Zeiss LSM 880 Operating Manual page 98

CHAPTER 1 - SYSTEM OPERATION
ZEISS Left Tool Area and Hardware Control Tools LSM 880
A beam splitter is chosen according to the selected laser line(s). It separates the excitation light from the
emission signal. These filters reflect the wavelength indicated and transmit all other wavelengths.
Make sure there is no filter in the reflector turret (None) or (for a scan head mounted to the rear port of a
inverted microscope) it is set to the mirror position (Rear).
For working in non descanned mode (multiphoton imaging) please refer to CHAPTER 5: MULTIPHOTON
LASER SCANNING MICROSCOPY WITH LSM FAMILY SYSTEMS.
Lambda Mode
The Lambda mode is chosen for imaging heavily overlapping emission signals. When choosing this mode
by activating the Lambda mode button display changes to show the currently active laser line(s) and the
detection range of the QUASAR detector.
With this imaging mode, it is possible to acquire an intensity image displaying the intensity of the
fluorochrome(s) within a spectral bandwidth of 10 nm. According to the detection range that is set for
the QUASAR detector, a specific number of such images is acquired which is called a Lambda stack.
These image data provide the intensity information within the selected detection range for each pixel.
Therefore, the data allow deducing an emission spectrum for each pixel corresponding to the emission
spectrum of a specific dye.
This calculation is done by linear unmixing (see section Unmixing View) using the acquired Lambda
stack and allows to clearly separate even heavily overlapping emission signals.
A workflow similar to the Channel mode is applied. The number of scans changes with the hardware
(settings). A full spectrum can be acquired with one scan using a 32 channel Quasar detector with or
without the flanking PMTs and a spectral resolution of 8.9 nm or higher. For all other combinations like
using a three channels Quasar or a higher spectral resolution more scans are required for spectral
imaging. The spectral resolution is set in the Range column.
Online Fingerprinting
The imaging mode Online Fingerprinting is based on the acquisition of a Lambda stack. It requires
using already available emission spectra from the dyes which are used as markers in the imaged
specimen. The choice of the emission spectra and the setting of the detection range for the QUASAR
detector are defined in the loaded configuration or using the Imaging Setup tool.
Within the Use column the spectra of the dyes for unmixing are chosen /added.
The detection range is equal for all dyes. When changing it for one dye it will be changed for all other
dyes.
All other tools are controlled as described for the Channels mode.
For best results, the settings for Online Fingerprinting should match the settings of the Lamdba Mode
when the dyes were acquired for defining the reference spectra.
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