Analysis of the cytochrome distribution via linear and nonlinear Raman spectroscopy

Abstract

The cytochrome distribution in hyphal tip cells of Schizophyllum commune was visualized using resonance Raman mapping and CARS microscopy. For comparison, resonance Raman mapping and CARS imaging of cytochrome was also performed during branch formation and in completely developed central hyphae. Cytochrome, as an essential component of the electron transport chain in mitochondria, plays an important role in providing energy to actively growing mycelia. It could be shown that mitochondria at the growing hyphal tips and at branching regions are more active, i.e. contain more cytochrome, as compared to those in older parts of the hyphae. This finding is compatible with the idea of high energy consumption for biosynthesis and intracellular transport at the growing tip, while older hyphae have lower needs for ATP generated via the respiratory chain in mitochondria. To the best of our knowledge this is the first study reporting about the localization and distribution of cytochrome, as an indirect mitochondria localization within S. commune or other basidiomycetous fungi, by means of resonance Raman microspectroscopy and CARS microscopy. These Raman methods bear the potential of label-free in vivo mitochondria localization and investigation.

Toward in Vivo Chemical Imaging of Epicuticular Waxes

“…Furthermore, MALDI-TOF constitutes a destructive technique; therefore, MALDI-TOF will not be helpful imaging dynamical changes. A technique that is in principle capable of imaging the wax distribution in living leaves is Raman-based microscopy (Yu et al, 2008;Krafft et al, 2009aKrafft et al, , 2009bWalter et al, 2010). Raman scattering provides chemical fingerprint information, can be recorded in back-scattering direction (epidetection), and is generally not hampered by the presence of water in biological samples (Urlaub et al, 1998).…”

Multimodal imaging to study the morphochemistry of basal cell carcinoma

“…Raman spectroscopy and microspectroscopy is commonly applied for the characterization of the chemical composition of cells and tissue [21][22][23][24]. The chemical sensitivity of Raman spectroscopy stems from the fact that Raman scattering is sensitive to the vibrations of particular molecules and, hence, to the molecular structure of the analytes.…”

“…the presence of collagen structures, can be selectively recorded by SHG microscopy [19], single and twophoton fluorescence reveal the spatial distribution of native fluorophores in the sample [37,38]. Finally, the joint application of Raman and CARS microspectroscopy allows for complementary characterization of the chemical composition of the sample [21,24,39]. Therefore, the approach presented here will yield detailed insight into the morphochemistry of the BCC samples and pave the way for further studies aiming at the implementation of in-vivo multimodal imaging for the clinical detection and ultimately to the diagnosis of the disease.…”

Multimodal nonlinear microscopy: A powerful label-free method for supporting standard diagnostics on biological tissues

“…As pointed out in the introduction, morphological information on cells and collagen can be provided by TPF and SHG microscopy, respectively, whereas Raman-based techniques allows for a more detailed characterization of the chemicalmolecular composition of the tissue under investigation. 89,115,116 Hence, combining TPF and SHG microscopy with CARS microscopy allows providing both morphological and chemical information in tandem, realizing a morpho-chemical characterization of a biological specimen. The implementation of all these techniques in the same experimental setup is straightforward since the pump (or the Stokes) pulses for CARS can be used for exciting TPF and SHG.…”