IMMUNODEPLETION OF CONDENSIN FROM XENOPUS EGG EXTRACTS HSSΔcond.Δmockmock boiled beadsextracts cond. anti- cond. anti- α-tub 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 MICROSCOPY Based 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 MICROSCOPY Thin 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 shift This 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

TYPES OF LIGHT MICROSCOPY Bright-field Phase-contrast Differential-interference contrast (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 DETECTION Antibodies 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 MICROSCOPY Pixel size: 6.8 µm CCD chip dimension: 1360 x 1024 pixel Microscope magnification: 100x  6.8 µm/100 x1360=92.48 µm  6.8 µm/100 x 1024=69.63 µm  One image is µm in length and µm in height

Actin fibres in interphase cells Stained with Phalloidin-Fluorescein DNA (  DAPI stain) pseudocoloured in red 10 µm

Microtubules in interphase cell Stained with anti-tubulin antibodies and secondary fluorescein antibodies DNA (  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 PROLIFERATION Concanavalin 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