IMMUNODEPLETION OF CONDENSINFROM XENOPUS EGG EXTRACTS

HSS Δcond. Δmock mockboiled beadsextractscond.

anti-cond.

anti-α-tub.

- 170

- 130

- 100

Smc2 Smc4

FLUORESCENCE MICROSCOPY: VISUALISING FITC-STAINED SAMPLES

Excitation filter (488nm+/-20 nm) Emission filter (525nm+/-25 nm)

Dichroic beam splitter(>495 nm)

Camera/Eyepiece

FLUORESCENCE MICROSCOPY:MICROSCOPE SET-UP

Emission filter

Excitation filter

Beam splitter

EXAMPLE: BRIGHT-FIELD MICROSCOPY OF A STAINED SAMPLE

Kidney ducts stained with hematoxylin (blue, basic extracellular matrix) and eosin (pink, acidic nuclei)

Source: MBC

BRIGHT-FIELD MICROSCOPYBased on differential absorption of light by objects

Absorption: Decrease in the amplitude of a light wave (i.e. object gets darker)

Absorption may be wavelength-independent or wavelength-specific (e.g. chloroplasts are green under the microscope, the amplitude of all other wavelengths is reduced)

Objects visible by bright-field microscopy are called “amplitude objects”

PHASE-CONTRAST MICROSCOPYThin objects (e.g. single cells) don’t absorb sufficient light to be good “amplitude objects”However, all objects shift the phase of a passing light-beam by a fraction of their wavelength. They are called “phase objects”.Using special optics this (invisible) phase shift can be converted into a (visible) amplitude shiftThis conversion is based in interference between the direct light beam and the phase-shifted light beam

EXAMPLE: PHASE CONTRAST

LIGHT PATH IN PHASE CONTRAST MICROSCOPY

Diffracted beamDirect beam

-1/4λ on the diffracted beam (passing through the specimen)

-1/4λ on the diffracted beam (passing through the retarder of the phase ring)

Net result: shift of 1/2λ of the diffracted beam results in negative interference between direct and diffracted beam apparent conversion of a “phase object” into an “amplitude object”

Δ1/4λ Δ1/4λ

TYPES OF LIGHT MICROSCOPY

Bright-field

Phase-contrast

Differential-interferencecontrast (DIC)

Source: MBC

Fluorochromes can be excited by a particular wavelength and emit light of a longer wavelength Stokes shift.

=heat

PRINCIPLE OF FLUORESCENCE

COMMON FLUOROCHROMES USED FOR BIOLOGICAL APPLICATIONS

PROBE DETECTIONAntibodies or nucleic acid probes can be conjugated to fluorescent dyes, such as FITC (fluorescein-isothiocyanate)

Fluorescent group

Reactive group for conjugation to other molecules via amine groups

SCALE BARS• all microscopic images must have a scale bar

• experimental determination of scale bar: take image of hemocytometer with squares of known dimensions (e.g. Thoma)

• calculate length from pixel as outlined below:

200 µm

CALCULATING MAGNIFICATION FOR DIGITAL MICROSCOPYPixel size: 6.8 µmCCD chip dimension: 1360 x 1024 pixelMicroscope magnification: 100x

6.8 µm/100 x1360=92.48 µm 6.8 µm/100 x 1024=69.63 µm

One image is 92.48 µm in length and 69.63 µm in height

Actin fibres in interphase cells

Stained with Phalloidin-FluoresceinDNA (DAPI stain) pseudocoloured in red

10 µm

Microtubules in interphase cell

Stained with anti-tubulin antibodies and secondary fluorescein antibodiesDNA (DAPI stain) pseudocoloured in red

10 µm

MACROPHAGE PHAGOCYTOSIS: SIGNALING THROUGH HETEROTRIMERIC G-PROTEINS

MACROPHAGE PHAGOCYTOSIS: CHEMOKINES ACT THROUGH HETEROTRIMERIC G-PROTEINS

Artificially activated by phorbol ester (mimics DAG)

19

Artificially elevated by ionomycin

DIACYLGLYCEROL AND PHORBOL MYRISTATE ACETATE

DAG

PMA

MACROPHAGE PHAGOCYTOSIS: E. COLI LIPOPOLYSACCHARIDE (LPS)

Lipid A

MACROPHAGE PHAGOCYTOSIS: LPS-INDUCED ACTIVATION CLUSTERS

LYMPHOCYTE PROLIFERATIONConcanavalin A:• Polyvalent lectin• α-D-mannosyl and α-D-

glycosyl binding• Mitogen• Polyclonal activation

(in contrast to antigen-mediated clonal expansion)

• Pleiotropic effects• Metabolic stimulation• Receptor clustering

(lipid raft)?

T-CELL ACTIVATION