Huang: Microscope Protocol

Setting up

1. Turn on microscope control box, piezo stage insert and fluorescence box (FB should not be turned on and off rapidly, if warm should not be turned on for 15 minutes – check schedule to see if anyone is on after you and call to check)
2. Turn camera on
3. Heating unit (do not change controls, 2 and 4 are supposed to be best), temperature control unit (nominal is temperature is set to) takes about an hour to warm up and to equalize. Make sure doors are shut! Can turn it on over the web.
4. Check all 4 doors that are closed to reduce temperature fluctuations.
5. Open MetaMorph
6. Focus first 10x, 40x and should be fine for 100x
7. Before changing sample MAKE SURE objective is all the way down.

MetaMorph

1. Aquire - Acquire
2. Show live

• Can change illumination directly and hit F2 Stop live and then Show live again.
• Can change autoexposure (2-5 ms seems to work pretty well – sensitive camera).
• Save ‘Live’: save with y_m_d + very informative description have each exp in its own folder. Make sure you are in your own folder before you start saving. (it will automatically save info described under annotate but it’s better to have a quick sense of what is in the picture). Can also set the save path.
• Special is where all the gain information is. Want gain set to 1. EM gain sends photon count to photon multiplier before it gets sent to CCD should be set to 100 if can’t see anything can set it higher (it will improve things only up to a point).
• On main screen:

On the stage there is a piezo set at -50 for automatic focusing: travels between 0 and -100.

How to acquire multidimensional images

1. Apps -> Multidimensional acquisition

• Journals are programs that teach you how to do stuff
• Can save and load states
• Stream is if want to send data out really fast.
• On Stage can add positions
• Binning how many pixels
• Set Autofocus to directional average

Microscope

• There are 4 buttons behind the focus wheel on the left : from top bright field, phase, fluorescence and fluorescence+phase

Can also turn to fluorescence with 6 controls at front, it will say on the screen which one is which (GFP, CFP, YFP, Texas Red, Dapi).

• 3 buttons on the right: Zup set Zdown
• The position of the objective relative to the point you define is on the screen.
• Can press twice Zup+Set and will set to 0.
• To change the focus wheel from fine to coarse press button on the left of stage controller.
• On stage controller back wheel is for z axis, front top is y and bottom x.
• To go up in resolution press the Up button on the stage controller Down to go down. The buttons on the right side change from fine to coarse.
• There are 6 buttons in front of focus wheel on the left of the microscope change the aperture (can’t change in phase), diafragm and intensity (top 2 buttons, goes max to 20) of the light.

Turning system off

1. Turn off microscope control box, piezo stage insert and fluorescence box (FB should not be turned on and off rapidly, if warm should not be turned on for 15 minutes – check schedule to see if anyone is on after you and call to check)
2. Turn camera off
3. Turn off heating unit then wait 15 minutes and turn off temperature control unit.