Andor XD Spinning Disk Confocal Microscope

Power-up procedure

Only turn on the things you need.

1. If you will be using fluorescence for eyepiece visualization, turn on the Prior arc lamp, otherwise leave it off.

2. Switch on the power strip. This will turn on the Andor controller and lasers, microscope controller, microscope, cameras, the Yokogawa spinnining disk module, ASI stage controller, and the motorized dichroics.

3. If you are going to use the onstage incubator, switch on the main power of the Tokai Hit control unit. Turn on the heaters: top heater, stage heater, and/or lens heater that you need. Before you put on the lens heater, you should first turn the lens to position using the Olympus keypad (or in MetaMorph if the program is already open) to avoid tangling of the wire. You don't need the lens heater for dry objectives.

The onstage incubator comes with an adaptor for one 35mm dish or one slide. The incubator has a water reservior built-in. To keep the chamber humidified during acquisition, first sit the incubator on the stage, then fill the reservior with deionized water from a water bottle (supplied). Be very careful not to spill water in the microscope. To ensure safe clean up after the experiment, aspirate the water away with the vacuum aspirator by the system before removing from the stage. Then clean the reservoir with paper towels.

4. Turn on the computer, log in, start MetaMorph, and sign in the logbook.

The Microscope

Nearly everything on the microscope is motorized so can be controlled through MetaMorph.

Objectives

Mag
NA
Immersion
DIC
Working Distance
10x
0.3
Dry
DIC
10 mm
20x
0.5
Dry
DIC
2.1 mm
40x
1.3
Oil
DIC
0.2 mm
60x
1.3
Silicon Oil
DIC
0.3 mm
60x
1.3
Water
DIC
0.28 mm
100x
1.4
Oil
DIC
0.13 mm

To view your sample down the eyepieces

Pull the laser safety slider to the right . . .

laser safety slider and light intensity

  • From 'Illum' at the top left in MetaMorph, select 'Eyes/Orca - DIC' or other fluorescent illumination for 'Eyes'
  • Click the shutter icon next to the illumination list to open/close the shutter (always keep exposure to a minimum)
    illum, shutter and objective
  • Brightfield light intensity can be controlled by the 'up' and 'down' buttons on the front of the microscope. Fluorescent light intensity is controlled by the dial on the arc lamp.
  • Focus knobs are on both sides of the microscope. You can choose between  fine (F) and coarse (C) focusing by pushing the lower green button. The button labeled with 'ESC' is used to lower down or raise up the objective with one push. 
    focus knob
  • X-Y stage movement is controlled through the joystick on the ASI controller. Push the button on top of the handle to choose speed for 'f'ast' or 'slow' movement.

    ASI joystick

Image acquisition using MetaMorph

Acquisition through the "Multi Dimensional Acquisition" dialog box

  • Slide the laser safety slider to the left. You should feel the click.
  • Open the MDA dialog box for Timelapse, Multiple Wavelength, and Z Series for 3D acquisition. Choose the functions you need. Leave the 'Use Dual Z Moters' selected. Hardware auto focus is optional (see procedure below).

MDA

Main - Select the things you want to do and then go through each tab:

Saving

  • Select directory on D: drive (if you are acquring at a very high frame rate (eg streaming), use the hard-drive and then move the data afterwards to your server/external drive)
  • Choose a base file name (all the images will be named basename-t1z1 etc)
  • (Optional) Check the increment base file name box if your experiments share the same base name
  • Your images are automatically saved along with acquisition.

Timelapse

  • Choose number of time points, duration and interval (one will always be a consequence of the other two)

Wavelength

  • Illumination -  select the illumination you want to use from the dropdown illumination list (e.g. Confocal 405, Confocal 488, DIC confocal, etc.)
  • If using EM gain, choose Digitizer of '14-bit (3 MHz, EM Gain)'. Set EM Gain (maximal 300). Be aware, the higher EM gain, the more noise in your images.
  • Set exposure time - minimum possible to get a reasonable image
  • Press the full chip button before you start to make sure the entire camera chip is used
  • Test the settings using the 'Live' and 'Snap' buttons at the bottom of the box (hover the mouse over the icons to find out which is which)
  • The laser power has been set at 20% for all lasers as default. To change the laser power, click 'Laser Power Control' on the taskbar and open the control box. Adjust the laser power and choose the minimal laser output possible.
  • Laser power
  • Optimize the acquisition condition for each channel.

Stage

  • Multiple positions can be saved as a position list. Please delete or override the default 'Position 1'.
stage positions

Set Focus: You are able to use 'Hardware Auto Focus' in time-lapse experiment. For current objectives on the microscope, only 20x (dry), 40x (oil), 60x (silicon oil), and 100x (oil) objectives work.

To do 'Harware Auto Focus': (1) Enable the function in 'Main' page; (2) locate an imaging position, click 'Set Focus'. The system will set the focus and 'beep' once; if it beeps 3 times, 'Set Focus' fails; (3) add the position in the list; (4) repeat steps (2) and (3) for all positions.

