L16 Fluorescence Microscopy

what are the disadvantages of brightfield imaging

• limited ability to make out intracellular organelles

• impossible to identify individual proteins/processes

define luminescence

emission of light by a substance not resulting from heat

what are the types of photoluminescence

• luminescence (bio/chemi)

• phosphoresence

give examples of bio-luminescence

• luciferase- firefly tails

• aequorin -jellyfish

give examples of chemi-luminescence

glow-sticks

define phosphorescence

slow emission of light that has been previously absorbed by a substance

whats the speed of phosphorescence

SLOW (ms to hours)

give an example of phosphorescence

watch hands

when is light emission for phosphorescence

light emission is after illumination

when is light emission for fluorescence

light emission is during illumination

define fluorescence

emission of light by a substance that has absorbed light

whats the speed of fluorescence

fast

• 0.5 to 20 ns

define autofluorescence

fluorescence from naturally occurring molecules in your sample

give examples of auto-fluorescence

many cells/proteins

what is fluorescein

universal fluorescent dye used in engine coolant and opticians eye drops

give examples of stuff that gives fluorescence under UV

• quinine

• beta-carboline

what diagram shows fluorescence energy conversion

Jablonski energy diagram

what state is fluorescent emission of light from

S₁ excited state

how do we see fluorescence

• the widefield fluorescence microscope

• confocal microscope

what filter do we use to see fluorescence

dichronic filter block

how does a dichronic mirror work

• reflects below a specific wavelength

• transmits above it

what filters are used for fluorescence

• excitation filter

• dichronic mirror

• emission filter

what does a dichronic mirror do

reflects below-transmits above

what is the equation to solve d (lateral resolving power)

wavelength of light/ 2x numerical aperture of lens

define super resolution microscopy

imaging beyond the diffraction limits of normal microscopy

what are the three major concepts to overcome the resolution limit

• structured illumination (SIM)

• Stimulated emission depletion (STED) - super resolution imaging

• Localisation (STORM and PALM)

describe STED super resolution imaging

• up to 60nm X-Y resolution (~4x improvement)

• up to 130nm Z resolution (~5nm improvement)

• fixed samples

what are the disadvantages/problems with sample preparation

• get the probe to the target

• only label the target

• overcome any sample autofluorescence -best signal to noise ratio

• phototoxicity -live-cell consideration

• photobleaching -some dyes are more resistant than others

why is it a problem to overcome any sample autofluorescence

best signal to noise ratio

why is phototoxicity a problem

live-cell consideration

why is photobleaching a problem

some dyes are more resistant than others

how do we get a dye to target small-molecule probes

• dye chemistry

• antibodies-immunofluorescence

what do fluorescent proteins do

genetically manipulate target proteins to express a fluorescent tag

give an example of genetically manipulating target proteins to express a fluorescent tag

GFP

describe dye chemistry

• live-cell imaging applications

• get through cell membrane

• only become fluorescent in certain environments

• accumulate in certain organelles

• very easy to use and visualise

what dye is used for tubulin

tubulin tracker green

what dye is used for ER

ER tracker red

what dye is used for DNA

hoechst (blue)

what dye is used for plasma membrane

cell mask deep red

what are the problems/disadvantages of organellar specific probes

• limited retention time in the cell/organelle

• limited targets

• specificity

• toxicity

what is used for fixation

formaldehyde

what is used for permeabilisation

mild detergent

what is used for blocking

excess of ‘non-specific’ protein

what is used for primary antibody

± direct label

what is used for secondary antibody

fluorescence

what are the problems of sample labelling

• small molecule chemical probes

• immunofluorescence techniques

what are the problems of small molecule chemical probes

• many cannot be fixed

• few specifically target individual proteins

what are the problems of immunofluorescence techniques

• difficult to use with live cells -only cell surface proteins are visible

• getting label to target requires ‘permeabilisation’

