Automated high throughput whole slide imaging using area sensors, flash light illumination and solid state light engine.

BACKGROUND: Whole Slide Imagers or digital slide scanners have developed very rapidly in the last 8 years and went through three generations. Third generation instruments have just reached the market which have the stability and throughput to be used for routine clinical work. We describe in this article the technical background and reasoning behind engineering decisions we made during the development of 3DHISTECH's 3rd generation combined brightfield and fluorescent scanner. MATERIALS AND METHODS: The Panoramic 250 FLASH utilizes Plan-Apochromat 20x and 40x objectives, a 2 megapixel 3CCD camera for brightfield and a monochrome scientific CMOS camera for fluorescent scanning. A solid state light engine for fluorescent and a strobe light for bright field illumination are used. RESULTS: The system can scan 1cm2 including focusing at 45x resolution in 1 minute. It can scan a well stained DAPI, FITC, TRIC, 1cm2 fluorescent slide in 11 minutes. It can load and scan 250 slides in walk away mode. CONCLUSION: Using the latest camera technology and electronics, state of the art computer and standard microscope optical components high throughput high quality whole slide imaging is feasible and is sufficient for most of the routine diagnostic work. Extended depth of field and Z-stack scanning is possible with the use of area scan technology.

Automated multichannel fluorescent whole slide imaging and its application for cytometry.

Slide-based image cytometry (SBC) has several advantages over flow cytometry but it is not widely used because of its low throughput, complicated workflow, and high price. Fully automated microscopes became affordable with the advent of whole slide imaging (WSI) and they can be transformed into a cytometer. A MIRAX MIDI automated whole slide imager was used with metal-halide and light emitting diode (LED)-based fluorescent illumination, filter block changer, and a cooled monochrome charge coupled device camera. The MIRAX control software was further developed for fluorescent sample detection, autofocusing, multichannel digitization, and signal correction due to nonuniform illumination. Fluorescent calibration beads were used to verify the linearity of the system. The HistoQuant software package of the MIRAX viewer was used for image segmentation and quantitative analysis. The data was displayed by the histogram, scatter plot, and gallery functions of the same program. Fluorescent samples can be reliably detected, focused, and scanned. The measured integrated fluorescence showed linearity with exposure time and staining intensity. Automated fluorescent WSI with stable LED illumination and high-quality homogeneous fluorescent slides can be used conveniently for SBC.