Fluorescence microscopy

Report
IMMUNODEPLETION OF CONDENSIN
FROM XENOPUS EGG EXTRACTS
extracts
HSS Δcond. Δmock
boiled beads
cond. mock
anticond.
Smc4 
Smc2 
- 170
- 130
- 100
antiα-tub.
FLUORESCENCE MICROSCOPY:
VISUALISING FITC-STAINED SAMPLES
Camera/Eyepiece
Excitation filter (488nm+/-20 nm)
Emission filter (525nm+/-25 nm)
Dichroic beam splitter(>495 nm)
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
-1/4λ on the diffracted beam
(passing through the retarder of the
phase ring)
-1/4λ on the diffracted beam
(passing through the specimen)
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”
Direct beam
Diffracted beam
Δ1/4λ
Δ1/4λ
TYPES OF LIGHT MICROSCOPY
Bright-field
Phase-contrast
Differential-interference
contrast (DIC)
Source: MBC
PRINCIPLE OF
FLUORESCENCE
Fluorochromes can be excited by a particular wavelength and emit
light of a longer wavelength  Stokes shift.
=heat
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 92.48 µm in length and 69.63 µm in height
Actin fibres in interphase cells
10 µm
Stained with Phalloidin-Fluorescein
DNA (DAPI stain) pseudocoloured in red
Microtubules in interphase cell
10 µm
Stained with anti-tubulin antibodies and secondary fluorescein antibodies
DNA (DAPI stain) pseudocoloured in red
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: LPSINDUCED ACTIVATION CLUSTERS
LYMPHOCYTE PROLIFERATION
Concanavalin A:
• Polyvalent lectin
• α-D-mannosyl and αD-glycosyl binding
• Mitogen
• Polyclonal activation
(in contrast to
antigen-mediated
clonal expansion)
• Pleiotropic effects
• Metabolic stimulation
• Receptor clustering
(lipid raft)?
T-CELL ACTIVATION

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