TRAINING MANUAL. Olympus FV1000

TRAINING MANUAL Olympus FV1000 September 2014

TABLE OF CONTENTS A. Start-Up Procedure... 1 B. Visual Observation under the Microscope... 1 C. Image Acquisition... 4 A brief Overview of the Settings... 4 Image Acquisition for Single dye... 6 Image Acquisition for Double and Triple labelled samples... 8 Image Acquisition for samples labelled with more than 3 fluorophores... 9 Adding the DIC or Bright Field Channel... 10 Z-stacks... 11 D. Reload the Image Parameters... 13 E. Improving your Image... 13 F. Other Important Functions...17 G. Shut Down Procedure... 18 H. Appendix I : Tile Images... 19 OlympusTraining Manual

A. Start-Up Procedure 1. Turn on the mercury burner (#1) 3 2. Turn the Microscope Box on (#2) 3. Turn the Scan Head box on (#3) 4. Turn the Stage box on - back right (#4) 1 2 4 7 5. Turn the Diode laser power switch on (it has to be on even if you are not using this laser) 6. Turn the Argon laser power switch and key on 5 6 7. Turn the HeNe1 laser key on (this is the 543 laser) 8. Turn the computer on and login 9. Double click in the software icon and login again B. Visual Observation under the Microscope Focus Knob - there are 2 buttons right beside this knob: ESC = puts the objective all the way down Focus = toggles between fine and coarse mode for the focus focus up and down these buttons control the intensity of the transmitted light OlympusTraining Manual 1

B. Visual Observation under the Microscope continued... You can select an objective lens and a fluorescent filter cube by using the hand switch. You can also use the microscope controller box in the software to choose the objective. PS: if you can't find the microscope controller window, go to DEVICE in the main tab of the Olympus Fluoview software. Open the drop down menu and select "Microscope controller" Here are the specifications of the objectives in this system: Please note that the only oil objective in this system is the 60X. All the others are dry. To load your sample: 1- choose the objective you want to use 2- open the doors of the chamber 3- move the microscope's arm back (be careful not to go too far otherwise the condensor will hit the chamber) 4- lower the objective before loading your sample to avoid damaging the objective. Use the functions focus up and down in the microscope to do that 5- load your sample 6- move the stage around using the stage controller at your left side 7- move the microscope s arm back To focus your sample: 1-If you want to use transmitted light to focus your sample, select the transmitted light button in the image acquisition control window OlympusTraining Manual 2

B. Visual Observation under the Microscope continued... To focus your sample continued: 2- if you want to acquire a DIC image of your sample, make sure you match the DIC prism with the objective used. Position #3 = 20X Position #4 = 40X Position #5 = 63X Just rotate the prisms to put the right one in place You don't need to worry about this if you are only observing your sample 3- clockwise rotation of the focus knob will move the objective down and counter-clockwise will move it up. Right bellow, to the side, of the focus knob, there is a button to toggle between fine and coarse mode for the focus. 4- To control the intensity of the transmitted light, change the voltage settings in the software 5- if you want to focus your sample using fluorescence light, select the EPI lamp button in the image acquisition control window 6- now, select the filter cube you want to use (according to your fluorophore) in the hand switch controller (left side of the microscope) or in the microscope controller window 7- open the fluorescence light shutter to let the light comes through the objective (to open, move the slider to the left) 8- you can control the intensity of the light by opening or closing this shutter 9- use the hand switch controller to switch between different filters 10- to turn the light off, close the shutter or click the EPI lamp button again OlympusTraining Manual 3

C. Image Acquisition A brief overview of the settings: 1- Acquisition Setting, Data manager and Explorer Windows Scan mode (unidirectional and bidirectional) Number of pixels Scan speed (the speed the laser dwells in each pixel). As lower as better the image, however higher the bleaching. As a starting point you can use 4.0us/Pixel In the Area you can control the zoom and rotation of your Zoom in function Laser output adjustment The time scan tab controls the parameters for time lapse In the Microscope tab you can select the objective you want to use, set the parameters for Z stack and change the focus (bigger arrow heads are for coarse focus adjustment and fine arrow heads are for fine focus change) The data manager window contains all the image information. You can right click on the image to save it. Use the explorer window to find and organize your files OlympusTraining Manual 4

