Wittrup:Deltavision deconvolution microscope

Startup

General Start-up Procedure

1. Turn on lamp and log in time.

2. Turn on power strip adjacent to Linux system in front

3. Press power for windows CPU (top shelf left) – appears as if ‘on’ with green light, but must be turned on manually

4. Press power for Linux CPU (middle shelf left)

5. Remove slides and obstructions from objective

6. Flip top console to 2 (windows computer) and initialize (press F1, then 'Y')

7. Flip back to computer 1 (Linux)

8. Sign on using login: Athena username

9. Enter password: Athena password

10. Open Softworx software

11. Turn on “Room in Use” light (switch on right side of door)

General Notes: If the Windows computer has been turned on (you can check this by switching over to computer 2 on the CompuSwitch. If it does not switch over, the computer is probably off), you may have to restart the software if there is a problem. To do this, hit escape, then double-click on the DV Instrument Controller icon. There may be some on-screen instructions to initialize the equipment (such as pressing F1, etc.).

Running SoftWorx

Once all the computers are turned on and the microscope is initialized, make sure the CompuSwitch is on computer 1 (Linux). Double click on the Start softWoRx icon. (If you are only analyzing previous images, you may go to “Data Folder” and open these directly.) Go to File . . . Acquire to open up the main control windows. The filter monitor appears that shows you the current filters for the excitation module (EX), emission filter for camera (EM), and eyepiece filter (EP). It also shows you the status of the neutral density (ND) filter.

The Resolve 3D window allows you to change and operate most of the microscope from this window. The top lines allow you to change the excitation, emission, and neutral density filters. (The eyepiece filter must be changed manually. This is done by moving the wheel located on top of the eyepiece box. All the other filters automatically change when the eyepiece is changed.) In general, you will have all the filters set the same. The next line allows you to change your exposure time (given in seconds). This and the neutral density filter are the two main controls for adjusting the brightness of your sample (discussed more in next section). The lines below the exposure time adjust the resolution. In general, you will have the resolution set to 1024x1024 (the highest) and Bin set to 1X1 (no “binning”). If your sample is still not bright enough after adjusting the ND and exposure time, you may lower the image size and increase the bin number. This will lump pixels together to increase the overall brightness at the expense of resolution. You will also find the lens magnification below the image size. The lens must be set manually to that which is being used. To change the objective, manually turn the wheel underneath the table, then adjust the magnification box. The controls below these adjustments allow you to move the field of view and depth. These will be discussed more under the “Viewing Samples” section.

Viewing Samples

First, choose the magnification that you would like to use. The 20x lens is an air lense, meaning no oil is needed and slide should be wiped clean first. If you are using the 63x or 100x objective, check to make sure there is no residual oil on the lens. Only use lens paper to wipe the lens (Kimwipes may scratch!). If this does not do a sufficient job, you may wipe the lens with a Q-tip soak in chloroform. Place a tiny drop of the appropriate oil onto the lens. (The computer can suggest an appropriate oil if you know the “working distance,” the thickness of you sample. Beside the Lens selection button in the Resolve 3D window, click on “Info.” Put in the appropriate info, and the recommended refractive index will be displayed.)

This is an inverted microscope, so samples are viewed through the “bottom,” meaning the cover-slip should be placed face down. Containers with cover-slip bottoms may be purchased to avoid moving the cells from their original container to a slide. Other containers may absorb all or some of the fluorescent light.

Bring the objective up to the slide, allowing it to touch. When first adjusting the focus, move the objective downward to obtain focus. Although it is more difficult to break slides with an inverted scope, care should be taken not to push the objective up onto the slide which could break.

Adjust the filters to the proper wavelength using the eyepiece wheel located on top of the eyepiece box. The other filters will automatically adjust. You may want to lower the neutral density when looking for your sample initially. The metal rods on the lower right side of the microscope switch back and forth between the camera and eyepiece. It is easiest to leave the lower rod pulled outward at all times and push the top rod in for viewing through the eyepiece; push the rod in to view using the camera.

Looking through the eyepiece, you can adjust the focus with the manual focus wheel. If you do not see anything, you may want to scan your sample for cells first. (Also, check to make sure the filters are properly set, the top rod is pushed in to view through the eyepiece, and a 2nd rod, located right beside the right eyepiece, is pulled out.) This can be done using the joystick. Located above the joystick are 3 buttons to control the speed of scrolling: slow, medium, and fast. Once you have located an area of interest, you then switch over to camera imaging. When you are not looking through the eyepiece, you always want to keep the excitation shutter closed. This will keep the light from killing your cells while you’re not looking!