Display: Default is okay for most things, for high frame rates they should be minimized to not slow down the computer

Summary: An overview of the settings for your experiment

Settings and configuration can (and should) be saved through 'Save State' function and reloaded ('Load State') in future experiments.

Press the 'Acquire' button to start your multidimensional acquisition.

Modifying settings during acquisition

The most recent image/stack of each channel will be displayed as you acquire the images.

A different acquisition dialogue box shows up, in which information about time points, stage positions and Z positions will be display in real time. In addition, the acquisition can be paused for user to change time interval, to update stage position ('Set to current'), or to take the chance to apply treatment to the samples. 

'Resume' button continues acquisition. To finish the experiment, click 'Cancel' button anytime or hit 'Esc' on the keyboard.

duke lmcf pause and cancel mda

Acquisition through the "Acquire" dialog box

This is for simple images of your samples. It can be opened from the task bar 'Acquire' or from the dropdown menu, Acquire> Acquire

MM Acquire

  • Set the display to "Autoscale" - the triangles on the image histogram show the scale min and max.
  • The image gamma - the relationship between measured and displayed brightness. 1=linear
  • Exposure Time - Type in value (or choose AutoExpose)
  • Full chip uses entire CCD camera, sub-arrays produce smaller files and may be faster
  • Select illumination from the 'Illum' list, press 'Show Live' to see the specimen through the camera - it may be necessary to focus or move the stage slightly
  • Clicking the folder icon allows you to set whether each image starts in a new window or overwrites the previous image (handy for optimizing your image, be careful not to lose your data though)
  • External shutter linked to camera should be [Current Shutter]

Press the 'Acquire' button to capture the image

Saving images

Simply click 'Save' and name your file. Images will be saved as TIFF images with all the original data. Not all programs will open these images. Photoshop will open the files as 16-bit images, you will need to adjust the levels ON A COPY of the file. ImageJ and FIJI can open them also.

(A) The original source images are grey-scale images, although they are displayed in color. To save the images in color, open the 'Overlay Images' functional box from the task bar, add an image (from the 'Image' drop-down menu) to a color channel and click 'Apply', or add images to more than one color channels to generate a merged image.

(B) To save your images as 8-bit grey-scale files (compatible with all programs)

  • Press 'Copy to 8-bit Image' button on the task bar
  • Select the desired image from the 'Image:' drop-down menu
  • Press Copy and Save by going to File >Save as (make sure the file name is different otherwise you will overwrite the original)

Pseudocolored and 8-bit converted images should not be used for fluorescent intensity-based analysis and quantification.

Review multidimensional data

(A) Tell metamorph which images you want to build. Press "select base file" in the main 'Review Multi Dimensional Data' window which gives you this box -

duke lmcf review MDA select files
  • Select directory on D: drive - point to your folder with the images
  • Select data set - choose the individual experiement you want to look at
  • Press 'View'

(B) Select the wavelengths, stage positions, Z-slices and timepoints you want to view.

review multi dimensional data-2
  • Select the stage position (one at a time here) and channels you want (probably all of them)
  • Right clicking in the boxes selects that image. Right click on the row/column heading selects the entire row/column
  • You can choose multi-colour overlay (under display), projection, or best focus if wanted.
  • Use the play and reverse arrows to view the timecouse etc.
  • Load Images loads the select images to a single file which can be saved as one or exported to avi or quicktime (Stack/Make Movie from the top menu).

Time and scale stamps

(A) Most of the time, a time stamp is required in a timelapse movie. Under 'Display' tab, find 'Graphics' --> 'Date/Time'. For most time-lapse experiment, 'Elapsed time' is used. The time format can be set to days, hours, minutes, seconds, milliseconds, or in combination. 'Stamp' the time on the current image or all images if 'all planes' is selected.
time stamp

(B) The Scale stamp is also found in 'Display --> Graphics'. Stamp a scale bar on the image(s) similarly to 'Date and Time'.
If you work on your images on one of LMCF workstations and want to add a scale bar on the images, you first need to go to 'Measure'-->'Calibrate Distance', and from the dialogue box to 'Load the (calibration) File' for the Andor spinning disk confocal microscope. Click the objective used for imaging and 'Apply' the calibration to the images.

calibrate distance

Finishing and shutting down

Check the booking calendar when you finish your session. Record your use of the system using the CoreResearch booking system

If there is somebody using the arc lamp after you in 1 hour: Clean the objective(s) you have used using lens tissue and cotton tipped applicators, close metamorph, log-off the computer. If the next user will not use the fluorescence source, the arc lamp can be switched off even if the rest of the system is on.

If there is nobody booking after you within 1 hour: Shutdown the computer and switch off the system. If you have used the lens heater, make sure to take off the lens heater from the objective. Otherwise the wire will be tangled on the lenses when the next user will use another objective lens. Cover the microscope.