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Journal of Biomedical Optics

Multiphoton excitation spectra in biological samples
Author(s): Mary E. Dickinson; Eva Simbuerger; Bernhard Zimmermann; Christopher W. Waters; Scott E. Fraser
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Paper Abstract

Multiphoton microscopy is becoming a popular mode of live and fixed cell imaging. This mode of imaging offers several advantages due to the fact that fluorochrome excitation is a nonlinear event resulting in excitation only at the plane of focus. Multiphoton excitation is enhanced by the use of ultrafast lasers emitting in the near IR, offering better depth penetration coupled with efficient excitation. Because these lasers, such as titanium:sapphire lasers, offer tunable output it is possible to use them to collect multiphoton excitation spectra. We use the software-tunable Coherent Chameleon laser coupled to the Zeiss LSM 510 META NLO to acquire x-y images of biological samples at multiple excitation wavelengths, creating excitation lambda stacks. The mean intensity of pixels within the image plotted versus excitation wavelength reveals the excitation spectra. Excitation lambda stacks can be separated into individual images corresponding to the signal from different dyes using linear unmixing algorithms in much the same way that emission fingerprinting can be used to generate crosstalk free channels from emission lambda stacks using the META detector. We show how this technique can be used to eliminate autofluorescence and to produce crosstalk-free images of dyes with very close overlap in their emission spectra that cannot be separated using emission fingerprinting. Moreover, excitation finger- printing can be performed using nondescanned detectors (NDDs), offering more flexibility for eliminating autofluorescence or crosstalk between fluorochromes when imaging deep within the sample. Thus, excitation fingerprinting complements and extends the functions offered by the META detector and emission fingerprinting. We correct biases in the laser and microscope transmission to acquire realistic multiphoton excitation spectra for fluorochromes within cells using the microscope, which enables the optimization of the excitation wavelength for single and multilabel experiments and provides a means for studying the influence of the biological environment on nonlinear excitation.

Paper Details

Date Published: 1 July 2003
PDF: 10 pages
J. Biomed. Opt. 8(3) doi: 10.1117/1.1583734
Published in: Journal of Biomedical Optics Volume 8, Issue 3
Show Author Affiliations
Mary E. Dickinson, California Institute of Technology (United States)
Eva Simbuerger, Carl Zeiss Jena GmbH (Germany)
Bernhard Zimmermann, Carl Zeiss Jena GmbH (Germany)
Christopher W. Waters, California Institute of Technology (United States)
Scott E. Fraser, California Institute of Technology (United States)


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