Autologous Bone Marrow Transplantation - Blog Science Connections

436 Digital Image Analysis System
added to the well. After centrifugation, medium was aspirated via the outlet
port, and 70% methanol was added to the inlet port. After 10 minutes, the
slide was carefully removed from the CCB and covered with a mounting
medium (Aquamount, Lerner Lab., New Haven, CT) and a coverslip.
RESULTS
The configuration of the system is outlined in Figure 1. Cinder control of
the microcomputer, the scanning stage moves the slide in increments of one
field of vision (for a 25 X objective lens). In order to ensure that every area of
the cytospin was digitized and analyzed by the microcomputer, the scan was
programmed with overlaps in each field of vision. The computer digitizes
each area to be analyzed into a 512 x 480 pixel image in the PC vision
graphics memory. A threshold algorithm is applied to the image so that any
pixels brighter than the defined threshold are set to 1 and any below the
threshold are set to 0. This speeds analysis of the image for positive events.
Next, the image is scanned for positive events; it was determined that
scanning every eighth pixel increased scanning speed and provided enough
overlap to avoid missing any cell. The scanning program is capable of
digitizing and analyzing a field of vision for positive events in 0.61 seconds.
When a positive event is detected, the coordinates of the event on the
slide are stored in a file on the cartridge disk drive. After scanning the entire
cytopreparation, the computer then "plays back" each positive event under
ISIT VIDEO
CAMERA
PC VISION
DIGITIZER
FLUORESCENCE
MICROSCOPE 51 2
RS
SCANNING
STAGE
232
X
480
JOYSTICK
IBM PC AT
MICROCOMPUTER
MOUSE
Figure 1. Diagram of digital image analysis system for detecting minimal disease using
fluorescent probes.