em~620: Texas Red, Alexa 594 (Use the 543 nm line on our confocal) The Far Red Dyes Ex~650; Em~670: Cy5, Alexa 633, Alexa 647, TOTO-3(DNA), Draq5 (DNA) (Use the 633nm line on our confocal) Other dyes that work on our confocal: CFP (use the 458 nm line), YFP (use the 514 nm line) 109

110 Absorption and Emisson of some fluorescence dyes ex em Alexa Fluor Alexa Fluor Alexa Fluor Alexa Fluor Alexa Fluor Alexa Fluor Alexa Fluor Alexa Fluor Alexa Fluor ex em Fluorescein X-Rhodamine Texas Red ex em Cy Cy3 (512); ;(615) Cy ;(640) Cy5 (625); Cy

111 Choice of colors Two-channel pictures (fluorescent double staining, DNA chips) usually use red and green to show each channel. This causes two problems for red-green blind people. 1. Impossible to understand which part is labeled with green and which part with red. 2: Impossible to distinguish the region of colocalization. For colorblind people, yellow (red plus green) looks the same as bright green. Simulation of how colorblinds see this picture. Suggested solution: Substitute Magenta for Red. Magenta is the equal mixture of red and blue. Although colorblind people have difficulty recognizing the red component, they can easily recognize the blue hue. The region of double positive becomes white, which is easily distinguishable for colorblinds. from: by Masataka Okabe and Kei Ito 111

112 Beampath Configuration none nothing in the path, the beam will travel on straight plate lets beam travel straight like none but has elements which insure correct beam guidance, will be set by the program when necessary. Mirror deflects light of all wavelength. Use to guide the beam to selected detectors LP xxx longpass filter. Will let all light of a wavelengh larger then xxx (here 530nm) through and block all light of a shorter wavelength. Used to determine specifity of detector. BP xxx-yyy bandpass filter. Will let light within the range specified (here nm) though and block all light of a different wavelength. Used to determine specifity of detector. 112

113 Beampath Configuration NFT xxx (secondary beam splitter) light with shorter wavelength then xxx (here 570nm) is deflected, light with a longer wavelength passes unaffected. It is used to direct emitted fluorescent light to different detectors. HFT xxx(/yyy/ ). (primary beam splitter) deflects the indicated laser lines (here 458 and 545) but lets all other wavelength pass through. It is used to deflect the laser onto the specimen and allows the emitted fluorescent light to pass through. Detector. There are three channels available for a fluorescent signal plus one for a brightfield/dic image (ChD). Check tickbox to activate detector. The detector sees only light intensity, not wavelength, choose the proper beam splitters and filters to make it wavelength specific. It also means that the color you see is a pseudocolor which you can define (click on color field to open 113 window). Choose any which you find useful.

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