Protein chromophores that can be activated to initiate fluorescence emission from a quiescent state (a process known as photoactivation), or that are capable of being optically converted from one fluorescence emission bandwidth to another (photoconversion), represent perhaps the most promising approach to the in vivo investigation of protein lifetimes, transport, and turnover rates. Appropriately termed molecular or optical highlighters, photoactivated fluorescent proteins generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength. Photoconversion optical highlighters, on the other hand, undergo a change in the fluorescence emission bandwidth profile upon optically-induced changes to the chromophore. This interactive tutorial explores the optical conversion of several useful highlighter probes and simulates how these proteins would be viewed in an actual confocal microscope.

The tutorial initializes with a randomly selected specimen composed of cell cartoon drawings at 20x magnification appearing in the image window. A higher magnification view of a single cell (100x) can be selected using the radio buttons in the lower right-hand corner of the control panel. The virtual cells are initially being imaged using laser excitation at wavelengths appropriate for the optical highlighter fluorescent protein appearing in the pull-down menu text box. In cases where photoactivation or sub-cellular localization highlighters are selected from the menu, specific portions of the cartoon (or in some cases, the entire cell) that lack a fluorescent probe label appear in grayscale. Optical highlighters available in the tutorial are localized either to the mitochondria, nucleus, or the complete cell (nucleus and cytoplasm). The Imaging Laser, which can be toggled on and off using the check box (default is on) in the control panel, is pre-set to the optimum wavelength for observing the selected probe (as listed in Table 1). When this laser is turned off, the cell cartoons appearing in the window simulate the appearance of a differential interference contrast (DIC) grayscale image.

In order to operate the tutorial, use the pull-down menu to select an optical highlighter and a localization target (entire cell, nucleus, or mitochondria). Next, activate one of the three Region of Interest selection buttons (rectangular, circular/elliptical, or free-hand) in the upper left-hand portion of the control panel. The mouse cursor is used to define a specific region by dragging around the area of interest with one of the available selection tools. For example, to generate a rectangular or elliptical selection area, click on the left mouse button and drag the cursor until the desired size is achieved. The free-hand selection tool should be used to create a path having the initial and final points separated by less than 5 pixels to ensure formation of an enclosed boundary. After being created, the selection boundary can be relocated anywhere within the image window by dragging with the mouse (a crossed double-headed arrow icon appears when the cursor is placed within a selection boundary).

After selecting a region of interest, click on the photoconversion laser button (PCL) to simulate illumination of the specimen with the appropriate wavelengths, and then observe the transition of fluorescence intensity. The laser wavelength selection pull-down menu contains only a single value for most of the optical highlighters, however two laser choices (543 and 457 nanometers) are available to photoconvert or quench, respectively, the kindling fluorescent protein. At any point during operation of the tutorial, the Reset button can be clicked to reinitialize the selected specimen.