Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix

Abstract

By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.

Correlative cathodoluminescence electron microscopy bioimaging: towards single protein labelling with ultrastructural context

“…CCLEM could also be employed as label-free method, when imaging auto-cathodoluminescence of tissues and cells. [115][116][117][118] "ColorEM" or analytical electron microscopy converts greyscale analytical information into false color-coded images, and can be used to identify biological features based on elemental composition. 119,120 ColorEM techniques include energy dispersive X-ray analysis, electron energy loss spectroscopy, and correlative cathodoluminescence electron microscopy, which are reviewed in great detail by Pirozzi et al 120 The interaction between an incident electron beam and the sample generates additional signals, as described previously.…”

“…All the above described methods are based on primary electron beam interaction with the sample and have a potential for label-free detection. 118,120,122 In combination with STEM, all the methods have nanometric spatial resolution and complement each other, as the resolution is mostly determined by the sample thickness and electron beam spot size. 120,130 Highenergy STEM-EELS imaging can induce significant beam damage, 132 while EDX and CL are less invasive due to the lower beam energies.…”

“…However, this heavily relies on the development of suitable labels and the sample properties. Current CCLEM studies on biological samples indicate a resolution of approximately 20-50 nm, 61,67,118 in the order of a typical nanoparticle-label size 82 and on par with several of the SR microscopy techniques.…”

Lanthanide Cathodophores for Multicolor Electron Microscopy

“…Specifically, we sought to achieve multicolor detection of single cathodophores on the surface of a cultured mammalian cell. Here, a critical limitation was the auto-cathodoluminescence (auto-CL) from cells, which, by analogy with autofluorescence in fluorescence imaging, is the intrinsic luminescence of unlabeled cells excited with the electron beam 5,17 . Fixed HEK293T cells exhibited broadband auto-CL emission (SI Fig.…”

Cathodoluminescence Nanoscopy of 3D Plasmonic Networks

“…Thus, in either technique it is possible to map the local electric fields with nanoscale resolution (for further information, see the Supporting Information). ,,,, As EELS is measured in transmission, it is not suitable for thick 3D samples such as those studied here (with a thickness of a few micrometers).…”