SP5 Image Acquisition

Image Acquisition: First Steps

The SP5 confocal is controlled through the Leica Application Suite: Advanced Fluorescence (LAS AF) software. The same basic software suite is also used to control the TCS SPE confocal, and a number of other Leica microscope systems not available at the MRC LMCB. Versions of the software are available for offline analysis of data; see facility manager Andrew Vaughan for details.

User Interface

The LAS AF user interface consists of a viewer display window where images are displayed; and a working area, with main menu bar and arrow symbols for selecting the operating step (see an image of the Working Area here). By default LAS AF opens at the Acquire operating step.

Laser Initialisation

The system's lasers will have to be turned on in the Configuration window after initial system start-up, otherwise illumination of the sample will not be possible. See the Start-up page for details.

The Acquire Operating Step

Make sure that the operating step arrow is set to Acquire (below).

The Acquire view consists of the Experiment and Acquisition tabs and the Beam Path Settings work area (see User Interface - Working Area).

Acquisition Mode

The Acquisition Mode panel can contain up to four controls:

• The drop-down menu on the left allows the user to select from a range of 2D, 3D, 4D (e.g. xyzt) and 5D (xyzλt) scanning modes.
• The seq. button switches between single (simultaneous) and sequential scanning modes. The default setting is single; if sequential mode is activated the button turns from black to brown.
• The Tile Scan and Mark & Find buttons on the right of the panel activate advanced settings that use the microscope's motorised stage. If the stage is not initialised during start-up of the software these buttons will not appear.

Note

If you can't see the controls you're looking for, their panel may have been hidden. Panels can be hidden by clicking the arrow icon in their top right corner (below):

Clicking the arrow causes the panel to 'collapse' into a single line of text. A panel can be expanded again by finding its collapsed form and clicking on the arrow icon to expand it.

The question mark icon provides help about the features accessible through the panel.

XY Scan Settings

Scan settings common to all acquisition modes can be adjusted from within the above panel.

Format

The scan format specifies the 2-dimensional array of points that the scanner will acquire. The format corresponds to the number of pixels forming the image on the computer monitor; provided the ratio has been set to 1:1 in the image viewer. Format is one of the settings (together with Zoom, Pinhole and Objective) that is of principal importance in defining resolution.

Pinhole

The pinhole slider appears at the bottom of the XY panel when the Pinhole button is clicked.

The pinhole (or iris) is located in front of the confocal microscope's photo-multiplier tube (PMT) detectors and is responsible for reducing the amount of out-of-focus light that enters the detectors. In general, the smaller the pinhole the less out-of-focus light there is contaminating the image; but for any defined set of conditions there is a pinhole size below which no more stray light is excluded. This pinhole size is defined in part by the chosen objective lens and is represented by a value of 1 Airy Unit.

Clicking the Airy 1 button will set the pinhole to optimum resolution for the current set of conditions. The drop-down menu on the left allows the user to change the pinhole units from Airy units to microns, and back.

Increases in the size of the pinhole have a greater detrimental effect on resolution in Z than on resolution in X and Y - by a factor of a square.

Speed

The SP5 is a laser scanning confocal microscope (LSCM). LSCMs gradually build an image from multiple dots scanned line-by-line to create a frame; this is in contrast to a camera, which acquires a full frame in a single exposure. The speed drop-down menu defines the line frequency of the scan. A line frequency of 400 Hz means that 400 lines are acquired per second.

Bidirectional X

The standard scanning mode is unidirectional. In this mode the scanner acquires fluorescence data while scanning in one direction (e.g. left to right) and then rapidly 'flies back' without acquiring data in the opposite direction. In bidirectional mode the scanner acquires data in both directions, shortening the time it takes to scan a frame.

Zoom Factor

The mirror that scans the laser across the sample can be programmed to scan in the same format (e.g. 512 x 512) but over a smaller area. The area scanned is the Image Size. Since the format is unchanged each of the dots that make up the image now represents a smaller area; this is reflected in a change in the Pixel Size. Increasing the scan format (e.g. to 1024 x 1024) increases the number of dots in the image area and correspondingly decreases the pixel size independently of the zoom factor (and image size). The image size and pixel size are also dependent on the objective lens being used.

The image size (and hence the scanned area) can be reduced using the zoom factor slider. In addition, the Zoom in check box activates the Region of Interest (ROI) tool. This allows an ROI to be drawn on the current image or live scan; which the scanner then zooms into. A third method of zooming involves configuring one of the control panel knobs as a zoom control and using this to zoom in and out.

Once the scanned area has been zoomed it can be panned vertically, horizontally and diagonally using the arrow buttons. Clicking the centre circle centres the scanner.

Averaging & Accumulation

Acquisition and averaging of multiple identical image frames reduces the contribution of random noise in the image, which can be a feature when the system is being operated at high gain levels. Averaging can be done on a line-by-line or frame-by-frame basis and the number of frames to be averaged is selectable from drop-down lists.

Note

Averaging is usually unavailable in the live scanning mode, but line averaging can be performed live if switched on in the Configuration view. The fact that multiple images must be acquired means that the live scan will proceed more slowly if this setting is activated.
The relationship between signal and noise means that the following relationships hold true:

Note that the improvement in the images as a result of increases in the signal to noise ratio remain relatively modest; with the benefits decreasing as large numbers of frames are averaged. If the increase in photo-bleaching seen with multiple acquisitions is taken into account then there is not much to be gained by averaging a great many times. The image below illustrates the smoothing affect averaging has on an image.

Images can be accumulated (i.e. summed) rather than averaged. This increases the intensity of images acquired from very dim samples. One drawback is that the background and noise are also summed, so accumulation can be combined with averaging to generate a compromise between intensity and high signal to noise ratio.

The Auto Gain feature automatically adjusts the gain level to increase contrast in the image. It does this by automatically selecting a narrow ROI in the middle of the field and scanning it several times at increasing and decreasing gain levels until the correct contrast is achieved; it then acquires an image. If the brightness of the sample and or the laser power are low, the use of auto gain can result in a rather noisy image.

Rotation

The scan mirror can be rotated as well as panned (see zoom factor, above). Rotation can be performed by moving the slider, or by using the rotation setting on the Control Panel.

Important

The mirror cannot be rotated through 360°. Rotation of the scanner beyond its limit can damage the machine.