C. Image Acquisition continued... A brief overview of the settings continued: 2- Image Acquisition Control Window Transmitted light observation (for visual observation) Fluorescence observation (for visual observation) Scan buttons select depth for Z-stacks and time for time series Dye List Optical Path Diagram Scanner setting (for photobleaching) Adjustment for each channel (do not change the gain) Pinhole adjustment Image information (for pixel size and resolution Sequential mode (for up to 3 channels). For more than that use the virtual channel under the dye list 3-2D or 3D View window Kalman filter = for line and frame average Light intensity adjustment for halogen bulb OlympusTraining Manual In this window, you can merge the channels and reconstruct the Z-stack. You can also find information about the distribution of the fluorescence intensity of the pixels in your image (histogram) and change the colour of specific channels. If you can t visualize the channels separately, click on the arrow head (left corner of the window). A new window will open. Click on Ch to separate the channels and Z to visualize all the stacks in the gallery mode. 5

C. Image Acquisition continued... Image Acquisition for Single Dye: 1- Click on the Dyelist button at the Image Acquisition control window. 2- Choose your dye from the available list. If you already have a dye in the Selected Dyes area, make sure you click ALL CLEAR before adding new ones (unless you will use the same dye) 3- Double click on the dye to add to the Selected Dye tab 4- After adding all the desired dyes, click on APPLY 5- Close the Dyelist window To check and/or modify the light path configuration of your dye, click on the Light and Path button Here you will find all the options for setting the light path configurations. You can change the filters, mirrors and channels if you want. Please note that there's no spectral detectors in this system (no channel S). For a fast, low resolution scan, you can use the Focus x2 or Focus x4 scanning mode. If you want to use this mode to adjust your detector settings (HV and offset), you MUST select AutoHV in the Acquisition Setting windows. With this function selected, as the scan speed becomes slower (for the final image), the saturation level will be maintained. If you forget to select this function, when you press XY for the final image you will see that your image is saturated. As the scan speed is much slower in the final image, the laser spends more time in each pixel, acquiring more signal. If you don't have the auto HV to compensate for that your image will be saturated. Another option would be adjusting the settings of your image while scanning in the XY REPEAT mode. This mode uses the parameters that you set for the final image and it will reflect exactly how your final image will look like. OlympusTraining Manual 6

C. Image Acquisition continued... Image Acquisition for Single Dye continued: Adjust the sensitivity of the detector by changing the HV and offset values. The HV will change the brightness of your image and the offset will reduce the noise (background). Press keybord Ctrl+H key to visualize the dynamic range of the detector. The maximum intensity of your image is 4096 (12bit) image. Intensities above that is shown in red and intensities bellow 0 is shown in blue. If you are planning to quantify this image you want NO RED and SOME BLUE If your signal is too bright, you can also adjust the laser power in the Image acquisition control or in the acquistion setting windows DO NOT CHANGE THE GAIN. Leave it as 1. To stop the scanning, click STOP Before scan your final image (XY), make sure to define: a- the right scan time (you can start with 4us/pixel) b- the right aspect ratio (recommended: 1024X1024) c- the right zoom (see instructions on improving your image topic) OlympusTraining Manual 7

C. Image Acquisition continued... Image acquisition for double and triple labelled samples: If you have fluororphores that overlap, you MUST use sequential scan mode. 1-Go to the Dyelist panel and add all the fluorophores you want to image (even if they overlap) 2- In the Image Acquisition Control window, select sequential. You can choose to switch between tracks using line or frame mode. If you choose "line", all the lasers will illuminate your sample at the same time and the system will switch between groups line by line. You will visualize all channels almost at the same time. If you choose "frame", the lasers will illuminate your sample separately (each laser at a time) and the system will switch between groups after a full channel has been scanned. 3- To save time during acquisition, you can group the fluorophores that don't overlap to be scanned at the same time. For example, you can drag Alexa Fluor 633 from group 3 to group 1 to be scanned together with Alexa Fluor 488. Then you leave the overlaping fluorophore in a separate group. 1 2 3 OlympusTraining Manual 8

C. Image Acquisition continued... Image acquisition for double and triple labelled samples continued... 4-To adjust the HV and offset for each channel: a- if you have each fluorophore in a separate group, you can start scanning the image and adjust each channel b- if you have combined fluorophores groups, you MUST select the group before doing the adjustments. Otherwise you will keep changing the parameters and not seeing the effect until you select that group 6- After checking and adjusting all your parameters, click XY for the final sequential image Image acquisition for samples labelled with more than 3 fluorophores: As we only have 3 photodetectors in the system, you will have to create a virtual channel in order to use the same channel for 2 fluorophores. 1- Open the DyeList window and select all the fluorophores you want to use. 2- Now, click on Virtual channels scan on the DyeList window. The Virtual channel controller window is automatically open. 3- Each phase represents a sequential track. Drag the fluorophores to different phases, grouping the ones that don t overlap. 4- To visualize the fluorophores in each phase, you must select the phase on the Virtual Channel Controller. Once you select the phase, you can then adjust the parameter for the dyes in that phase. 5-After doing all the adjustments, click START to scan all the phases. ** If you are acquiring a Z-stack using virtual channels, make sure to check the parameter for the Z-stacks in all phases. They must be the same. OlympusTraining Manual 9

