Axial resolution of confocal Raman microscopes: Gaussian beam theory and practice

A straightforward and transparent model, based on Gaussian beam optics, for the axial r0 resolution of a confocal microscope is presented. A confocal Raman microscope was used to determine the axial confocality in practice. The axial response of a thin planar object was measured for three different objectives, two pinhole sizes and a slit. The results show that, in the case of a confocal configuration, the response calculated with the model provides a good prediction of the axial resolution of the confocal microscope

Application of Raman Microspectroscopic and Raman imaging techniques for cell biological studies

Raman spectroscopy is being used to study biological molecules for some three decades now. Thanks to continuing advances in instrumentation more and more applications have become feasible in which molecules are studied in situ, and this has enabled Raman spectroscopy to enter the realms of biomedicine and cell biology [1-5].\ud Here we will describe some of the recent work carried out in our laboratory, concerning studies of human white blood cells and further instrumentational developments

Raman microspectroscopic and Raman imaging:New techniques for cell biological and biomedical research

Door haar grote molecuulspecifiteit kan Ramanspectroscopie gebruikt worden voor het herkennen/aantonen van moleculen in een preparaat. Het is ook mogelijk om de secundaire structuur van biologische macromoleculen zoals DNA en eiwitten te bestuderen en informatie te verkrijgen over interakties tussen moleculen. Dit alles zonder het gebruik van labels of probes. Enkele voorbeelden daarvan worden besproken

Line-scan Raman microspectrometry for biological applications

A high-resolution confocal line-scan Raman microscope was developed for the study of biological samples such as cells and chromosomes. With the illumination of a line on the sample, all the spectral information of the line is recorded. The line-scan microscope was attained by the introduction of one scanning mirror, a slit, and two cylindrical lenses in a standard confocal Raman microscope. The resolution, for an ideal sample, is 0.5 μm in the lateral direction and 3.5 μm in the axial direction. Line-scan experiments were performed to demonstrate the applicability of the instrument for biological samples. Line-scan images of calcium phosphate bone implant coatings and of the banding pattern of polytene chromosomes are presented

Time gated fluorescence lifetime imaging and micro-volume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca2+, or pO2 and pCO2. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity