Keck Microscopy Facility
|
Keck Microscopy Facility |
|
DELTAVISION OPERATING INSTRUCTIONS
BE SURE and leave yourself enough time at the end of your session to transfer and remove your files from the acquisition computer. If you have troubles with the system and Greg is not available, returning the system to the default configuration is a good way to try and get back on track.
Applied Precision's web site has more information on image restoration (deconvolution) with the Deltavision. The Keck Center web site has information regarding specimen preparation for getting optimal results from deconvolution.
The
manuals in the room are very helpful.
SIGN IN ON THE LOG SHEET
1) Turn on the mercury lamp power supply (1).
2) Open up the panel door below the acquisition computer.
3) Turn on the power strip (4) next to the Photometrics box.
4) Turn on the PC (5) and close the panel door.
5) Select "1" on the switch box (6). Wait for Windows 98 to load.
6) Double click on the DV Instrument Controller icon (not the folder).
7) The initialization stops to confirm that the objective turret is lowered prior to initializing the stage to ensure that the objectives are not damaged. Follow the instructions on the screen.
8) Wait until the screen reads "Starting normal operation...."
9) Select "2" on the switch box (6). Move the mouse if the screen is dark.
10) If you see a log-in screen, click on the Keck icon to log on as "keck" - you'll need to get the password from Greg.
11)
Double click the SoftWoRx icon on the desktop.
SELECTING OBJECTIVES.
The objectives are labeled with a white tape label in a conspicuous position. If you do not see a label, tell Greg. The high-mag oil lenses are used to collect images for deconvolution. Use the other objectives for surveying your specimen. Note that the 60X has the same resolving power as the 100X, but transmits more light -- it is often a better choice.
Note that the 40X and 100X lenses have an adjustment collar -- it should be rotated full clockwise.
There is also a 60X water lens, which is an excellent choice for live-cell imaging, or anytime you need to image something that is not right next to the coverglass. See Greg if you want to use it.
Focus the oculars first (skip this section if you don't care if the oculars are at the same focal plane as the camera).
1) The knob on the right side of the microscope should be set to the eye, not SPL or SPR.
2) Turn on the transmitted light by hitting the TRANS button on the remote keypad.
3) Set The lower binocular wheel to the blank position (use the picture above the condenser as a key).
4) Look through the eyepieces and focus each one so that the cross hairs in the middle of the field of view are in focus and appear as double lines. If you don't see the cross hairs, check step 16 of the default configuration.
5) Turn off the transmitted light by hitting the TRANS button on the remote keypad.
Now for the Specimen
1) Under the SoftWoRx File menu, select Acquire (Resolve3D)...
2) Select an appropriate excitation filter in the Resolve3D window. The emission filter will automatically change to match the exciter. Use a set of filters for a probe that should be in all or most of the cells. Don't try to bring the specimen into focus looking for a probe that may not be there! You can also use DIC (link coming) for initially focusing on your specimen.
3) Set an appropriate emission filter with the lower binocular wheel (use the picture above the condenser as a key).
4) When using oil immersion objectives use the type DF Cargille immersion oil.
5) Apply a drop of oil to the cover glass of your specimen.
6) Put the slide on the stage, cover slip down, and secure it with stage clips or tape.
7) Set the stage speed to fast on the remote keypad and center the oil droplet over the objective using the joy stick on the keypad. Then use the course focus to bring the objective into contact with the oil.
8) Open the shutter by hitting the EX button on the remote keypad.
If no light is visible at the sample, check the following steps from the default configuration:
9) Make sure that the knob on the right side of the microscope is on the eye, not on SPL or SPR.
10) While looking through the eyepieces, slowly continue to raise the objectives with the course focusing knob until your specimen is in focus.
11) Change the speed of the stage to medium on the remote keypad and use the joystick to move around on the slide and find an interesting field. You made need to change the speed to slow to get what you want in the center of the field of view. Focus on the plane of interest.
12) Close the shutter by hitting the EX button on the remote keypad.
13) Turn the knob on the right side of the microscope to SPL.
SETTING THE AQUISITION PARAMETERS
1) Confirm that an appropriate excitation/emission filter set is selected in SoftWorx.
2) Click the Lens button in Resolve3D and select the appropriate lens from the drop down menu.
3) Click on "Find Exposure". The system will take successively longer exposures while updating the Image Collection window until the target intensity is reached. The default target intensity is 3600 gray levels. The calculated exposure time will appear in the Exp box. This is the exposure time that will be used when you click the "Acquire" button. You can change the exposure by entering a number in the Exp box. You can also change the target intensity by clicking on the "Settings" button. Click here (link coming) for a short tutorial on 12-bit imaging and selecting exposure times.
Note that there is a bug in the program -- the first time in your imaging session that the "Find Exposure" button is clicked, you may see a "Camera saturated" error message followed by an "Insufficient response" error message. Just repeat the "Find Exposure" operation.
4) The minimum and maximum intensity of the last image in the Image Collection window will be displayed in the Min and Max boxes. Use these values to evaluate the exposure. Avoid saturation (gray level of 4095)!
5) Center the object of interest in the field of view using the "Center Object" function. The Lens and Aux Mag must be set correctly to give the correct pixel size.
6) Reduce the size of the data set by using a minimal region of the CCD chip. Click on the Size box and hold down the mouse button to scroll down the range of image sizes and see each format on the last image in the Data Collection Window. Select the minimum size that will include your object of interest. This will not change the resolution of the image.