C. Image Acquisition continued... Image acquisition for samples labelled with more than 3 fluorophores continued... Phase 1" Phase 2" Adding the DIC or Bright Field Channel Please refer to page 3 for DIC prism. For regular bright field image, make sure to change the position of the prism slider to BF (position 1). 1- On the TD1 channel, select the laser that you want to use for this channel. It can be any of the laser lines. 2- Then, activate the TD1 and adjust the HV and offset parameters 3-Click on Fast2X or XY repeat to scan a preview image and adjust the parameters OlympusTraining Manual 10

C. Image Acquisition continued... Z-stacks Use the Microscope tab in the Acquisition Settings window to set the StepSize values, bottom and top of your image. 1- Hit clear start and end 2- Start scanning 3- Change the focus all the way down (clock wise in the focus knob). You can use the arrow head buttons to shift the focal point bigger moves 1.0um smaller move 0.1um 4- Once you find the lower limit (bottom) of your image, click SET 0 and END SET. This will be your 0um position 5- Now start changing the focus in the opposite direction (counter clockwise to go up or use arrowhead buttons). You can visualize how far you are going into the sample by looking at the depth 6- Once you find the upper limit of your image (top), click START SET 7- Press the Stop button to stop scanning 8- Enter the StepSize. Optimized values can be referred to by using the Op button. 6 to shift the focal point 5 1 4 OlympusTraining Manual 11

C. Image Acquisition continued... Z-stacks continued... 9- Adjust: a- scan speed b- aspect ratio c- zoom d- HV and offset for each channel 10- Select Depth on Image Acquisition control window 11- Click on XYZ button to scan the series 12- Click on SeriesDone. A 2D ViewLiveImage is displayed on the window bar for the series that have been acquired. To reconstruct the Z-series 1- Click on button to select the type of projection To save the display image, right-click on the image, select Save Display and save the image with a new name. OlympusTraining Manual 12

D. Reload the image parameters 1- Go to File and open an exiting image 2- Click on to open the 2D control Panel 3- Click on to reload the parameters E. Improving your image Proper Pixel Size: The best aspect ratio and zoom depend on the resolution you want to achieve. Open the Image Information window and check the optical resolution of the system for the conditions you have. The optical resolution value for X,Y and Z will change when you change the objective and the wavelength. The Pixel size is the size of each pixel in your image using the parameters that you set. According to the Nyquist sampling Theorem, in order to image all the details of an object the pixel size should be between 2 to 2.5 times smaller than the object. For example, if your optical resolution is 200nm, your pixel size should be between 75 to 100nm. If the pixels in your image are bigger than 100nm, you may lose details. If it is smaller than 75nm you will be over sampling without gaining additional information. You can change the pixel size by changing the zoom or the aspect ratio. Roughly you want your pixel size to be half of your optical resolution value. OlympusTraining Manual 13

E. Improving your image continued... Proper Pixel Size Nyquist Criterion requires a sampling frequency of at least 2x the highest spatial frequency to accurately preserve the spatial resolution in the resulting digital image Too few pixels - pixel size is too large for maximal resolution Too many pixels - too many pixels (oversampling) does not add spatial resolution Optimal # of pixels 2-2.5x the resolution limit Example: R=0.61 /NA = (0.61x488nm)/1.4 =~200nm R/2 = 100nm R/2.5 = 75nm Optimal pixel size is 75nm-100nm Pixel too big 300nm Pixel = resolution limit 200nm Pixel < resolution limit Optical Image 200nm 300nm 200nm 100nm 100nm 200nm Digital Image OlympusTraining Manual 14

To open this window, click on on the Image Acquisition window Resolution of the system based on objective and wavelength used Resolution using your parameters (should be about half of the optical resolution) 512 x 512 190nm pixel size Pixel too big 988 x 988 100 nm pixel size Pixel < resolution limit OlympusTraining Manual 15

