Visualization of proteins inside acrylamide and other gels usually relies on different staining methods. To omit the protein-staining procedure, we visualized unstained proteins inside acrylamide gels by laser excitation with ultraviolet (UV) light (280 nm, 35 mJ/cm 2 ) and directly detected native UV fluorescence. In one-dimensional gels, a detection limit as low as 1 ng for bovine serum albumin and 5 ng for other proteins with a linear dynamic range (2.7 orders of magnitude) comparable to state of the art fluorescent dyes could be achieved. In addition, the application of this method to 20 µg of a whole cell lysate separated in a two-dimensional gel showed more than 600 spots. Since protein labeling always represents a serious obstacle in protein identification technologies, the working efficiency with our procedure can be considered as a significant improvement for protein visualization and reproducibility in proteomics.Currently, two-dimensional gel electrophoresis (2-DE) represents the technology most widely used to separate complex protein mixtures for subsequent differential comparison (proteomics). Proteins are separated according to their isoelectric point (pI) in the first dimension and according to their apparent molecular mass in the second dimension. This method was first introduced by Klose and O'Farrell in 1975. 1,2 Continuous improvements in mass spectrometry (MS) over the last 10 years routinely allow protein spot identification in 2-DE. For proteomics research, 2-DE gels of different states (e.g., healthy vs diseased states) are acquired and compared in order to investigate biochemical processes (e.g., disease-causing mechanisms). The image analysis of several dozens of gels is a bottleneck in proteomics because of limitations in reproducibility of 2-DE and staining processes.Currently, different staining methods for the visualization of proteins in a gel have been established. Staining after separation: Silver staining is the most sensitive standard detection method (1 ng per band), 3 but it is accompanied by problems, such as chemical modifications of the proteins. 4 Furthermore, the staining and destaining procedures often result in a loss of protein and, therefore, in a loss of sensitivity for mass spectrometrical analysis. Additionally, each protein has an individual staining behavior due to its compositional properties. 5 Another drawback is the low dynamic range of this staining method. Staining with Coomassie Brillant Blue G-250 (CBB) is widely used because it does not interfere with further MS analysis, however, at the cost of a lower detection sensitivity (20-60-fold). 3 In contrast, labeling methods with fluorescent dyes are easier to handle and offer an improved dynamic range, but they are cost-intensive. 6 Staining before separation: Labeling of proteins with fluorescent dyes before separation is a critical process, because the isoelectric point and the molecular mass of the proteins can be changed by this method as a result of the covalent modification. Radioactive labeling ( 14 ...

Microsecond laser pulses are currently being investigated in a new ophthalmic procedure for treatment of disorders associated with the retinal pigment epithelium (RPE). The precise mechanism for microsecond laser-induced RPE damage, however, has not been determined. We have previously shown that short pulse laser irradiation in the nanosecond to picosecond time domain causes transient microbubble formation around melanin granules in pigmented cells. Nanosecond time-resolved microscopy was previously used to visualize the intracellular cavitation dynamics. However, this technique is difficult to use with microsecond laser exposures, especially when multiple laser pulses are applied in a rapid sequence as in the clinical setting. Here we describe a simple pump-probe method for detecting transient light scattering signal from individual RPE cells when they are irradiated with nanosecond and microsecond laser pulses. For single 12 ns pulses the threshold for bubble detection was the same as the ED(50) threshold for cell death. For 6 micros pulse duration the threshold for bubble detection was about 10% higher than the threshold for cell death. With repetitive pulse trains at 500 Hz the ED(50) decreased about 25% for 10 and 100 pulses. Cells die when a single bubble was detected in a multiple pulse sequence.

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Absorption ofpulsed laser radiation by individual melanosomes produces localized heating and microcavitation bubble formation around the particles, with transient bubble lifetimes of a few hundred nanoseconds. Intracellular cavitation in the retinal pigment epithelium (REP) leads to prompt cell death. Threshold laser fluence for cavitation bubble formation and RPE cell damage was measured as the exposure spot size was varied from 20 to 200 im using a RPE tissue explant model. The threshold energy for cell killing decreased with decreasing spot diameter but the fluence (energy/area) was nearly constant for all spotsizes. How this compares with data from in vivo animal studies is discussed.

A new immunoaffinity solid phase extraction of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide is described. An immunoadsorber was applied which was created for the first time by the immobilisation of specific antibodies (polyclonal, host: rabbit) by the sol-gel method. The extraction method in combination with high performance liquid chromatography-fluorescence determination has been validated and shown to be applicable to blood samples of heroin victims in a low concentration range. Blood extracts were essentially free of interfering matrix components when compared to C8-extracts. Additionally, a novel, sensitive and selective detection system for wavelength-resolved analysis of laser-induced fluorescence coupled to HPLC was developed. The analytes were excited with a frequency tripled Ti:Sa laser (lambda=244 nm quasi cw). The total emission spectrum was recorded with a detection system consisting of an imaging spectrograph and a back-illuminated CCD camera. This technique of detection, combined with an extended optical path (at least 6 mm could be illuminated by the laser), resulted in an optimal fluorescence intensity of the analytes. The method permitted the analysis of morphine, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in a low concentration range and could be applied to a complex matrix such as postmortem blood samples because analyte peaks could be discriminated from matrix peaks by their characteristic emission spectra.

PURPOSESelective targeting of the Retinal Pigment Epithelium (RPE), by either applying trains of microsecond laser pulses or, in our approach, by repetitively scanning a tightly focused spot across the retina, achieves destruction of RPE cells while avoiding damage to the overlying photoreceptors. Both techniques have been demonstrated as attractive methods for the treatment of retinal diseases that are caused by a dysfunction of the RPE. Because the lesions are ophthalmoscopically invisible, an online control system that monitors cell death during irradiation is essential to ensure efficient and selective treatment in a clinical application. MATERIALS AND METHODSBubble formation inside the RPE cells has been shown to be the cell damage mechanism for nano-and picosecond pulses. We built an optical system to investigate whether the same mechanism extends into the microsecond regime. The system detects changes in backscattered light of the irradiating beam during exposure. Samples of young bovine eyes were exposed in vitro using single pulses ranging from 3 µs to 50 µs. Using the viability assay calcein-AM the ED 50 threshold for cell death was determined and compared to the threshold for bubble formation. We also set up a detection system on our slit lamp adapted scanning system in order to determine the feasibility of monitoring threshold RPE damage during selective laser treatment in vivo. RESULTS AND DISCUSSIONIntracellular cavitation was detected as a transient increase in backscattering signal, either of an external probe beam or of the irradiation beam itself. Monitoring with the irradiation beam is both simpler and more sensitive. We found the threshold for bubble formation to coincide with the threshold for cell damage for pulse durations up to 20 µs, suggesting that cavitation is the main mechanism of cell damage. For pulse widths longer than 20 µs, the cell damage mechanism appears to be increasingly thermal as the two thresholds diverge. We conclude that bubble detection can be used to monitor therapeutic endpoint for pulse durations up to 20 µs (or equivalent dwell time in a scanning approach). We have integrated a detection module into our slit lamp adapted laser scanner in order to determine threshold RPE damage during selective laser treatment in vivo. KEYWORDSSelective retinal photocoagulation, retinal pigment epithelium, melanosomes, cavitation, bubble formation, laser scanner, slit lamp, online detection of cell death, back scatter CORRESPONDENCE Clemens Alt Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/21/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Proc. of SPIE Vol. 4951 53 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/21/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx

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