Transcript
Index. Page 2. How to switch on and additional information Page 3. Opening and starting Micro-Manager Page 4. Desktop explanation and basic slide viewing. Explanation of Main window components Page 5. Initial scanning set up. Acquire single channel. Fast Live Mode to help with focussing (F8) Page 6 to page 7. Multi Channel Scans. Acquire-Focus Offset (F10) to get difficult multichannel images in focus. Saving scanning conditions. Page 8. Acquiring multiple channel images Page 9. Saving and adjusting of images after acquisition Page 10. Saving, adjusting, setting scale bar and converting to 8bit Page 11. AutoExposure and Acquire. Brightfield scans. Page 12. RGB acquisition. Page 13. Appendix. One time setup for Micro-Manager Page 14. One time setup for Micro-Manager. Options changes, syncing exposures Page 15. Fast launch setup. Switch light path from eyes to camera Page 16. Filter wheel Page 17 to Page 18. AutoStretch. Creating a shortcut to your data area Page 19. Creating a shortcut to your data area. Setting up autosave to this area Page 20. Setting up autosave to this area Page 21. Scalebar Page 22 to Page 23. Alternative ways to save data Page 24. Profile viewing of acquired image Page 25. Fast-Live mode (F6). Acquire-Focus Offset (F10) Page 26. Acquire-Focus Offset (F10)
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Micro-Manager for Limelight. Manual can be downloaded at http://www.igmm-imaging.com/equipment/Epifluorescence
Do not use the microscope unless you have been trained by a facility member If this is your first login on this machine, go to the Appendix at the back of this document to see initial, one-off changes you need to make to the software. (p13)
Please email if you have any questions;
[email protected]
Camera Shutter control
PC
Fluorescence source
Switch on the following; Turn on PC – Camera – on top of the camera (If required) and the power supply in the box to the side Fluorescence power source (If required) Filter wheel/Shutter control White light on right side of microscope (If required).
Any additional help can be obtained by pasting the following link into your favourite browser.
https://micro-manager.org/wiki/Micro-Manager_User's_Guide 2
Log in with University of Edinburgh username.
Micro-Manager is already installed on the PC but it is faster to create a shortcut launcher on the Taskbar to start it. (Explanation in pictures, page 15) Now click on the Micro-Manager icon to open the software, the following windows will open. Make sure that the Limelight config file has been loaded. (If not, go to p13 for instructions.) NB: A window may appear asking you to register, ignore by clicking ‘Later’ or ‘Never’ and carry on.
No config file loaded. Must be Limelight.cfg. Go to p13 for instructions
By clicking OK, Micro-Manager is fully launched and it will start communicating with all the peripherals; this means there will be some noise from the various filter wheels as the system registers. The resultant desktop will look like this, if it doesn’t then close the application and ensure that all the peripherals on page two of this document are SWITCHED ON.
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This box is ImageJ, which can be used for postacquisition features
Function keys controlling channels, acquisition and exposure
Main Micro-Manager window for acquiring images, lens setting and MDA
To view your slide, place your slide on the microscope stage, ensure that the light is going to the eyepieces (p15) , the correct filter wheel is selected (p16), select the objective required and then, using the function keys, select the channel you require, i.e. F3 for FITC in functions keys box (3 Colour) to check your slides. A Live window will be opened on the screen but will show nothing as there is no light going to the camera. Once you have found a something you wish to take an image of, move the light from the eyepieces to the camera (p15) and what you saw by eye will appear on the screen, assuming the exposure value is high enough. When not viewing the sample, press F1 to close the shutter (This will reduce the amount of bleaching of your sample as you setup on the desktop), The main window components are as labelled below.
Start/Stop live window
Multi-Dimensional Acquisition (MDA) Acquire more than one channel simultaneously
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Exposure value for channel selected
Channel selected Gain – can be used to increase or decrease exposure time Set lens used to acquire – scalebar can be set
Initial scanning set up.
