Digital Station #1

Olympus IX70 inverted microscope

System Specification Standard Configuration (custom configuration available upon request) Microscope Olympus IX70 inverted Objectives *10x N.A. 0.30 Phase 1 *20x N.A .0.40 Phase 1 *40x N.A. 0.60 Phase 2 LWD (for use with substrates thicker than coverslips) *40x for use with coverslips available by request *60x N.A. 1.4 Phase 3 (green) Condensers *Long Working Distance (N.A. 0.55) *(Short Working Distance (N.A. 1.00) in cabinet on wall) *Extra Long WD (N.A. 0.3) for microinjection available by request Fluorescence Filters *Dapi, FITC, Rhodamine, CY5 *Alternative Sets for CFP, YFP, and others by request Computer Dimension 4500 with CD/DVD burner, Zip drive, USB ports, Firewire, Internet file transfer Camera Sensicam QE cooled CCD camera Software IP Lab; Photoshop CS2; ImageJ; Excel Automated Hardware LUDL excitation filter wheels; ASI emission filter wheel; ASI focus motor; Uniblitz shutter for transmitted light

New As Of July 2007

Turn on System

• SIGN INTO THE LOG BOOK !!
• Turn on mercury lamp (at right, on shelf). THIS MUST GO ON FIRST.
• Check if the computer is on by wiggling the mouse or pressing the shift key.  If it's off, turn it on with the round button on the front.
• Turn on the power strip.
  
  This will supply power to the camera, shutters and transmitted light source.
• Log in to the common User account.
• Run IP Lab Spectrum [Double click the  icon on desktop or go to Start Menu]

Viewing Samples by Eye

• Rotate beam splitter to eye only position.
• To look in bright field or phase press F2 to open/close the transmitted light shutter.
• To look in fluorescence press F1 to open/close the fluorescent light shutter.
  For the simplest operation, both software filter settings should be set to #1 and the fluorescent filter turret for single probe imaging should be set manually in the microscope.

Transmitted Light

• Check the following settings:
• The condenser labeled "long working distance" (0.55 NA condenser) should be on the microscope.
• Note: Short and very long working distance condensers are available for this microscope. Please see the AIF staff if you need one of these other condensers.
• Turn the condenser turret to the appropriate setting (BF, Ph1, Ph2, Ph3, DIC).
• Halogen lamp diffusion filter is in place (others out of light path).
• Be sure that the fluorescence (electric) shutter is closed. Press F1 to close it.
• Turn the filter wheet turret to the "Blue" filter.
• Insert the photo frame by pulling out the slider on the left of the microscope above the focus knob.
• Adjust brightness with knob on halogen lamp power source.
• Focus the eye pieces on the cross hairs:
  • Focus the right eye first by adjusting the knurled ring.
  • Focus the left eye second. You must hold the base of the eye piece with one hand and turn the top with your other hand to adjust.
  • The right occular adjusts easily, while the left is stiff. Please see an AIF staff member if you’re having trouble getting the left occular focused.
• Place your sample on the stage and focus (see instructions below).

Focusing:

• While looking through eyepieces, you may use the coarse focus when on microscope.
• DO NOT USE FINE FOCUS KNOB ON SCOPE.
• YOU MUST FINE FOCUS WITH THE FOCUS MOTOR! There are three settings on the fine focus motor. Adjust with tiny lever on front of the controller box.

Phase Contrast

• Open the condenser diaphragm all the way.
• Turn the condenser turret to the appropriate setting:
  • Ph1 - 10x and 20x objectives
  • Ph2 - 40x objective
  • Ph3 - 60x objective (green label only)
• Focus your sample
• Align the microscope for Koehler Illumination

Koehler Illumination

• Be sure that the sample is in focus!
• Close the luminous field diaphragm moderately (top lever in light path).
• Focus the diaphragm image by raising and/or lowering the condenser.
• Center the diaphragm image using the two large screws at the front of the condenser.
• Open the luminous field diaphragm until it is at the edge of your field of view.

