FISH Automation 소개 2007. 09. 06 진단검사의학과 강지연
Fluorescence in situ Hybridization 검사하고자 하는 표본에 찾고자 하는 probe를 반응시킨 후 특정부위의 유전자나 염색체의 결실, 추가, 증폭, 재배열을 형광현미경으로 관찰하 는 방법 Specimen treatment Harvest / Slide preparation 및 경검 Front End FISH 1 st day Pretreatment § Dehydration 암실에서 진행 § DNA-Probe 첨가 § Denaturation § Hybridization § 2 nd day (암실에서 진행) Cover slip 제거 § Washing (Wash 1, Wash 2, Wash 3) § Counter stain: DAPI 부착 §
Back End FISH Analysis by fluorescence microscopy dual color, dual fusion break apart
FISH Automation - Overview o o Front End Automation q Automates slide/specimen pretreatment and assay q Can be used for FISH, G-Banding, IHC & others q Two Configurations (1) VP 2000 and Thermo. Brite™ (2) Xmatrx Back End Automation q Automated slide scanning & enumeration q Meta. Systems q Other Applications in process
Denaturation & Hybridization Automation Choices Front-End Back-End Slide Pre-treatment & Washing Slide Scanning & Enumeration VP 2000 Slide preparation Thermo. Brite Pre-treatment, Washing, Denaturation, Hybridization & Final Cover slip * In Development metasystems Xmatrx*
Front End FISH VP 2000 Update n n n n Automates FISH Assay Steps: q FISH Pretreatment q Specimen Staining Up to 50 slides simultaneously 3 heated reagent basins Ventilated closed system General purpose system accommodates most slide-based assays: histology, G-banding, etc Increase FISH throughput Increase consistency of results Decrease labor costs
Front-End FISH Thermo. Brite™ Slide Hybridization & Denaturation System Eliminates separate denaturation 40 definable programs – LCD screen 12 slide capacity – guides hold slides Humidity cards Superior temperature uniformity Rapid temperature ramp
Front-End FISH XMATRX Automation of ALL FISH assay steps Pre-treatment, Probe, Coverslip application, Washing, Denaturation, Hybridization, Cover Slip Removal, Wash, Counter stain & Final Cover slip Throughput q Up to 40 slides per run q Up to 80 assays per run Random access, up to 8 different assays in one run Continuous access, slides can be added or removed during the run Full reagent management capability Applies, removes & reapplies cover slips PC with easy to use software
Front-End FISH A Fully Automated FISH System
Back-End FISH Carl Zeiss Microscope Axio Imager. M 1/Z 1 Motorized stand Automatic Focussing Maximum variability Control by PC or touch screen The best fluorescence with higher contrast
Back-End FISH Metafer - Slide Scanning System
Metafer Concept CCD Camera Metafer Motorized stage (x, y) real-time image analysis and microscope control Motorized microscope (z) PC Win 9 x 2000 / NT
Metafer Concept • Microscope slide is scanned field by field at lowest possible magnification without gaps • Objects of interest are automatically identified ; gallery images, object features and positions are recorded
Metafer Advantages of Metafer High speed Slide Scanning (70 mm/s) High quality image capture during the scan High throughput and capacity multi-fluorochrome capability from UV to IR - very few objects available for analysis - statistical analysis of many objects
Metafer Applications MSearch : metaphase finder - Micronucleus analysis - Automatic analysis of dicentrics Meta. Cyte : automated FISH analysis / spot counting - spot counting and translocation analysis - amplification analysis in tissue section - Comet scan - High content screening RCDetect : rare cell detection - minimum residual disease detection
Metafer - MSearch : metaphase finder
Metafer – Msearch : metaphase finder Fast metaphase finding in transmitted and fluorescent light
Metafer - Meta. Cyte : automated FISH analysis
Metafer – Slide Feeder
Back-End FISH Isis - Fluoresc. Imaging System
Isis Fluoresc. Imaging System – features and benefits Fast and easy capture Powerful image enhancement Various color display mode, measurement function Database manegement as Ikaros Additional Isis modules q Color karyotyping module q m. FISH and m. BAND module q CGH(Comparative Genome Hybridization) module q Telomere measurement module
Isis Fluoresc. Imaging System – Capture Focus adjustment live on screen Automatic exposure control If with Carl Zeiss Axio Imager. M 1 or Z 1 microscope - automatic filter change - focus stack capture
Summary Improve workflow efficiency q Increase throughput; enable labs to do more with the same resources q Reduce “hands-on” time; free up resources and simplifies workflow q Increase assay quality and reproducibility Multiple solutions; different levels of automation Applications to our laboratory(metasystems) q Rapid finding of metaphase & FISH interphase q Good assay quality of FISH analysis q Pannel FISH q Urovision
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