High resolution multimode digital imaging system for mitosis studies in vivo and in vitro.

Many questions about the mechanisms of mitotic spindle assembly, chromosome movement, and chromosome segregation can be answered by quantitative measurements of digital images obtained from several optical modes. For example, epifluorescent detection of X-rhodamine-labeled tubulin reveals spindle microtubule organization, whereas DAPI or other DNA-binding dyes stain the chromosomes in live preparations where these structures are often invisible to phase or DIC methods (l-4). Photoactivated fluorophores bound to tubulin can be used to measure microtubule assembly dynamics in real time (1,2), and other fluorescent probes can be used to track the dynamics of membranes, other organelles, or molecular complexes within the spindle (5). In transparent specimens, the movements of chromosomes and centrosomes can be recorded with transmitted light if phase contrast or DIC optics are employed (6) while polarization methods can reveal spindle fiber microtubule dynamics and provide a quantitative measure of microtubule assembly and orientation (6). In addition to real-time recording, a digital fluorescence microscope system can also provide 3-D structural detail from stacks of optical sections when the specimens are stained with specific molecular probes. The high resolution, multimodal digital microscope system that we have constructed for our mitosis studies is diagrammed in Figure 1. Optical components of the Nikon FXA microscope stand were chosen to provide diffraction-limited resolution in transmitted and fluorescence modes for images projected onto a cooled charge coupled device (CCD) camera (Fig. 2C). Cooled CCD cameras have several advantages for digital imaging over unintensified and intensified video detectors, including dynamic range, linearity, low noise, and little geometrical distortion. The Hamamatsu C4880 was chosen because it has two readout modes: a fast scan mode (up to 7 frames/s) which is useful for focusing, and a slow scan mode ( 12 bit/pixel, 500,000 bytes/s readout rate), which gives the most useful dynamic range (4000 grey levels above a noise floor of about 50 f 10). The MetaMorph digital imaging system is programmed to control image acquisition from the CCD camera. We have found that the central area (300 X 300 pixels) of the 1000 X 1000 pixel detector provides sufficient resolution and field of view for our mitosis images while reducing the time required to process the digital images and the amount of digital storage required (180 KBytes/image). An important criterion in our mitosis studies is that all the epiand trans-illumination images be aligned and in focus at the same position on the CCD detector. This was accomplished with a single Chroma filter cube containing a multiple bandpass dichromatic mirror and emission filters designed for the DAPI (blue), fluorescein (green), and X-rhodamine (red) emission wavelengths. Excitation intensity and wavelength are selected by a MetalTek stepper motor controlled dual filter wheel, where one g-position wheel holds a series of neutral density filters, and the other holds narrow bandpass filters for the different excitation wavelengths. Other filters and iris diaphragms in the epiand trans-illumination paths are used to provide further manual control of illumination intensity and field of illumination (Fig. 1). Focus position along the z-axis is controlled by a Ludl stepper motor attached to the Nikon FXA fine focus. The MetaMorph digital imaging system has programs and journal scripts that control for either time-lapse or z-axis stepping,