*Contrast (for brightfield and DIC) can be adjusted using condenser diaphragm. (lever on the left of the Long Working Distance condenser).
**The Koehler Illumination should be checked and adjusted every time you switch objective lenses or if you are imaging live cells, every time you change dishes .

Click here for an illustrated version of Koehler Illumination.

Halogen Lamp Filters

• Diffusion filter: always use
• IF 550 filter: Use with CCD camera (transmitted light images)
• ND 16 filter: neutral density filter; use to reduce light intensity
• There is also a 1.5X "booster" slider on the right side of microscope that increases the magnification by 1.5x.

Epi-fluorescence

For the simpliest operation, which is one fluorescent probe at a time, both Ludl filter wheels must be set to filter position #1. You then set the filter manually on the microscope.

• Press F1 to open/close fluorescence shutter.
• Press F2 to open/close transmitted shutter.
• Be sure that the Analyzer and the Wollasten prism on the left and right side of the microscope, respectively are out of the light path.
• Turn the filter turret to the appropriate fluorescence filter.
• Focus your sample.
• Position your area of interest in the center of the field.

Image Acquistion

Two methods for taking pictures are mentioned.

For both methods, start here:

• Observe image as directed above.
• Turn the beam splitter to . This sends 80% light to the camera.
• Press F3 for a live preview of your specimen on the monitor.
• For transmitted light images (brightfield, phase, Nomarski), insert the IF550 filter into the light path and check that the turret is set to 'Blue'.
• Adjust the exposure time so that the maximum number in the bottom left of the screen is 3000 - 4000.
• Remember this exposure time.

Method 1(Quick Snap):

• Make sure the microscope is set to .
• Use Shift + F11 to set the exposure time and binning (image size) for keys F9, F10, F11, and F12.
• Press the appropriate key to take the picture.
• Save images as .TIFF format.

Method 2 :

• To collect a transmitted light image (brightfield, phase, Nomarski), press Shift + F2.
• Press Shift + F1 to acquire a fluorescence image.
• Enter the exposure time and binning
  • Note: To collect a 1X1 bin image, multiply calibrated exposure time (ms) by 4.
• Press Enter or click OK to collect the image.
• The final image should have a maximum number in box at bottom left of window between 30,000 - 60,000. If it is smaller than this, image manipulation will be hampered.
• Save images as .TIFF format.

To Collect a Z-Series

Use the 6D acquire or Multimode Acquire commands within IPLab.

Enter the values for:

• exposure time in ms
• Z step size; +0.5 step goes a half micrometer up per step and -1 goes a full micrometer down per step down.
  
• binning
• number of images

Microinjection

We may put a microinjection system on this microscope if required.

At the End of Your Session

Return the microscope to it's default settings

• Long working distance condenser on the microscope
• Use lens paper ONLY to clean oil immersion objectives
• 10x objective in the light path
• Filter wheel turret set to 'Blue' filter
• 1.5x 'booster' pushed in

After microinjection

• When you're done, remove needle from the microinjection holder.
• You must disgard all needles, tips, pipettes in the red barrel located underneath the counter.
• Please dispose of biological material appropriately.
• Report any spills to AIF staff. ONLY STAFF CAN CLEAN LENS ON THE MICROSCOPE.

Shutdown

• Copy your files from the hard drive by writing a CD or Zip disk, transferring to RestStop, or use a USB jump drive. If you have any questions about our policy on data left on our computer, please read our policy web page.
• Exit all running software
• Log out of computer account
  
• If noone is waiting to use the microscope immediately after you, turn off the powerstrip that turns off the camera and shutters.
• If it is a weekday or if somebody is scheduled to be in within 2 hours after you, leave the Mercury Lamp on, otherwise turn the lamp off.
• If you used it, leave the Short Working Distance condenser in the cabinet and the Long Working Distance condenser on the microscope.
• Clean up area AND take your belongings with you. Slides or dishes left on the microscope will be discarded without notice.
• Sign out of logbook.

Other Links

• Adding spatial scales