| | Excitation Fingerprinting | |  |
| | |
What is Excitation Fingerprinting?
This is another innovative method Carl Zeiss provides for the unambiguous separation of fluorescence signals.
The automated image acquisition is the basis for the recording of excitation spectra. Suitable acquisition parameters provided the excitation spectrum first supplies information about the near-infrared excitation wavelength that is most efficient for the dye used. The total intensity of the fluorescence signal changes with the excitation wavelength. It is recorded and results in a profile that is a dye-specific, identifying characteristic, same as the emission spectrum. The method is available for multiphoton microscopy and permits the separation even of fluorescences originating from tissue layers deep below the surface, since any available detector, including non-descanned ones, can be used for signal registration. |  Overview
 Multifluorescence
 Emission Fingerprinting
Excitation Fingerprinting
MultiChannel Unmixing
Multitracking
Online Fingerprinting
Automatic Component Extraction
appropriate products
LSM 710 NLO
|
| |
Excitation spectra of fluorescent proteins, recorded with LSM 710 . |
| |
How does Excitation Fingerprinting work?
The first step in Excitation Fingerprinting is the acquisition of an image stack, in which the excitation wavelength is automatically changed from image to image as preselected by the user. Accordingly, each single image of the stack differs by the intensity of the fluorescence signal. The graph of intensity as a function of the excitation wavelength yields the excitation spectrum. The spectra can be saved and used for identifying the respective fluorescence signals. By means of Linear Unmixing, a mathematical algorithm, the dye components existing in the image are precisely computed and separately displayed for each pixel, based on the given spectra. The results are highly informative and reliable images which illustrate the distribution of the various fluorescent tags in the specimen. |
| |
 |  |
Zebra fish with fluorescent labeling
of neurons | Separation of label and autofluorescence with Excitation Fingerprinting |
|
| |
Easy generation of excitation spectra
Identifying the optimum excitation wavelength for a fluorochrome is greatly facilitated by an automated procedure of recording the emission signal at the different wavelengths of the multiphoton laser: The excitation spectrum is obtained from the image stack. A method that is unbeatable for speed and ease!
With calibrated settings of the AOM, the images are acquired at a constant laser power for specimen excitation. The excitation spectra can then be compared to data on multiphoton cross-sections. |
| |
Excitation lambda stacks can be obtained automatically with a macro. |
| |