Equipment

Filters

Instructions for starting the system

Instructions for using the software

Instructions for shutting down the system

Problems







Equipment:

Nikon Diaphot 200 microscope

inverted
objectives available

Princeton Instrument camera

1300 X 1030
interline
cooled (-10oC) - do not set the camera for a lower temperature
12-bit
Pixel size is 6.7 X 6.7 microns
Peak sensitivity is at 500-550 nm.


Sutter Lambda 10-2 shutter and filter wheel (directly coupled to the microscope, fiber coupling available)

Hoffman Modulation Contrast is available for 10X and 40X.

Eppendorf transjector and injectman for injecting cells.




Filters:

The filters in the Sutter excitor filter wheel are:
Filter % Transmission Filter block
340 nm. - Fura
380 nm. - Fura
open 100% transmission Fret, FITC, TRITC
360 nm. - Fura, Triple (DAPI)
0.1 ND 80% transmission Fret, FITC, TRITC
closed 0% transmission
0.3 ND 50% transmission Fret, FITC, TRITC
570 nm. - Triple (Texas red)
0.6 ND 25% transmission Fret, FITC, TRITC
490 nm. - Triple (FITC, GFP)

Filter blocks

FURA block: dichroic (beamsplitter) and emission filter for dual FURA excitation. Chroma #72000 (no excitor filters in block).

TRIPLE block: dichroic (beamsplitter) and emission filters for DAPI, FITC and Texas Red Chroma #51006 (no excitor filters in block).

FITC block: excitor 480/30; beamsplitter 505 DCLP; emission 535/40 Chroma #31001

TRITC block: excitor 540/25; beamsplitter 565 DCLP; emission 605/55 Chroma #31002

FRET block: 440/20 nm excitor filter for CFP and a 460drlp dichroic (beamsplitter). This block has no emission filters - you must use the emission filter wheel which has filters for CFP and YFP. Since there is no emission filter in the block, do not look through the eyepieces with this block in place.


The filters in the Sutter emission filter wheel are:
Filter
closed
480/30 nm.
535/25 nm.
closed
closed
closed
closed
closed
closed
open


Instructions for starting the system:

1. Turn on the xenon lamp before anything else is turned on. Check the number of hours on the xenon lamp - let me know when it is at 400 hours.

2. Turn on the main switch and then turn on the equipment (left to right) that will be used. The shutter needs to be on to use just the software. Leave the focus motor off if it won't be used.

3. Turn on the computer at the Dell box.

4. Log on as keck/user if you plan to access other computers in the Keck Center. Otherwise it doesn't matter.

5. Start the MetaMorph software:

Meta Imaging Series folder:MM + filter wheel
MM+filter wheel +focus motor
MetaFluor




Instructions for using the software:

Set up the filters and optics:

1. Open "Devices/Illumination". Select Illumination Device #1. Open and close the shutter to make sure the Sutter is communicating with the software. If "Lambda-2 deivice not responding" comes up then push the reset button on the Lambda-2.

2. Select the filter cube on the microscope. The Fret, FITC (blue) and DAPI/FITC/Texas Red filter blocks are currently in the microscope.

3. Choose the excitor (wavelength) filter in the software:

Select the wavelength for excitation with the triple or fura block.
Choose open or neutral density filters if using the fret, FITC or TRITC blocks.

4. Choose the emission (intensity) filter:

Select the emission wavelength for the fret block.
Use the open position for all other blocks.

Select the objective and focus:

LOWER THE OBJECTIVE TURRET before changing objectives.
The fluor lenses are best for fluorescence.
Use a phase lens only if collecting a phase image or if you need the working distance.
Note whether the chosen objective requires oil, adjustment of the aperture or adjustment for the coverslip thickness.

Check that the light is going to the eyepieces and not the camera. Turn off the overhead lights. Open the shutter in the software - there should be light at the sample. If not, check the shutter behind the microscope. There are also two neutral density filters (without knobs) behind the microscope that are usually out.

1. Focus through the microscope then use the slider on the right side of the microscope to send the light to the camera.


Set up the acquisition parameters:

1. Select "Acquire from digital camera".

2. Define the acquisition settings - exposure time, regions, binning, auto scale, subtract background, and correct shading.

3. The camera may/not be parfocal with the microscope focus. Select "digital camera video control" to focus on the small monitor (quicker) or the focus in the "aquire from digital camera menu" to focus on the large computer screen.


Collect and Save images:

1. Expose and Transfer

2. Adjust exposure time and repeat if necessary

3. Save the image to a zip or jaz disk

4. Close the image.


Instructions for shutting down the system:

1. Exit the Meta software

2. Select shutdown from the Start menu at the bottom left corner of the computer screen.

3. Wait until the screen goes dark

4. Turn off the individual switches on the power strip from right to left (main switch last).

5. Turn off the xenon lamp

6. Cover the microscope only after the lamp(s) have cooled



Problems:

If the message "Lambda-2 not responding" comes up then push the reset button on the Sutter Lambda-2 box. In version 3.5 or 4.0, the program will look for the Ludl focus motor a few times. It eventually gives up and loads the program.

After selecting filters, when the shutter is opened in the software there should be light at the sample. If not, check the shutter behind the microscope. There are also two neutral density filters (without knobs) behind the microscope that are usually out.

Back to Keck Home Page