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| Content Provider | Springer Nature Link |
|---|---|
| Author | Brama, Elisabeth Peddie, Christopher J. Jones, Martin L. Domart, Marie Charlotte Snetkov, Xenia Way, Michael Larijani, Banafshe Collinson, Lucy M. |
| Copyright Year | 2015 |
| Abstract | Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dual-modality probes for ILEM when preserved using the in-resin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on re-creation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging strategies for standard FPs acting as dual-modality probes in a single ‘multi-colour’ integrated microscope system. |
| Starting Page | 179 |
| Ending Page | 188 |
| Page Count | 10 |
| File Format | |
| ISSN | 18646158 |
| Journal | Journal of Chemical Biology |
| Volume Number | 8 |
| Issue Number | 4 |
| e-ISSN | 18646166 |
| Language | English |
| Publisher | Springer Berlin Heidelberg |
| Publisher Date | 2015-07-08 |
| Publisher Place | Berlin, Heidelberg |
| Access Restriction | Subscribed |
| Subject Keyword | GFP YFP Fluorescent protein In-resin fluorescence Vacuum Integrated light and electron microscopy Physical Chemistry Biophysics and Biological Physics Cell Biology Pharmacology/Toxicology Biochemistry |
| Content Type | Text |
| Resource Type | Article |
| Subject | Cell Biology Biochemistry Biophysics |
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