Scanning timeline Mount slide Click Live or Click Channel i.e. FITC(F3) Live window opens Click AutoExpose (F9) to get exposure value Click Acquire (F11) to acquire image Click on Save icon, save as image stack 6. Click on Acquire (F11)
1. Click channel to see specimen on screen, i.e F3
4. Exposure value displayed. Can be manually changed
2. Live window opens
3. Click on AutoExpose(F9) to get non-saturated exposure value
7. Save 16bit, single channel image
5. After AutoExpose, window is no longer live. Click live to see changes/focus/etc
If happy with the image and focus, and only a single channel is required, then the Live window button can be clicked (No longer live) and then the save icon clicked to save the image. Alternatively, clicking Acquire (F11) can be clicked instead, creating a new image window which can be saved. Save the image as an Image Stack, a folder will be created with the name given and a 16Bit Tiff file within. Other save options are on p22. Very pale samples, with long exposures making focusing difficult, can be focused more easily using Fast Live Mode (F8). Instructions are on page 25. 5
Multiple Channel Scans. For multiple channels, these need to be selected before scanning can proceed. This is done by clicking on Multi-D Acq in the Micro-Manager Main window. By clicking on this, a new window appears. In this new window, the channels required can be chosen to reflect your experiment needs. As can be seen, there are no channels in the window so to start this process, first click on the ‘Channels’ box and then click on the ‘New’ button to add channels. Do this as many times as required, in this case 3.
3. Click on New to add channels to acquire. 1. Multi-Dimensional Acquisition (MDA)
2. Click on Channels and Channel in Channel Group box. As can be seen in the Exposure column, any exposures set with AutoExposure, or manually, are displayed here as well. To remove a channel from the list of those to be scanned, highlight/click on the row of that particular one and click ‘Remove’.
Unwanted channels can be removed by clicking on that line and clicking Remove
Exposure selected before are displayed
Exposure synchronised with main window (See p.14) If, when focusing on each channel, there is difficulty in getting each channel on the same focal plane, then click on F10 (Acquire-Focus Offset). For an explanation, go to page 26 of the manual.
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If the correct channel does not appear, simply clicking on a non-required channel in the Configuration column, choosing the correct channel from the list will correct this. If the colour of the chosen channel is wrong, simply click on the appropriate coloured box, choose the correct colour and click OK.
1. Select channel by clicking on box and selecting from the list
2. To change colour of final acquired image, click box and select from RGB tab in new window.
With the channels selected, the colours chosen, and the exposures set, these settings can be saved for re-using at a later date, especially if comparative image acquisition is being done. Simply click on Save as… in the Multi-Dimensional Acquisition window, choose somewhere in your data area to save it and click OK. When it comes to re-using these settings, simply click on Load… and open the saved file. Preset combinations of multiple channels can be loaded from \\igmm-smbhost\microscopeusers\Micro_Manager_Presets\
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Acquiring multiple images and saving as you go. With all the parameters now setup, click ‘Acquire!’ The system will now take 3 images, in this particular case, one for each channel selected and using the exposure values calculated before. Once the 3 images have been taken, they are merged into a single, composite image. Pressing F11 (Acquire) will do the same operation. If no channels were selected, pressing F11 would give a single image of the last channel selected or viewed.
Click on Acquire to capture multi-channel image.
Save images, can automatically save a scan to the pre-designated folder.
Composite 16Bit image of the 3 individual scans.
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Make sure Image stack file is saved
The images will be taken and saved in the folder selected in the Directory Root in the MultiDimensional Acquisition window, as pre-selected when ‘Save images’ was ticked in the MDA window. If the ‘Save images’ is left unticked, then the images can be saved as an image stack individually. The individual images/channels can be viewed by simply removing those scans not required in the Micro-Manager window. This is done by clicking the box next to the channel, removing as many or as few as required. Adjustments can be made to the image by altering the dynamic range. Default output of multiple scans is Composite but this can be changed to Greyscale or Color after the scan to view individual channels.
Change from composite to Color or Greyscale, ie. Color
Individual channels can be switched on/off by unticking
Adjustments to the image can be done, using the histogram next to that particular channel, by simply clicking and dragging the small triangles at the bottom and top of the graph. An automatic adjustment of the range of the graph can be done by clicking Auto, with further adjustment by the user or Full to get the full range from 0 to 16383 on the scale, which can again be adjusted by the user. Click Auto to automatically Or, adjust range manually by adjust image range dragging ‘triangles’.