what is the solution to sample labelling

fluorescent proteins

what is the target gene DNA manipulated to contain the code for

GFP

what does the host cell ‘transiently’ express

GFP-tagged gene

• or tagged gene gets incorporated into genome

how is DNA inserted into the cell

• Virus

• liposomes

• electroporation

• microinjection

what do fluorescent proteins cover

most of the visible spectrum

• eBFP 380/440 -mPlum 590/648

what do fluorescent proteins monitor events in

live cells

give an example of fluorescent proteins being used

visualisation of zebrafish micro-vessels

what is the zebrafish

a model organism used to study; developmental biology, cancer, genetics etc

what are the fusion construct problems of fluorescent proteins

• not native proteins

• strong promotors can ‘enhance’ signal

• transient transfections -higher expression

• may perturb protein function

what do we consider from over-expression artifacts

is the protein found in unexpected areas

what does dynamic imaging require

• chemical or genetic (eg GFP) tag to label organelles or proteins

• many images per second

what are the problems/disadvantages of dynamic imaging

• requires short exposure times

• bleaching issues

• toxicity/phototoxicity

• how do you know the proteins/organelles are active?

what is RFP

red fluorescent protein targeted to the mitochondria

what is interaction/association ‘colocalisation’ used to identify

• cells/organelles that co-express certain proteins

• the location of proteins (co-labelling cells/organelles with known markers)

what does colocalisation (interaction/association) microscopy only suggest

• we can only claim colocalisation down to the resolution of our microscope

• do proteins that share the same space interact?

what does FRET visualise

molecular association and interaction

what does FRET measure

the interaction and location of the interaction of two proteins or structures

what does FRET stand for

Forster Resonance Energy Transfer

what is labelled on FRET

• donor fluorophore

• acceptor fluorophore

what must the emission spectra of the donor in FRET be matched with

the excitation spectra of the acceptor

what is the typical FRET pair donor for fluorescent proteins

CFP

what is the typical FRET pair acceptor for fluorescent proteins

YFP

what is the typical FRET pair donor for fluorescent dyes

fluorescein

what is the typical FRET pair acceptor for fluorescent dyes

rhodamine

what takes place when the two structures in FRET become associated (<10nm)

energy transfer from the donor to the acceptor

give an example of what FRET is used to study

cAMP signalling

what is a problem of visualising cAMP signalling

no fluorescent cAMP ‘reporter’ molecule exists

• solution- use fret

no cAMP

FRET

+ cAMP

no FRET

what happens when DNA for CFP-R and YFP-C PKA subunits are introduced into the cell

cell expresses the fluorescent PKA subunits (a-c)

what happens when FRET in cAMP signalling goes blue

excitation

what does a single excitation produce in Fluo-3 (ion imaging)

a single emission

when does Fluo-3 only fluoresce in ion imaging

when bound to Ca2+

what happens when Fluo-3 is bound to Ca2+

a large increase in fluorescence

what are the problems/disadvantages of Fluo-3 ion imaging

• photobleaching

• difficult to accurately measure Ca2+ concentration

what is the excitation peak of Fluo-3

506nm

what is the emission peak of Fluo-3

526nm

is Fluo-3 ratiometric

no

what is the excitation peak of Fura-2

340nm and 380nm

what is the emission peak of Fura-2

510nm

is Fura-2 ratiometric

yes

in Fura 2, what is active and inactive?

media is inactive, cytoplasm is active

describe Fura-2

• dual excitation-single emission

• only fluoresces when bound to Ca2+

• large increase in fluorescence when bound to Ca2+

• ratiometric- easy to correct for photobleaching

• can be used to accurately measure Ca2+

what are the problems with Fura-2

• UV exposure

• Dual excitation

why is dual excitation in Fura-2 a problem

• slow

• specialised imaging equipment

describe the GcAMP- genetic Ca2+ inhibitor

• based on GFP, Calmodulin and M13 -single excitation single emission

• only fluoresces when bound to Ca2+

• large increase in fluorescence when bound to Ca2+

what is calmodulin

Ca2+ binding protein

what is M13

peptide sequence from myosin light chain kinase

describe ion imaging example of imaging elementary Ca2+ release events

• cardiac myocytes loaded with Fluo-3 Ca2+ indicator -intracellular Ca2+ signalling

• fast confocal imaging

describe ion imaging example of imaging inter-cellular Ca2+ wave

• skin cell monolayer loaded with Fluo-3 Ca2+ indicator

• Monolayer ‘wounded’ -intercellular Ca2+ signalling