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2-Photonen-Mikroskopie
iba 2PLSM Unit
2PLSM Unit
Chondrocytes on a collagen substratum - A: Image of the intrinsic fluorescence intensity; B: pseudo-color encoding distinguishes between the spectral properties of cells and the substratum (spectral unmixing, green: collagen substratum, red: chondrocytes)
Chondrocytes on a collagen substratum

Scientists use the two-photon excitation of fluorescent molecules (Jablonski-diagram) particularly for the fluorescence microscopy of three-dimensional cell populations and tissues. Aqueous media do not absorb energy at wavelengths around 800 nm. Furthermore, tissue absorbs less energy from the low-frequency excitation beam of a titanium-sapphire laser (about 790 nm, 5 - 15 mW in the focal area) than from a higher frequency beam. When scanning a biological sample layer by layer (two-photon laser scanning microscopy, 2PLSM) an up to 500 µm deep 3-D image of the sample emerges.

In our investigations at the iba Institute in Heiligenstadt (Thuringia, Germany), we analyze the intrinsic fluorescence of samples rather than samples tagged with fluorescence markers. This enables us to analyze in-line samples at any time throughout the incubation period without disturbing the state of the biological samples. CCD cameras with a shutter speed of about 0.5 - 2.5 seconds per object plane allow us to use the intrinsic fluorescence of the samples to render and interpret 3-D images of our samples.

The Advantages of the 2-Photon over the One-Photon Microscopy (e.g. CLSM)

  • Gentle, non-invasive analysis of cells and tissue
  • Efficient excitation of the cell/tissue-intrinsic fluorescence
  • 3-D scanning causes only minor photo damage to a sample
  • Minimal bleaching effects
  • The use of low frequency excitation allows the analysis of biological tissue and 3-D cell cultures to a depth of up to 1 mm
  • Excellent sensitivity because the entire fluorescence contributes to the imaging

Applications

  • Inline microscopy of 3-D samples in tissue engineering
  • Studies of cartilage
  • Inline biofilm analyses
  • The photon induced release of active agents
Flow chamber with inline 2PLSM and hydrostatic cell stimulation in cartilage tissue engineering
Flow chamber with inline 2PLSM
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