Image Acquisition using the Radiance 2100

The LaserSharp 2000 software supports user profiles. Double-click the LaserSharp 2000 icon; select your name from the drop-down list that appears and enter your password.

The LaserSharp 2000 interface consists of a main menu and three windows: the Experiment Browser; the Image Display Window and the Control Panel. The full interface can be seen here.

First Steps

Load a Method

Settings for acquiring single and multi-channel datasets, along with more exotic configurations, are stored in the form of methods. Preset methods can be accessed by clicking on 'Methods' in the main menu.

Recently used methods can be selected from the drop-down list. If the method you want isn't there, click on 'Methods...' to view an expanded list.

Click a method on this list and click the 'Load the Selected Method' icon (below) to configure the system with your desired settings.

Set the Objective Lens

Select the correct objective lens from the drop-down window in the Control Panel. This will ensure that subsequent images are correctly spatially calibrated.

Channels

The Channels section contains controls allowing the user to switch between simultaneous and sequential acquisition modes, as well as tools for setting laser power, iris size, gain and offset for each channel.

Simultaneous and Sequential Acquisition

The Radiance has three detectors and can acquire signals in up to three channels simultaneously. Alternatively signal can be acquired sequentially channel by channel. Simultaneous acquisition has the advantage of speed but may result in the detection of signal from more than one dye in a single detector. This can be caused either by co-excitation of two dyes by one laser, or by the cross-talk of dyes with similar emission spectra. Sequential acquisition alleviates this by only exciting with one laser at a time, and only collecting into one detector at a time. Sequential acquisition is slower than simultaneous acquisition.

The buttons at the top of the Channels section allow the user to switch between simultaneous and sequential settings. The image above shows the options available in a three-channel method. The buttons labelled FITC, TxRed and Cy5 allow access to the controls for the sequential settings.

Laser Power, Iris, Gain and Offset

Laser Power

The required laser line(s) can be selected using the tabs on the left. Power can be adjusted using the slider. Note that multiple lasers will be available in simultaneous mode, but only one will be available under any sequential setting. Note also that the argon laser has four lines at 457, 476, 488 and 514 nm, and all of these will appear in a sequential setting that uses the argon laser; make sure that you adjust the power of the correct line.

Increasing laser power will increase fluorescence emission from your sample up to a point, but it will also increase the rate of photobleaching, so very high laser powers are seldom desirable for most biological samples.

Iris

The iris (also known as the pinhole) is the aperture in front of the detector that eliminates out of focus light. The slider allows the user to increase or decrease the pinhole diameter; allowing more light to fall on the detector in the first case or improving resolution (especially axial resolution) in the second. There is an optimum resolution and therefore an optimum pinhole diameter associated with each combination of objective lens and wavelength. The optimum diameter for the current combination can be set by clicking the target icon below the iris slider.

Gain

Gain increases the signal from the detector. It can boost the signal from a dim sample but will also amplify noise, which manifests itself as speckles on the image. Increased levels of noise in the image can be smoothed by Kalman Averaging. The relationship between the slider position and the voltage applied to the detector is linear up to a value of 33.3%, after which the gain increase becomes non-linear and noise can increase at a greater rate. Try to keep gain below 33.3%.

Offset

The detector offset allows adjustment of the black level in the image. The actual level of black (zero signal) is arbitrarily set according to the detector and will not necessarily correspond to the dimmest features in your sample, which may be brighter due to autofluorescence and other phenomena. Offset allows the user to set the image black level to match the darkest features in the sample (ideally the background).