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Once adjusted, assuming the original unedited image was automatically saved to a folder, this image can be saved using the save macro in ImageJ. This will save it as a single file but with no metadata attached. Also, the 16bit image can be changed to an 8bit image (Viewable in all default viewers on PC’s and Mac’s or in Powerpoint) by clicking on the RGB button in ImageJ. Or, a scale bar can added – assuming the lens used was entered in the main window before taking the image –which will allow a scale bar to be set AND change the image from 16bit to 8bit. (p21)
Change to 8Bit
Set scale bar and change to 8Bit
Save image as a single TIFF file in 8Bit.
If Save Image was not ticked in the MDA window, the original 16Bit image can be saved by clicking on the save icon in the image window. Select the image stack option, enter the name required and click OK. This will then create a folder with the given name and the 16Bit TIFF composite image within. Any other adjustments can be made as described above and saved using the save button in ImageJ.
Click on save icon, and save as Image Stack file. This saves the 16bit image and the metadata file in a folder
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AutoExposure and Acquire. (F10) Once the image has been found that you wish to acquire, and if no comparison to other images are required, then the F8 function key can be used to take images. This Function creates new exposure values for every scan it does, which can make all the images appear to be at their best but means that no comparison, in terms of fluorescent or colourimetric output, can be carried out as the exposure rates will be different in every case, potentially.
Brightfield. (Num Lock) Brightfield imaging is done in exactly the same way as Fluorescence, apart from the filter wheel on the microscope should be turned to ‘Brightfield’ (Position 1), the brightfield lamp turned on at the side and the appropriate Brightfield Function key (Num Lock) used to view by eye AND on the screen. Once the image has been seen down the eyepieces, pull out the rod to move the light to the camera. The image will now appear in the Live View window on the screen. Adjust the exposure by entering a value into the Exposure window or by using AutoExposure (F9).
Once happy, if only a single channel is required, then click on Live to switch it off, then click on the save icon an save as an image stack (no separate images). If the lens has been set, then a scalebar can be applied or the RGB macro button can be clicked to change this 16bit image to an 8bit, then using the imageJ save macro to save as an individual file.
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RGB Acquisition
If the slide to be viewed with Brightfield requires colour camera image, for example an H&E stained slide, then the RGB function can be used for this. Once an area has been found to scan using Num Lock (View Brightfield), move the light path to the camera and open the Multi-Dimensional Acquisition window. NB: Increase the level of white light, as exposures can be longer than expected due to filters used. Now change the channel group to Colour Brightfield and then click New to create the required channels, i.e. Red, Green and Blue. Once done, click RGB AutoExpose and Acquire Space) and the three channels are first AutoExoposed and then 3 images are taken based on these values. If you wish to use your own manually entered exposure values, then once selected click Acquire.
1. Change to Colour Brightfield 3. Change colours to match channel
2. Click new 3 times for 3 channels
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Acquire to use manually entered exposure values.
Manually entered exposure values
Appendix.
The following instructions are for first time users of Micro-Manager on this particular PC. They should only need to be done on the first use but if issues arise later, may need to be repeated. When you log in for the first time, there are a couple of settings which need changing. The initial launch window will look similar to this. The window should contain the microscope name, in this case Limelight, so to load the required configuration file, click on the box with 3 dots . You now have to follow a set path to load the Limelight.cfg file. The path for this is
Computer C Drive Program Files Micro-Manager-1.4 Configs Limelight.cfg Now close the software, restart Micro-Manager and click OK if Limelight.cfg is now showing.
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Limelight.cfg config file should now be selected
Double click on Limelight.cfg
You only need to do this the first time you open Micro-Manager.
Finally, a couple of settings need to be changed to allow the image to be of a usable size and to allow some exposure settings to be visible in different windows. To do both of these, with Micro-Manager open, go to Tools Options and then tick the ‘Sync exposure between Main and MDA windows’ and change the ‘Preferred Image Window Zoom’ to 50%.
