CHAPTER 1 - SYSTEM OPERATIONLSM 880 Left Tool Area and Hardware Control Tools ZEISS10/2014 V_01 000000-2071-464 259(1) Manual Focusing on the Membrane• Acquire an image of the cell.• Place the focus above the cell and scan continuously.• Focus slowly down until you see a fluorescence signal.• Stop focusing.If the signal is too low for imaging, the membrane can still be detected using the Z Scan feature.(2) Focusing on the Membrane by a Z ScanThe Z Scan measures the count rate at previously defined x, y positions. It is performed without tablemovement, which is at the fixed position of the laser beam.• Acquire an image of the cell, focus through manually and select an equatorial section (middle of cell).Optional: Perform a Z-Stack that includes the membranes of the cell with the LSM and select anequatorial section.• Go to the Measure tool to Acquisition and the Positions submenu. Position the region of interest atthe site of the fixed laser beam in Current Position using the Crosshair or use the crossline in theLSM image to mark the position. In the latter case, Scanner must be active, since the XY-Stage willapproach the marked position only during a measurement!• Go to the X-Y-Z Scan tool and activate Z. The Z Scan display shows the current z-position of the laserbeam.• Select the Start and End Position and enter the Step Distance. If you have chosen an equatorialsection going 10 μm up and 10 mm down from the displayed current position are good values to startwith. Also a step distance of 0.5 μm should work well.5.3.19 SnapIf the Snap button is pressed, a measurement is triggered according to the System Configuration,Acquisition and Correlator settings.However, in regard to positions Snap triggers the measurement only at the highlighted or currentposition.5.3.20 Start ExperimentIf the Start Experiment button is pressed, a measurement is triggered according to the SystemConfiguration, Acquisition and Correlator settings.All selected positions will be approached sequentially.
