Confocal imaging using synchrotron radiation

• Published in: Journal of electron spectroscopy and related phenomena 1996-05, Vol.80, p.343-347
• Main Authors: Munro, I.H., Jones, G.R., Tobin, M., Shaw, DA, Levine, Y, Gerritsen, H, van der Oord, K, Rommerts, F
• Format: Article
• Language: English
• ISSN: 0368-2048; 1873-2526
• DOI: 10.1016/0368-2048(96)02988-X

The physical principles of confocal imaging in conjunction with synchrotron radiation from the 2 Gev synchrotron radiation source (the SRS) at Daresbury Laboratory have been combined in the construction & operation of a uniquely versatile microscope. This scanning microscope is designed primarily for the three dimensional imaging of biological material. The microscope operates at any wavelength in the range from 200 nm to ∼ 700 nm and achieves high lateral and axial resolution in the reflection contrast mode which is close to the theoretical limits of λ/3 and λ respectively. The microscope is used to achieve three dimensional imaging by ‘optical slicing’; a method which is considerably enhanced in usefulness for biological samples by the ability to simultaneously observe images both with reflection and fluorescence contrast. Recently sample contrast has been obtained using fluorescence polarisation imaging giving information about molecular (probe) orientation within the sample. The microscope is used also in a non scanning mode where a near diffraction limited volume is chosen within an appropriate site within the sample. It is then feasible to undertake “micro volume spectroscopy” and to measure fluorescence lifetime, polarisations intensity and spectrum as a function of time yielding a powerful armoury of “chemical mapping” techniques to study the internal structures of cells and of membrane transport mechanisms using intrinsic or extrinsic fluorescence probes.