Leica TCS SP8 SMD, Руководство пользователя

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F(L)CS Data Acquisition
18 F(L)CS Data Acquisition
With the TCS SP8 SMD, you can carry out point measurements and FCS and FLCS data
acquisitions are possible.
With sensitive detectors such as APDs, FCS measurements can be carried out. In all point
measurements, the fluorescence is recorded at a certain position in the specimen and
analyzed in the SymPhoTime software.
If no pulsed lasers are attached to the system or if lifetime information is not needed,
continuous wave lasers from TCS SP8 can be used for the fluorescence excitation.
18.1 Preparing the FCS Measurement
This chapter includes steps that are helpful in preparing for an FCS measurement, not the
description of the FCS measurement itself. That information can be found in
Chapter 18.2 to
Chapter 18.4 and in Chapter 19 .
18.1.1 Selecting an Objective
• Select a suitable objective: Since with FCS variations the focus size and shape have a
particularly significant impact, we recommend using water immersion objectives for in
vitro assays and in vivo experiments.
• For good FCS results, adjust the cover slip thickness with the correction ring (if available
with the objective used).
• Select an objective with a high numerical aperture in order to maintain the smallest
possible observation volume and yet to collect as many emitting photons as possible.
We recommend the water immersion objective HCX PL APO 63x/1.2W CORR CS
0.14 to 0.18
with manual or motorized correction ring. This objective functions
exclusively with specimen slides or (glass-bottomed) measurement chambers that are
140
μ m to 180 μ m thick (e.g. cover slip types #1.0 or #1.5).
18.1.2 Calibrating the Positioning Accuracy of the FCS Measuring Point
The measuring point is positioned via the scanner. Positioning accuracy can be optimized
using a calibration function. Repeat this procedure about every 3 days. If you determine that
the system is stable, you can also increase the time between fine-tuning sessions.
Use a bleach specimen with fixed fluorescence that is capable of being excited with a
wavelength in the visible range and can also be photobleached with higher laser intensity.
The bleach specimen may be a chroma slide (fluorescent dye that has been allowed to dry
onto a cover slip), or you can mark the top side of a cover slip with a fluorescent marker.
1. Switch the confocal system on and start the LAS AF software as described in the
instructions in Chapter 14.1 . Select the conventional mode.
2. Activate the lasers in the Configuration step, Lasers button. Set the Gain for the argon
laser to a value between 20 and 30 %.