Molekulare Diagnostik
Microscopy
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
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Molekulare Diagnostik
Microscopy Magnification, Contrast, Resolution • Light Microscopy (200 nm) – Brightfield (To visualize structures → stain with dyes, can only be used on fixed (dead) cells) – Fluorescent (Fluorescent dyes “glow” against dark background, Cells may be living or dead) – Advanced: – Phase contrast (Permits observation of transparent living cells, standard phase contrast and differential interference contrast) – Confocal (gives 3D image)
• Electron Microscopy (1 nm) – Transmission (TEM) – Scanning (SEM) Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
2
Molekulare Diagnostik
The optical microscope
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
3
Molekulare Diagnostik
Specimen preparation for brightfield microscopy Most cells are colorless or transparent. Tissues are sectioned. Therefore fixing and staining of cells is often nessecary for brightfield imaging
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
4
Molekulare Diagnostik
Fluorescent microscopy
• • • • •
Institute for Genomics and Bioinformatics, TU Graz / Austria
Permits localization of specific cellular molecules Fluorescent dyes “glow” against dark background Dye may be indirectly or directly associated with the cellular molecule Multiple fluorescent dyes may be used simultaneously Cells may be fixed or living
Dr. Andreas Prokesch
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Molekulare Diagnostik
Immunofluorescense microscopy
3T3-L1 cells d8
DAPI
DAPI
Anti-Pparγ
Anti-Calnexin(ER)
Anti-Arxes
Anti-Arxes
Merge
Merge
bar: 10µm
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
6
Molekulare Diagnostik
Advanced light microscopy –phase contrast • •
Permits observation of transparent living cells Light phase shifts induced by specimen are used to generate contrast – Phase contrast (refracted and unrefracted light) – Differential interference contrast (two light beams)
Brightfield, DIC, and PC image of the same cells Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
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Molekulare Diagnostik
Advanced light microscopy – confocal imaging z Confocal Scanning Microscopy y Generates focused images of living cells (one optical section per image)
yCan look inside thick specimens (eggs, embryos, tissues)
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
8
Molekulare Diagnostik
Advanced light microscopy – confocal imaging Several (confocal) optical sections can be deconvoluted into one sharp 3D image
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
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Molekulare Diagnostik
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
10
Molekulare Diagnostik
Karyotyping • Conventional karyotyping (Cytogenetics) • Fluorescent in-situ hybridization (FISH) • Spectral karyotyping (Sky)
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
11
Molekulare Diagnostik
Specimens used Peripheral blood
Bone marrow
Cultured skin fibroblast or epithelial cells
Prenatal diagnostics
Tumor biopsy Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
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Molekulare Diagnostik
Conventional karyotyping Incubate 3-4 days Culture in a growth medium and stimulate mitosis
Cut out the chromosomes and arrange into a karyotype Institute for Genomics and Bioinformatics, TU Graz / Austria
Peak though the microscope and photograph the chromosomes
Add chemical to stop mitosis in metaphase
Lymphocytes are harvested and treated with hypotonic solution
Swollen cells are fixed, dropped in a glass slide, dried and stained Dr. Andreas Prokesch
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Molekulare Diagnostik
Conventional karyotyping
MALE KARYOTYPE Institute for Genomics and Bioinformatics, TU Graz / Austria
FEMALE KARYOTYPE Dr. Andreas Prokesch
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Molekulare Diagnostik
Conventional karyotyping – Sex chromosome abberations Genotype
Gender
Syndrome
Physical Traits
XY
Male
None-normal
XXY XXYY XXXY
Male
Klinefelter’s Syndrome
sterility, small testicles, breast enlargement
XYY
Male
XYY Syndrome
normal male traits
XX
Female
None-normal
XO
Female
Turner Syndrome
sex organs don’t mature at adolescence, sterility, short stature
XXX
Female
Trisomy X
tall stature, learning disabilities, limited fertility
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
15
Molekulare Diagnostik
Fluorescent in-situ hybridization (FISH)
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
16
Molekulare Diagnostik
Fluorescent in-situ hybridization (FISH) Green: Chromosome 13 Red: Chromosome 21
Normal Institute for Genomics and Bioinformatics, TU Graz / Austria
Down syndrome Dr. Andreas Prokesch
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Molekulare Diagnostik
Spectral karyotyping (SKY) and multiple fluoescent hybridization (M-FISH) By mixing combinations of five fluorophores and using special imaging software one can distinguish all 23 chromosomes by chromosome-specific colors
Advantages: • Mapping of chromosomal breakpoints • Detection of subtle translocations • Characterization of complex rearrangements
Disadvantages: • Very expensive equipments • The technique is labor intensive • Does not detect structural rearrangements within a single chromosome • Limited resolution (~15 Mbp)
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
18
Molekulare Diagnostik
FISH in molecular diagnostics • • • • •
Detection of heritable diseases Prenatal diagnostics Tumor cell genome Detection of microdeletions (Cri-du-chat-syndrome, Williams syndrome, Kallmann syndrome...) Other chromosomal rearrangements (e.g. Philadelphia chromosome = reciprocal translocation chr9 and chr22)
Institute for Genomics and Bioinformatics, TU Graz / Austria
Dr. Andreas Prokesch
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