Leica Confocal Instructions: Software Setup

	Keck Imaging Center	Leica Confocal Instructions: Software Setup
Keck Center Home

Start the Software

Once the computer has booted up, press Ctrl + Alt + Del to get the Logon window.

Enter your User Name and Password.

If you're going to be producing large z-series or time series, check the hard drive space first. Double-click "My Computer" on the desktop. Right click on D: and select "Properties" from the list. Let Greg know if there is less than 1 gigabyte on D:

Double click on the Leica Confocal Software icon

Configure the Beam Path Settings

1) Click the "Beam" button:

2) Select an initial setting from the list:

This will be a general setting to get you started. The "Leica" menu has settings for common fluorophores, organized by excitation wavelength(s).

Double-click on a setting from the list that matches the fluorophore you want to image.

If the fluorophore you are using isn't listed then select one that is the same color. This will set basic parameters.

Settings you save will be under the "User" menu.

3) Check the excitation:

The AOTF sets the excitation wavelength and intensity.

The box should be active (checked).

For fixed tissue, start with the laser(s) to be used at 100% EXCEPT for the HeNe laser (633nm) which should be set at 33%.

Use these sliders to attenuate the excitation light intensity as needed -- you will rarely leave them at 100%.

Laser lines not in use should be at 0%.

3) Check the dichroic:

The dichroic mirror reflects the excitation light onto the specimen. Click on the green diagonal to get a list of options.

RSP500 -- single labels (488 nm excitation).
DD488/568 -- double labels (488 and 561 nm excitation) or single labels (561 nm excitation).
RSP465 -- single labels using 458 nm excitation.
Substrat -- multiphoton imaging.
TD488/568/633 -- triple labels (all three lasers), double labels (561 and 633 nm), or single labels (633 nm)

4) Check the detection parameters:

Activate the PMT(s) to be used (6). Use PMT 2 for single labels, it is the most sensitive PMT.

Select the fluorophore to be imaged with each PMT from the drop-down list (1). The emission spectra of that fluorophore will be shown on the spectral display (2). If your fluorophore is not on the list, choose one that is similar. Ask Greg to help you to add your fluorophore to the list.

Set the detection band width (3) to collect as much of the emission spectrum as possible. You can drag either end of the bar, or move the entire bar up or down the spectrum. Double click on the bar to get the numerical values for the detection band width (you can also set the band width limits in that window). Note that the laser line is indicated on the spectral display. Be sure and keep the detection band width 10 nm away from the laser line.

Select a Look-Up Table (LUT) from the list displayed

Configure Imaging Parameters

Mode: select xyz for normal scanning.

Format: defines the pixel dimensions of the image. Note that the scanned area does not change, so changing the format will change the pixel size.

64 x 64
128 x 128
256 x 256
512 x 512 - start here
1024 x 1024

Speed:

high (live imaging or specimens that are photosensitive)
medium (most samples) -start here
low (to increase the intensity i.e. longer dwell time/pixel)

Pinhole:

Click the pinhole button, then on "Airy 1". This is the best compromise of signal vs. axial resolution.

Zoom:

Start at a zoom of 1. Note that the pixel format does not change, so changing the zoom will change the pixel size.

Bidirectional scanning:

Leave set for unidirectional scanning (image). Bidirectional scanning is for live imaging or specimens that are photosensitive.

Contact Greg Martin at gvm@u.washington.edu for additional information. Suggestions and comments are always welcome!

Keck Imaging Center