E. Improving your image continued... Averaging The signal-to-noise ratio can be substantially improved by slowing the scan speed and averaging over several scans Average Averaging removes noise detected by the PMT. In general, the low the Gain is set to, the less sensitive the detector is and the less noise detected by the PMT. Therefore, a lower Gain setting requires less averaging. Line Average - each line the # of times indicated and then the average signal of each scan for each pixel is calculated Frame Average - the entire frame is scanned the # of times indicated and then the average signal of each scan for each pixel is calculated select your line average by clicking on "Kalman" under filter mode (Image acquisition window). Image acquisition is repeated to the specific number of times to provide an averaged image. This function helps reducing the noise and roughness of the image but it increases the scan time. OlympusTraining Manual 16

F. Other Important Functions Saving the image: Make sure to save it as OIB format (see File format bellow) To save your image you have 3 options: 1-Go to File - SAVE AS - select your folder in GONAD and save your image 2-close the image. When you do that, you will see a pop up window asking what to do with the image. Click on "Save and Close". Select your folder in GONAD and save your image 3-Right click on the image icon on the Data Manager window and select SAVE AS. select your folder in GONAD and save your image File Format: OIF = creates a folder which contains a 16-bit TIFF image and an "accessory file" with the image information OIB = creates a single file with the original image and information Zooming: If you want to image an specific region of your image, click on Zoom scan (Aquisition Setting window) and draw an ROI on your image. Check your pixel size to make sure it is still about half of your optical resolution. If its too small, you have 2 options: 1- decrease the zoom factor 2- decrease the aspect ratio: click on the Zoom scan again to reset the zoom - change the pixel format of your image (for example from 1024X1024 to 512X512) - scan the image again - select Zoom scan again and draw your ROI Changing the colour of the channels: 1-click on the LUT settings tab in the Live view window 2-select the channel you want to change the colour in the channel drop down bar 3-select the new colour OlympusTraining Manual 17

G. Shut Down Procedure 1- Save all your images 2- Before shutting everything down, check the online calendar to see if there is anybody coming after you. If the next user is coming within 1 hour after you, close the software, log off and leave the other components on. If the next user is coming with more than 1 hour after you, proceed with the shut down steps listed bellow. 3- Turn the keys in both lasers off. Wait until it the lasers cool down (about 5min) 4- Remove your sample, clean the objective and press ESC to bring the objective all the way down 5-Close the software and shut computer off 6-Close the shutter of the fluorescence lamp (all the way to the right) 7- Turn components 1-4 off 8- Close the doors of the microscope chamber 9- After 5 min cool down, turn both switches in the laser boxes off (one in the black middle box and the other in the lower gray box) 10- Sign the user sheet OlympusTraining Manual 18

H. APPENDIX I Tile Images: For Tile Images, you must first adjust the image parameters for your sample following the steps of this manual (light path configuration, laser power, HV, offset, zoom, pixel size and scan speed). Go to the main tab of the software - DEVICES - and open MULTI-AREA TIME LAPSE A warning window will pop-up. Please hit CANCEL. Two new windows will open: The Multi-Area Time Lapse Controller Window Go to OPTIONS - PREFERENCES Click on the Mosaic icon to open the Mosaic Outline window If the Registered Point List Window doesn t open automatically when you click on Multi-Area Time Lapse, then click on this icon Under INDEX, type how much overlap between the tiles you want (you can use any value from 90 to 100%). I have tested 95% and it worked well. You must do this step before registering the positions (unless you are not changing this value). OlympusTraining Manual 19

H. APPENDIX I Tile Images continued... To mark the positions, use the transmitted light or EPI lamp. Under the microscope, move your slide to the edge where your sample starts. Mark this position on the Mosaic Outline window. Move to the next edge and mark the position. You should do this until you form a square (4 positions) or a different geometric shape that includes all the edges of your sample. As you mark the positions, you can see geometric shape forming at the Multi-Area Time Lapse Controller Window. If all the positions are fine, click on ADD GROUP CLICK ON YES The positions you selected are now registered at the Registered Point List Window If you want to delete a position, highlight the position and click on the garbage bin icon. If you want to delete all the positions, click on NEW and don t save the old positions. OlympusTraining Manual 20

H. APPENDIX I Tile Images continued... Go to OPTIONS - FILE AND FOLDER Choose where you want to save your files. Suggestion: since the tile files are quite large, I suggest creating a folder on Desktop to save your images and then transferring to gonad after finishing. Please don t forget to remove your files from this computer. Define the name of the subfolder Before start the tile, go to Focus2X or XY REPEAT to make sure your sample is in focus and all the parameters are fine. Now, click on READY on the Registered Point List Window and Image Stitching If your file is too large, you may have to do this step in the another computer and even using another software (ImageJ). Go to DEVICES - MULTI-AREA TIME LAPSE VIEWER Open the MATL_Mosaic document Click on Actions - Stitch OlympusTraining Manual 21