(You may also locate your cells directly with the camera. The easiest way to do this is to use the “continuously acquire button located under “File” in the Resolve 3D window. This will continuously take pictures as you adjust the field of view. You may also use the 4 arrows located in the middle of the Resolve 3D window to adjust the x-y plane, or you can use the joystick.)

First, pull the top rod out to switch over to the camera. There are 3 buttons that can be used to acquire an image. This first is located on the keypad in the lower left corner; the second button is labeled acquire and is on the top left corner of the Resolve 3D window in Linux. The third button is in the same window, but it is a camera along the right hand side, halfway down the window. Press one of these to acquire an image.

For very bright images, a window may pop up telling you the image is saturated. This means you need to change the neutral density filter or lower the exposure time. Otherwise, you will lose resolution between the saturated pixels.

Once you have an image on screen, you want to adjust the brightness. To the left of the green light towards the bottom of the Resolve 3D window are the min, max, and mean pixel values for your image. A good target for the max value is around 1000. You can change this by adjusting the neutral density filters and changing the exposure time. If you still have trouble getting a bright image, you may have to use the “binning” function to increase the brightness, although this lowers resolution. If the green light (mentioned above) is red, there is a problem with the microscope.

You now have acquired an image that you would like to analyze. To do this, you want to take a “Z-stack,” which consists of multiple pictures (~100) taken at different focal planes in the z-axis. A computer program can then look at these images and adjust for light that originated from outside the focal plane, which blurs the image (deconvolution). With this optimized data, you may look at any of the focal planes in higher resolution, obtain better 2D pictures by taking the brightest pixels from each plane (not quantitative), or construct a 3D image of your sample using the volume viewer.

Taking a “Z-stack”

A Z-stack is a series of pictures taken along the z-axis. Each picture slightly varies the focal plane capturing a picture at a different “depth.” In order to take a Z-stack, you need to tell the computer where the “top” and “bottom” of your image is located. This can be done using the controls in the Resolve 3D window. In the middle of this window you will see a set of four arrows. (These arrows can be used to adjust the x-y field of view from the computer, without using the joystick.) Below this is a diagram of the x-y plane. This window traces your field of view. For more advanced uses, you may mark different points in your field of view to revisit them later. For now, we assume you have already located the x-y position of interest. Along the right-hand side of the x-y plane diagram is a schematic showing your position in the z-field. This is what you will adjust to find the top and bottom of your sample.

With your image in view on the screen, click on the up arrow several times (3 or 4) and click acquire (the picture of the camera just above the up arrow is convenient). Note: the up arrow is not in the set of 4 arrows in the middle of the screen. These adjust the x and y coordinates. The up and down arrows are located along the right hand side of the screen.) You are trying to locate the top of your sample where the picture just goes out of focus. If the picture is still in focus, click the up arrow more and click acquire. Keep repeating until the image is just out of focus. At this point, click on the button with a line at the top with an ‘x’ through it. (Holding the cursor over the buttons without clicking brings up an explanation of each button.) This marks the top of your sample. Click the down arrow several times and click acquire. Repeat this until the sample goes out of focus at the bottom, then click on the button with a line at the bottom with an ‘x’ through it. This marks the bottom of your sample. Click on the button next to the camera with arrows pointing towards the middle, then click Acquire. This takes an image in the center of your sample, which should be in good focus. This is the starting point for your Z-stack

Click on the “Experiment” button to the left of the “Acquire” button at the top of the Resolve 3D screen. The brings up a “Design/Run Experiment” window. Click on “Experiment Designer.” Another window opens up with a place for a file name at the top followed by Sectioning Setup and other adjustment windows.

Type in a file name at the top. If “middle of sample” is not chosen from the list of options under sectioning setup, choose it. (With the inverted microscope, the “top” and “bottom” are ambiguous, so starting from the middle is easier. The “top” of the sample is actually closer to the objective, so it is the bottom with reference to the room.) Click on “Get thickness” which imports the distance between the top and bottom of your sample. You can adjust the section spacing or the number of sections if need be. Section thicknesses of 0.15 to 0.2 microns are pretty good. 100 or more sections is common.

Under wavelength setup, click on a gray box to the left to activate a line. Activate a line for each wavelength you want to measure. Under excitation filter, adjust this to the proper wavelength. Verify the information in this line is correct.

The other adjustments are for more advanced experiments. If you want to do time-lapse imaging or take multiple z-stacks simultaneously (visiting different points), the other controls allow you to do this. Go to the top of this window (Resolve 3D Experiment Designer) and go to File . . . Save. You may then close this window.

In the Design/Run Experiment window, click on “Do It” in the lower left-hand corner. It will ask you to name the Z-stack that you are going to take. From there, the computer will follow the instructions and take the number of images that you requested. The Z-stack will be saved under the name you provided followed by “_R3D.dv”.

