Molekulare Diagnostik. Microscopy. Institute for Genomics and Bioinformatics, TU Graz / Austria

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1 Microscopy 1

2 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) 2

3 The optical microscope 3

4 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 4

5 Fluorescent microscopy 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 5

6 Immunofluorescense microscopy DAPI Anti-Pparγ Anti-Arxes Merge 3T3-L1 cells d8 DAPI Anti-Calnexin(ER) Anti-Arxes Merge bar: 10µm 6

7 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 7

8 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) 8

9 Molekulare Diagnostik Advanced light microscopy confocal imaging Several (confocal) optical sections can be deconvoluted into one sharp 3D image 9

10 10

11 Karyotyping Conventional karyotyping (Cytogenetics) Fluorescent in-situ hybridization (FISH) Spectral karyotyping (Sky) 11

12 Specimens used Peripheral blood Cultured skin fibroblast or epithelial cells Bone marrow Prenatal diagnostics Tumor biopsy 12

13 Conventional karyotyping Incubate 3-4 days Add chemical to stop mitosis in metaphase Culture in a growth medium and stimulate mitosis Lymphocytes are harvested and treated with hypotonic solution Cut out the chromosomes and arrange into a karyotype Peak though the microscope and photograph the chromosomes Swollen cells are fixed, dropped in a glass slide, dried and stained 13

14 Conventional karyotyping MALE KARYOTYPE FEMALE KARYOTYPE 14

15 Conventional karyotyping Sex chromosome abberations Genotype Gender Syndrome Physical Traits XY Male None-normal XXY XXYY Male Klinefelter s Syndrome sterility, small testicles, breast enlargement XXXY 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 15

16 Fluorescent in-situ hybridization (FISH) 16

17 Fluorescent in-situ hybridization (FISH) Green: : Chromosome 13 Red: : Chromosome 21 Normal Down syndrome 17

18 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) 18

19 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) 19