EPIFLUORESCENCE &/OR BRIGHTFIELD MICROSCOPY

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EPIFLUORESCENCE &/OR BRIGHTFIELD MICROSCOPY TURN ON THE FOLLOWING EQUIPMENT The fluorescent light (if needed) The power strip for the microscope and accessories The CoolSNAP HQ camera on the right

1 EPIFLUORESCENCE &/OR BRIGHTFIELD MICROSCOPY TURN ON THE FOLLOWING EQUIPMENT The fluorescent light (if needed) The power strip for the microscope and accessories The CoolSNAP HQ camera on the right (Turn off the QuantEM camera on the left.) The computer Start the NIS Elements AR software CONFIGURE THE MICROSCOPE AND FIND YOUR FIELD OF INTEREST Choose your objective. We currently have both DIC and phase contrast objectives. If you are imaging solely in fluorescence, the DIC objectives are recommended. Place your cells on the microscope. Setup environmental control and turn on Perfect Focus, if needed. Turn the black knob on the front of the microscope to Bino. Push the mirror switch lever (silver slider on the TIRF attachment) to the right. Choose an optical configuration for the eyes. Open the EPI shutter for fluorescence or the DIA shutter for brightfield, and center your cells in the field of view. Choose the Magnification 1x or 1.5x (black knob on the lower right side of the microscope). Make sure the dropdown list on the top menu matches the magnification so your images will be calibrated correctly. Close the shutter. *You may want to initialize Stage (Devices Initiate Stage) Epifluorescence &/or Brightfield Page 1

SET UP FOR FLUORESCENCE Choose an optical configuration. Make sure the SEDAT filter is in position #1 in the filter turret if you are imaging DAPI, FITC, TRITC or CY5.

2 SET UP FOR FLUORESCENCE Choose an optical configuration. Make sure the SEDAT filter is in position #1 in the filter turret if you are imaging DAPI, FITC, TRITC or CY5. Use neutral density (ND) filters whenever possible to protect live cells from photo damage. Move the DIC Analyzer out of the light path if it is not needed. SET UP FOR BRIGHTFIELD - KOEHLER ILLUMINATION Choose the BF optical configuration. Open the DIA shutter. Bring the cells into focus. Lower the field diaphragm lever on the dia-illuminator until the field diaphragm image appears in the field of view. Turn the condenser focus knob to bring the field diaphragm image into focus. Adjust the field diaphragm lever until the field diaphragm image is almost as large as the field of view. Turn the two condenser centering screws to move the field diaphragm image to the center of the field of view. Move the aperture diaphragm lever on the condenser to set the opening to ~75% of the NA of the objective. Move the field diaphragm lever on the dia-illuminator until the size of the field diaphragm image is slightly larger than the field of view. SET UP FOR PHASE CONTRAST IMAGING Choose a phase contrast objective. Turn the condenser turret to the module with the same Ph code as the objective. Turn the aperture diaphragm lever on the condenser to the right to open the aperture diaphragm fully. Set the eyepiece tube turret to position B and turn the Bertrand lens focusing screw to bring the annular diaphragm image into focus. Using the 2 yellow screwdrivers, turn the 2 annular diaphragm centering screws on the condenser module until the annular diaphragm image is centered with the phase plate image. Epifluorescence &/or Brightfield Page 2

SET UP FOR DIC IMAGING Choose a DIC objective Turn the condenser turret to the module with the DIC code.

3 Return the eyepiece tube turret to position O. Remove the NCB11 filter on the dia-illuminator and move the GIF filter into the optical path to improve the contrast. SET UP FOR DIC IMAGING Choose a DIC objective Turn the condenser turret to the module with the DIC code. Slide the analyzer into place at the base of the objective turret (slide to the left). Slide the polarizer (located above the condenser turret) into position (slide to the left). Adjust the lever on the polarizer to adjust the level of image sharpness. Epifluorescence &/or Brightfield Page 3

CHOOSE A WIDE FIELD OPTICAL CONFIGURATION To view your sample through the eye pieces, select either brightfield or one of the fluorescent settings at the top of the OC panel.

Wide field To switch to the camera mode, select a wide field optical configuration from the OC Panel TL TIRF In the TE2000 Pad window, check that the light path goes to the bottom port.

4 CHOOSE A WIDE FIELD OPTICAL CONFIGURATION To view your sample through the eye pieces, select either brightfield or one of the fluorescent settings at the top of the OC panel. Eyes Make sure then lightpath goes to the top eye piece port. Wide field To switch to the camera mode, select a wide field optical configuration from the OC Panel TL TIRF In the TE2000 Pad window, check that the light path goes to the bottom port. Sweptfield Faux RGB SETUP THE CAMERA Click on LIVE (the green triangle on the top menu) to get a live view from the camera. In the CoolSNAP HQ Settings window, choose an exposure time. This camera has a 1392x1040 pixel array, so binning may be useful to increase signal. Start with 4x calibrated gain for fluorescence or 1x for brightfield. In the LUTs window, adjust the scale (or use Auto Scale) for better visualization. Epifluorescence &/or Brightfield Page 4

SETUP FOR MULTIDIMENTIONAL (ND) ACQUISITION Set up experiment parameters in the Time, XY Pos, Lambda and Large Image tabs of the

The program then calcuates how many loops are requred based upon the time parameters you have chosen.

1 XY Position: You can choose multiple areas with in your dish to image.

5 SETUP FOR MULTIDIMENTIONAL (ND) ACQUISITION Set up experiment parameters in the Time, XY Pos, Lambda and Large Image tabs of the ND Acquisition window. Time: Choose the time interval between when images are taken Choose the duration of the movie. The program then calcuates how many loops are requred based upon the time parameters you have chosen. Stage Accuracy settings: -Go to Devices Manage Devices XY Drive Device Parameters -Change the accuracy setting from Open to 0.1 XY Position: You can choose multiple areas with in your dish to image. Find an area/cell you want to image Make sure perfect focus is turned on. Focus the image. Then click on the box under image position. Repeat for each area in the dish you want to film. Epifluorescence &/or Brightfield Page 5

RUNNING THE EXPERIMENT Once the experiment is defined, save the ND file to the D drive. Please be sure to make a new folder with your name on it.

6 Lamba: The lambda tab allows you to choose the optical configuration you want to use. Under the T position column you can opt to image that channel at every time point, every Nth time point, or just at the beginning. RUNNING THE EXPERIMENT Once the experiment is defined, save the ND file to the D drive. Please be sure to make a new folder with your name on it. Choose a file name subsequent experimental runs will be labeled as N+1. The 1 time loop option will run your ND experiment through 1 time interval. This will allow you to determine if all the parameters and settings are correct before you start the longer experiment in its entirty. When you are ready, select Run Now. WHEN YOU ARE DONE Close Elements, and move files off the D Drive onto the network drive. Shut down the computer. Turn off the fluorescent light and the microscope (power strip). Return the magnification to 1x. Cover the microscope. Clean up. Epifluorescence &/or Brightfield Page 6

7 Nikon TE2000 Microscope Epifluorescence &/or Brightfield Page 7

Nikon E800 Operating Instructions.

Nikon E800 Operating Instructions. You can request electronic copies of this manual by contacting lshats@jhsph.edu Copies are also available on the JHU MMI Department web site. Please send your comments