Deconvolving

Due to the wave nature of light, when it passes by tiny objects, the light changes course. This affects the resolution of a microscope on small length scales. Deconvolution is a mathematical function that tries to solve where the light originally came from before it changed course. It uses the information from the different focal planes of the Z-stack to determine this. It is an iterative program, and you can change the number of iterations.

With the raw data Z-stack window open, got to the main window (softWoRx 3.2.3). Under “Process,” choose Deconvolve. Drag the window number (located just above the brightness meter “sun” on the left-hand side) into the input box. This should import all the data for you. The output file is automatically named after the raw data file with a “_R3D_D3D.dv” extension. Under OTF File, choose the file that contains the Zeiss microscope at the magnification level you used. There is no 20x file for Zeiss microscopes. You may use a 20x for Olympus, but this may not give optimal results. You may also change the number of cycles, 7 – 10 being typical. The more cycles you have, the clearer your image will be. Click on “Do It” at the bottom of the screen.

Depending on the number of sections and iterations, this may take a long time. After the computer is done deconvolving, you may close the two windows. Go to Data Folder in the main operating window and find the file you just created with the “_R3D_D3D.dv” extension. Double click to open it. This is your deconvolved Z-stack. If you open the original file and compare, you will notice the deconvolved file has better resolution. The scroll bar along the left of the picture allows you to scroll up and down through the Z-stack.

The next thing you will want to do is adjust the brightness. Click on the brightness “sun” below the window number. The newly opened window allows you to adjust the brightness. You will typically see a peak (of yellow) near the left-hand side in the blue box. This is mostly background, so you want to drag the left white box until it is on the right-hand side of this peak. This should eliminate most of the background. Scroll through the Z-stack while leaving the brightness window open. You can drag the right hand box as close to the yellow on the left as you like. If you drag this box too far left, the brightest pixels will be saturated, and you will lose resolution. You can scroll through to make sure you do not have this set too far to the left. Imaging

The two basic imaging techniques I will discuss are volume viewing and projections. Quantitative techniques will not be discussed.

Volume Viewing

This takes the deconvoluted Z-stack and recreates a 3D image of your sample. To do this, go under “View” in the main window and choose “volume viewer.” This brings up a second window with an input line. Drag the window number of your deconvoluted image into the input line. Under viewing parameters, select max intensity as method. (Other methods can be used in various situations. For example, progressive is good for imaging surfaces.) Changing the number of projections will increase the file size and time to completion but creates a more fluid image. You many change the rotation to the desired axis and number of degrees. Click Do It. You will see the rotated images as the appear.

Once the program is completed, you will be able to scroll along the image, rotating it in space. The file is not saved automatically, so you will have to do it manually if you want to keep the file.

Quick Projection

This creates a 2D image from the Z-stack, although it is not quantitative. Go under File in the main window and choose Quick Projection. Drag the deconvolved image window number into the Input line. Select max intensity for the Method, and click Do It. The program scans through the Z-stacks selecting the max intensity at each x-y coordinate to create the image.

Measuring Distances

There are several ways to measure distances in the images. To directly calculate the distance between two points, you can click on Measure in the main window. Select Distance, which brings up another window. Drag the window number into the line next to “Window” and click on the two different points in your image. The program calculates the distance in micrometers.

There is one last point about saving data. If you are doing quantitative analysis, it is best to save the data as grayscale. There are more shades of gray, so saving as grayscale retains more detail. The computer has a CD burner which you can use to transport files.

Saving Data to CD

1. Select “Utilities” menu on the main window

2. Choose “archive data to CD”

3. Click on the “Files” button

3. Drag files from data folder to CD burner window

4. Place blank CD in CD burner (on Linux system)

5. Click “Create CD”

General Shut-down Procedure

Clean up the your samples and the general microscope area! When you are done looking at your sample (after taking a Z-stack), wipe the oil off the lens with lens paper. If oil remains on the lens and builds up, it can leak inside of the objective and ruin it.

Shut-down:

1. Make sure lamp has been running for at least 1 hour before shutting down. If someone is using the microscope right after you, please confirm that they will still be using it before leaving the lamp on (especially before the weekend).

2. Logout on the lamp log sheet.

3. Turn the power strip off. Log off on the Linux computer. For shorter periods, you may leave the computers running.

4. To turn off the computers: on the Linux system, go to the footprint in the lower left-hand corner and click: logout . . . shutdown.

5. Flip computer switch (CompuSwitch) to computer 2 (windows).

6. Hit ESC (and y) to quit program, then got to “Start” and click Shutdown.

7. Go to start and click shut-down.

8. Turn power off manually for Linux system once says power down. Will not turn off automatically like windows system.