Set Window Zoom to 50%. The tick box to Sync Exposure
Tools Options….
If a separate metadata file is required, as opposed to the one embedded within the 16Bit image, then tick the ‘Create metadata.txt file box’. The metadata file contains all the information about the image, the hardware and software used to capture the image and more. This information can be read and used by software, such as ImageJ.
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Fast launch setup for Micro-Manager
If you choose to ‘Pin’ the shortcut to the Taskbar, the Desktop will now look like this. Once ‘pinned’, Micro-Manager can now be opened by clicking once on the icon.
Direction of light from slide to eyepieces or camera.
Image to eyepiece
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Image to camera
Filter wheel.
Ensure that the correct filter position has been selected on the microscope prior to selecting the channels. To do this, turn the filter wheel to position 1 (Brightfield), or position 7 or 8 depending on sample and fluorochromes to be viewed. And, that the bar is pushed in fully on the microscope to direct the light to the eyepieces.
Position 1 – White light Position 7 – FITC, Texas Red, DAPI Position 8 – TRITC, Cy5, FITC, DAPI
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Autostretch; When first scanning a sample, what can be seen down the eyepiece may not reflect, in terms of signal strength/output, what the camera will see and display on the desktop. As such, using the Autostretch function allows the software to ‘see’ the signal, compress the dynamic range and therefore make a weak signal appear stronger while using the exposure rate entered. This will then give the user a clue as to whether to increase the exposure rate, use the full dynamic range of the camera and get better results from the scan. Below is a standard scan with the Autostretch unticked , a low exposure rate maximum light level is 1,786. The image is hard to see as it is under-exposed.
and the
By clicking on the Autostretch , the scales and maximums are the same but the dynamic range has been ‘squashed’ up to only display that little region on the scale. The readings are roughly the same but the range is now MUCH smaller. And the image is now visible though ‘noisy’.
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By adjusting the exposure rate manually, or using F9 to AutoExpose, we can now get a much greater dynamic range without having to resort to using the Autostretch, and a clear, less ‘noisy’ image.
Creating a shortcut to your data area or other server. All images taken with Micro-Manager need to be saved, and this should be in a designated area in your Data area. It is easiest to create a shortcut to this area beforehand as this will make any subsequent saving much easier. To do this, open up Windows Explorer and click on Computer. Then right click inside the window, and click on ‘Add a netwotrk location’.
Click on Computer
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Click on Add a network location
In the next window, click Next and then click on ‘Choose a custom network location’ before clicking on ‘Next’ again. In the next window, type in the path to the location of your data area, in this case \\igmm-smbhost\hmorriso\data In this case it is ‘hmorriso’ but yours will be the login name you used to login to this PC. Once done, click Next.
In the next window, give the new shortcut a name , then click Next, and then click Finish in the next window. When Windows Explorer is opened again, there will be a new shortcut.
This shortcut to your Data area can now be entered into the autosave in Multi-Dimensional Acqusition by clicking on Save Images , then clicking on the box next to the Directory Root. Click on Computer and then click on the shortcut, in this case ‘Harris_Data_Area’ . The new area which opens is your data area, now create a folder in this area by clicking on the New Folder Icon , and then giving this new folder a name, in this case Micro_Manager_Save_Folder. Now click on Open.
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Auto-Save In the MDA window, the Save Images box can be ticked so that every image you acquire is automatically saved to a pre-determined folder with a univeral file name of your making. Each succesive file acquired is given the same file name with a sequential number added to the folder and image to differentiate it from the previous scan.
1. Tick Save images box.
3. Assign a name for each image. Software add number after name to differentiate between images
2. Select a pathname to save images to a folder or destination
4. Save images as Image Stack
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Scale Bar.
NB: Lens used to scan must be entered into Micro-Manager BEFORE scanning or scalebar will be using incorrect pixel values.
If a scalebar is required, this can be done by going into ImageJ Analyse Tools Scale Bar
Once happy with the scalebar, if you want to save the image WITH the scalebar, then you must ‘flatten’ the image to keep it. Do this by going to ImageJ Image Overlay Flatten. Then save the image in the format required.
