title graphic AIF graphic

complexexperiment.jpg (47821 bytes)Background:

Digital Station #5 was purchased with funds on a Shared Instrumentation grant in 2002 to meet the needs of high speed digital imaging of dual labeled particles moving in cells (e.g. CFP/YFP or GFP/TexasRed) and to perform ratio FRET (especially CFP/YFP).  Instrument is also used for long (6 to 18 hours) time lapse sequences.  Example publications with data from this instrument:


Sensicam QE air cooled CCD camera; I.P. Lab Spectrum running on a PC; Sutter DG4 for excitation; ASI filter wheel for emission; alternate DualView for emission; Uniblitz shutter for transmitted; piezo for 100um travel of focus; ASI electronically controlled stage; forced air or air/CO2 heated chamber.


Unlike other microscopes in the AIF, Digital Station #5 does not have a single standard configuration you can expect to find the system in every time you begin to work.  Therefore, you need to check that the instrument is configured for what you need.

Before Turning On, Install the Microscope objective lens.

You may not turn the microscope nosepiece to change objective lenses.  The lens must be changed manually by unscrewing it and screwing in the correct objective.

Make sure all electric power to the microscope devices is OFF before changing the objective.  This is important because you do not want to break the piezo (the little box next to the objective) or the stage.
piezo.jpg (18316 bytes)

Tilt the condenser back (or remove it completely).
tip-condenser.jpg (14395 bytes)

Take the stage plate off.
remove-stage.jpg (33060 bytes)

Remove and replace the objective.
remove-objective.jpg (42760 bytes)

Put the stage plate back in.  There are metal springs in the back right corner.  Push the stage plate down at an angle into the corner springs and then press the plate flat into the stage.
clips.jpg (8916 bytes)

Note that the piezo is off center.  The top stage plate must be placed off center as pictured.
offcenter.jpg (18261 bytes)

Tilt the condenser back down (or put it back in).

Filter Blocks.

The microscope should always be left with a JP4 CFP/YFP filter for ratio FRET in turret position C. 

Any changes in the filter blocks will be written in a table on the wall.

This is the configuration as of 28 March 2008.  However, except for the JP4 CFP/YFP filter, this may not be the actual configuration the microscope is in!


Filter turret in microscope (as of Jan 30, 2008):

Turret Label Name    Excitation    Emission    Dichroic 


  (green tape)
for use by eye
(none) approx.
450 to 485
530 to 570 nm
(see Chroma website for spectra as ASCII)

  (red tape)
cannot use by eye!
(none) (none) 86002v2
(see Chroma website for spectra as ASCII)


DG4 Excitation Filters.

The standard configuration is

  1. CFP exciter Semrock 434/17
  2. YFP exciter Semrock 500/24
  3. Rhodamine (560/55).
  4. CFP/YFP dual or GFP/FITC excitation (480/40) or the filter you may swap out as needed for your use.

In early 2008 maintenance on the instrument was done.  The following changes have been made.

Component Old Date changed New
Emission filter in DualView Chroma 465/30 20080326 Chroma 470/20
Excitation filter in Sutter DG4 position1 Chroma 430/25 20080328 Semrock 434/17
Excitation filter in Sutter DG4 position2 Chroma 500/20 20080328 Semrock 500/24

Filters changed in the DG4 sliders should be labeled at the outer edge of the slider.
dg4-filters.jpg (14581 bytes)

Environmental Chamber.

(Instructions on separate page.  Click here.)

Basic Startup.

  1. Sign in log book.
  2. Sutter DG4 Xenon arc lamp must go on first.  The switches are on top of the unit.   There is supposed to be a stepstool kept in the room so you may reach the switches.
    1.  Switch on left for light bulb.  2.  Switch on right for electronics.
    dg4.jpg (12303 bytes)
  3. Computer.
  4. Main power strip.  This will send power to the external filter wheel, camera, piezo focusing device, halogen lamp for brightfield or phase contrast and the shutter for the halogen lamp.  If any of these devices were turned off with their own individual power switches, you will have to turn them on before running the software.
    power-switch.jpg (17756 bytes)
  5. Log in and run IPLab software.
  6. A menu will pop up within IPLab asking you to Set Preferred Directory.  Set the directories and then click OK.  If you click Cancel, the software will not operate properly.
  7. There are two different
    CCD-5-startup-query.jpg (36124 bytes)
  8. If using bright field or phase contrast, set up microscope for Kohler illumination.

To image normal full frame, the DualView unit between the microscope and camera must be set to "Full View" and have the filter blocks pulled out one click as pictured.
dualview.jpg (17016 bytes)

What is the DualView?  Dual-ViewBrochure-11_02.pdf and http://www.optical-insights.com/micro-imager.html
Official alignment instructions:  DualView-alignment.pdf

Dynamic range of the cooled CCD camera is 0 to 4095.  Images are not rescaled to 0 to 65520.

The function keys are clearly labeled.  If you are used to using other AIF instruments, you will need to note the different function keys here.
fkeys.gif (33085 bytes)



Basic Shutdown.

  1. Quit software.
  2. Copy all files to Reststop, some other computer out on the Internet, CD, DVD or USB memory device.
  3. Turn off one main power switch to shutters, filter wheels, etc..
  4. If nobody is coming to use microscope within a few hours, turn off heater.
  5. If nobody is coming in with next hour or so, turn off DG4.
  6. Clean up!  Oil objective should be cleaned only by dragging lens tissue lightly over the glass and careful wiping of the metal around the glass.
  7. If you changed any filters, return to original configuration.
  8. Shut down computer.
  9. Sign out log book.
  10. Analyze and publish data.

Ratio FRET on fixed material.

There are scripts for automating the collection of ratio FRET images.

Each time the script is run the following images are collected and stored in a directory named the time of image collection:

DualView alignment instructions.


filter-specs.gif (10548 bytes) Filter specs for CFP/YFP.

The filters we have for the DualView are:
505dcxr D465/30 HQ535/30m  (CFP/YFP)
565dcxr D480/40 D630/50  (CFP/RFP)

Michael's scribbled notes:
o Green tape of alignment slide up
o Koehler brightfield

<Shift>F10 is alignment script.
run script
hit return
between yellow lines
stop = <shift>F9

say No to get ratio image of grid.


These dual excitation and dual emission filters were removed from the 51009 F/Cy3 block for dual imaging of red and green dyes.
dual-green-red-filters.gif (24717 bytes)
The filter block was then installed in the microscope.
To excite green or red individually, either the exciter in DG4 #2 or #4 position is activated.
To image green or red individually, the correct filter position in the external ASI wheel is selected or the green/red DualView insert is used.
The image is displayed on the camera as follows when the DualView and camera are in the below configuration.
splitscreen-key.gif (693 bytes)

dualview.gif (18908 bytes) camera-orientation.gif (17443 bytes)

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