J.J. Duindam

Applications of Micro-Raman Imaging in Biomedical Research

Recent results are presented of the application of imaging micro-Raman spectrometers in cellular biophysics and biomedical research. Various micro-Raman spectrometers have been developed that are now routinely applied in these fields. Results are presented that were obtained with a linescan Raman microspectrometer and with a Raman imaging microscope. Applications of Raman linescan spectrometry concern the investigation of polytene chromosomes obtained from the salivary gland of Chironomus thummi thummi. The distribution of DNA and proteins was investigated in bands and interbands. In a second example of Raman linescan spectrometry, bone implants were investigated. These bone implants were coated with thin layers of materials that improve biocompatibility. The density, crystallinity and protein distribution can be investigated. Information from Raman imaging may help in selecting the proper materials for maximum biocompatibility. Raman imaging microscopy is used whenever two-dimensional spatial information is required on the distribution of molecules or molecular components. Raman images are presented of filipine and phenylalanine in human eye-lenses in and around radial shades. Raman imaging is a particularly important tool for the study of the distribution of non-fluorescent drugs inside living cells. This is illustrated for the non-fluorescent drug cobalt octacarboxyphthalocyanine.

Aging affects the conformation of cholesterol in the human eye lens.

The distribution of 3-beta-OH-cholesterol along the optical axis of 20 human eye lenses of different ages was measured using confocal Raman microspectroscopy in combination with the 3-beta-hydroxysteroid-specific probe filipin. This non-destructive technique allow a spatially very detailed study of the distribution of 3-beta-OH-cholesterol in individual lenses. It revealed age-related changes of the 3-beta-OH-cholesterol distribution along the optical axis. The 3-beta-OH-cholesterol was found to be distributed asymmetrically along the optical axis and this distribution was found to change with age. The relative 3-beta-OH-cholesterol content in the anterior part of the lens decreases significantly with increasing age compared with its equal posterior counterpart. Additionally all the lenses have a very low 3-beta-OH-cholesterol content in the central part of their nuclear region, the area where the primary lens fibres are located.

Acoustic characteristics of the eye lens

The purpose of this study is to characterize the eye lens (human and porcine) by acoustic measurements and to investigate whether relations exist with the local protein content. The acoustic measurements were performed with a ‘scanning acoustic microscope’ (SAM), operating at a frequency of 20 MHz. At this frequency the lateral resolution in the acoustic images was 150 mm. A double-transmission pulse-echo technique was employed to obtain acoustic parameter images of a central, 1-mm thick slice of the lenses. The two-dimensional images were derived by ultrasonic spectroscopy displaying the ultrasound velocity, the attenuation at 20 MHz and the slope of the attenuation coefficient between 17 and 213 MHz. The images were summarized by profiles along the optical and equatorial axes of these parameters.