A macro has been written that allows the setting of a scalebar and changing to 8Bit, or changing to 8Bit alone.
Set scale bar and change image to 8Bit.
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Alternative ways to save acquired images Images can be saved automatically, as described on page 7, and this data can be read in ImageJ along with the saved Metadata file (Metadata file contains all the information about that particular scan in terms of time, type, channels, exposures and subsequent comments). The raw data should always be saved and then any subsequent changes through adjustment saved as an additional file. The following are other methods of saving an image or stack. If saving as a stack, the metadata file is not a separate file but included with the saved stack. To read this info, open the Bio-Formats under Plugins Bioformats Import select OME-XML and this will open a separate window with the metadata in here.
Save icon
RGB icon
In the ImageJ window, there are additional tools which can be utilised. The SAVE icon in the ImageJ window can save your image or stack as a 16bit images in whatever folder you wish but by doing this you lose everything from the metadata file apart from the image calibration, which will allow the setting of a calibration bar (Assuming the correct lens was selected in the Micro-Manager window before scanning). To save your image as an 8bit file and then be readable in other viewers or Powerpoint, click on RGB to ‘flatten’ the image and then click on the SAVE icon. To change previously scanned files, drag the Stack file into ImageJ, go to Image Colour Stack to RGB. This will create a new, composite file which can now be saved as a TIFF image and read elsewhere. This can also be done if you choose not to automatically save your data after every scan. Once the series of channels has been scanned, instead of immediately saving the stack or series of channel images, the above can be done in ImageJ.
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A composite scan, 2 or 3 together, can be split into it’s separate channels by going to ImageJ and Image Stack Stack to Images. This will give 3 individual channel images. Any images acquired can simply be saved by going into ImageJ File Save As Tiff . This would apply to any images acquired at any point, removing the additional Folder issues.
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Profile viewing of acquired image The output values across the scan can be checked, if required, by clicking on Profile . Once clicked, the yellow line which appears at an angle across the image screen can be moved by placing the cursor on the dots at either end of the line , or the middle, clicked on and slide the mouse to position the line. This produces a histogram readout along the length of the line. This can be useful when determining uniformity of output, level of output/saturation, etc. Remember to click Refresh if the line is moved to another position.
This process is repeated for all the channels required for the experiment being done, those exposure rates seen are automatically recorded and readable by other parts of the Micro-Manager software. If the scans are being done to record differences, with regards treatments, then the exposure rate should remain unchanged throughout. If the images are only a record of the sample seen, or if there is to be no measurements taken as a comparison, then autoexposure could be used each time an image is taken, for example F8 (AutoExposure & Acquire)
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Fast-Live-Mode (F6) If your exposure time is very long, this makes focusing quite hard. So clicking the Fast_Live_Mode can make focusing easier by changing the Bin value, increasing the gain thus decreasing the exposure time. Once focused, exit the script and the lower exposure value used to focus will return to what it was previously Focus object, then click OK and repeat the for channels required
Acquire-Focus Offset (F10)
There are times when you are a scanning a slide which has multiple channels and when focusing the areas of interest, are in slightly different position within the depth of the sample. This can then make focusing on a single plane very difficult, given that perhaps a couple of channels are in focus while the third is not. To get around this, there is a script (F10) which allows each channel to be focused independently. It should be noted, this could give a false view of what is happening with the sample, giving the impression that all the signals are in the same depth position and are not.
Looking down at the sample with camera
Specimen with signal throughout at different depths within the sample, making focusing all channels difficult 25
Looking down at the sample with camera
Using Acquire-Focus Offset will give this ‘false’ impression of the signal being all in the same plane but will maintain all channels in focus.
The net result is a sample which looks in focus on all channels, but care should be taken when reporting that the signals are not all in the same plane throughout the depth of the sample. To use this function, first set up the Multi-Dimensional Acquire window with the channels you want to acquire, and set the correct exposures for each channel. Once done, now click on F10 (AcquireFocus Offset). A dialogue window will popup telling you to focus that channel, once done click OK and then you are told to focus the next channel. Once you click OK, the acquire will finish and your focused image will be displayed.
End result is a multi-channel image with all channels in focus.
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