7) Repeat steps 3 & 4 for each filter set that you are using.
1) Click here (link coming) for information about selecting the initial focal plane and collection strategies for the DeltaVision.
2) Select a focal plane. If the eyepieces were focused, the focal plane you selected by eye should be very close to what you see in the image collection window. To fine tune the focal plane, click the up or down arrows (the ones with a gray box in between them) in the Stage panel of Resolve 3D. The number set in the dZ box will be the step size (in um) for each click of the arrows. Use the arrows and the Acquire button to get to the desired focal plane.
3) Open the Experiment Designer by clicking on the "Experiment" button in Resolve3D, and then on the "Design..." button in the Resolve3D Experiment window.
4) Click on the "Scan Options" button and make sure that "Enable Fast Acquisition" is not checked. Then click "Done"
5) Input a value for the Optical Section Spacing. This will be the distance between image planes in the z-dimension. You can find the Recommended Z Step Size by clicking on the "Info" button next to the lens designation in the Resolve3D window.
6) Input a value for the Number of Optical Sections. The product of the Optical Section Spacing and the Number of Optical Sections is the total thickness of the sampled volume.
7) The default configuration is for the microscope to collect the image set with the current focal plane at the middle of the sampled volume. If you want the microscope to start the image set at the current focal plane, input 0 in the Z Start: box.
8) In the Wavelength Setup portion of the Experiment Designer, check a box at the left for each of the wavelengths you want to collect. Then select the appropriate EX Filter from the drop-down list for each wavelength. The Exposure, EM Filter and ND Filter settings will be automatically updated to reflect the Acquisition parameters you established in the resolve3D window.
9) Time-lapse is for doing time-lapse studies (no, really...). Point Visiting is for defining a number of points (cells) that can then all be sampled automatically. Panel Collection is for automatically imaging overlapping fields to image large areas. Ask Greg if you want to try any of these functions.
10) Select Save & Exit from the File menu. Don't save the experiment as a new file unless you know that you'll want to use all of the parameters unchanged at a later date. The default experiment, Resolve3D.exp, gets overwritten for each experiment.
11) Click "Run" in the Resolve3D Experiment window.
12) Click "Do It" in the Running Experiment window.
13) Give the file a name and click OK.
1) To look at an image set go to File --> View.... Click "Input" and select an image file (a .r3d file). The default folder for the image files is /usr/people/keck. If you can't find your image file, click "Settings" in the Resolve3D window and check the file path listed after "Directory". That file path is where your images were stored.
2) Click "Do It" in the View File window. A window will open with the image file shown in color and at the middle plane of the z-series. Scroll through the image set with the slider on the left side of the Image Window.
Files can be processed and viewed on the DVLinux computer during your scheduled time on the acquisition system (deltavision1). By transferring images to the Keck Facility server you can get them started deconvolving on the DVLinux computer while you collect another image set on deltavision1. The Multi-Dimensional workstation can also be used for deconvolving your files.
Deltavision1 is the SGI acquisition system. Do not leave images on this system, they will be deleted without warning at any time, and you will be charged $20.
Transfer your files to he Keck Facility server. From there they can be Deconvolved on either the DVLinux computer or the Multi-dimensional Workstation.
1)Double
click on the xdir icon 
2) From the "Connect" menu at the top of the xdir window, select "Connect to Remote":
3) Select 128.95.245.42 (your User Name). If your User Name does not appear on the list, select any of the 128.95.245.42 destinations and type in your User Name in the "User Name" field near the bottom of the window and hit "Connect".
4) Type in your password for the server and hit "OK". A new window will open which is your lab's folder on the Keck Facility server. Navigate to the folder you wish to put the files on by double-clicking on the folder you wish to open. To make a new folder on the server, go to the "Ops" drop-down list at the top of the window and select "Make Directory".
5) Highlight the files that you wish to transfer. Be sure that you keep the .r3d files and the .log files together. Then drag them to the destination folder on the remote computer.
6) Note that this action does not MOVE the files, but just COPIES them. Once your images have been copied to the destination computer, be sure and delete them from the acquisition computer. Select the files you just moved and click on the trash can icon in the XDir window.
7)
Image files may be left on the server
for up to one year. Any files on
these computers that are older
than three months may be deleted without warning.
Use DVLinux for archiving your files to CD or DVD. Click here for instructions.
All files should to be removed from the acquisition computer deltavision1. Files left on the acquisition computer will be deleted without warning at any time, and you will be charged $20. Move your files to KeckStorage. Be sure and then delete your files from deltavision1 by dragging them to the dumpster and EMPTY THE DUMPSTER by clicking on Desktop in the Tool chest and selecting "Empty Dumpster".
1) Check the schedule. If someone else is signed up, you can leave everything on.
2) Exit the Deltavision/softWoRx software.
3) Select the "1" position on the switch box by the monitor (Raritan CompuSwitch).
4) Hit the "Esc" button twice to exit the program.
5) From the Start menu (bottom left of the screen) select "Shut Down". At "What do you want the computer to do?" select "Shut down".
6) Open the panel below the computer and turn off the switch on the power strip
7) Turn off the mercury lamp.
8) Clean oil off the objectives.
9) Cover the microscope! If you neglect to cover the microscope you will be charged $20.
Contact Greg Martin at gvm@u.washington.edu for additional information. Suggestions and comments are always welcome!
© 2007 Keck Microscopy Facility, University of Washington