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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