Microscopy - Graz University of Technology · Microscopy. Molekulare Diagnostik Institute for Genomics and Bioinformatics, TU Graz / Austria Dr. Andreas Prokesch 2 Microscopy Magnification,

Molekulare Diagnostik

1Institute for Genomics and Bioinformatics, TU Graz / Austria Dr. Andreas Prokesch

Microscopy



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)



The optical microscope



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



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



Immunofluorescense microscopy3T

3-L1

cel

ls d

8

DAPI Anti-ArxesAnti-Calnexin(ER) Merge

DAPI Anti-ArxesAnti-Pparγ Merge

bar: 10µm



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



Advanced light microscopy – confocal imaging

z Confocal Scanning Microscopyy Generates focused images of living cells

(one optical section per image)yCan look inside thick specimens (eggs, embryos, tissues)



Advanced light microscopy – confocal imaging

Several (confocal) optical sections can be deconvoluted into one sharp 3D image





KaryotypingKaryotyping

•• Conventional karyotyping (Cytogenetics)Conventional karyotyping (Cytogenetics)•• Fluorescent inFluorescent in--situ hybridization (FISH)situ hybridization (FISH)•• Spectral karyotyping (Sky)Spectral karyotyping (Sky)



Specimens used

Peripheral blood Cultured skin fibroblast or epithelial cells

Bone marrow Prenatal diagnostics

Tumor biopsy



Conventional karyotyping

Incubate 3-4 days

Culture in a growth medium and stimulate mitosis

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

Peak though the microscope and photograph the chromosomes

Cut out the chromosomes and arrange into a karyotype



Conventional karyotyping

MALE KARYOTYPE FEMALE KARYOTYPE



Genotype Gender Syndrome Physical Traits

XY Male None-normal

XXYXXYYXXXY

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

Conventional karyotyping –Sex chromosome abberations



Fluorescent in-situ hybridization (FISH)



Fluorescent in-situ hybridization (FISH)

NormalNormal Down syndromeDown syndrome

GreenGreen: Chromosome 13: Chromosome 13RedRed: Chromosome 21: Chromosome 21



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)



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)

Documents

Microscopy - Graz University of Technology · Microscopy. Molekulare Diagnostik Institute for Genomics and Bioinformatics, TU Graz / Austria Dr. Andreas Prokesch 2 Microscopy Magnification,