Live cell imaging
Why live cell imaging?
• Live cell analysis provides direct spatial and temporal
information
• Planning your experiment
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The markers/fluorophores
The cell’s environment
Practical aspects of the experiment: the microscope
Photodamage
• Applications of live cell imaging
Select your markers carefully
You only see a limited
number of
molecules/fluorophores
2 to3 channels in live cell
imaging
Fluorophores
• Usually tag: GFP, mCherry, Venus, dTomato, etc…
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Transient transfections
Overexpression
Inducible expression
Endogenous levels of plasmid at endogenous promoter
What you need to do
• Keep the cells happy
• Optimize your experiment to get the
most out of it
• Limit photodamage (cells will change
their behavior)
Key components
• Preparation and holding of the cell
specimen
• Temperature and CO2 control
• Microscope
• Light: wavelength, intensity
• Image acquisition
• Type of live-cell imaging experiments
Unhappy cells
Contamination in cells will affect
your experiment
And Mycoplasma!
Media types in human cells
• Need FBS
• DMEM/RPMI: culture media, contains phenol
red, which causes background fluorescence!
• CO2-independent media –for long
experiments
• Leibowitz L15 media, no phenol red!
Holders
Must have a #1.5 coverslip (0.17mm thick)
Maintaining live cells on the
microscope
• Tight control of the environment is critical for
successful live-cell imaging
• Heat within the specimen chamber or chamber holder
• Warm air stream over the stage
• Enclose the stage area/whole microscope
• Use CO2-independent media
• Use CO2 source
Heated objectives
• Alternatively, need to heat the chamber and
lense for 2-4hrs as lenses expand with heat
• Microscope also needs to be stable
Your microscope: temperature
control
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Heat within the chamber holder
Warm air stream over the stage
Enclose the stage area
Enclose the entire microscope
Your microsocope
• Active correction:
– Autofocus-Not ideal: extra light exposure and
change plane in x, y, z
– Active Z position monitoring: Nikon and Zeiss
• Long term focus stability-important for
time lapse work, not as important for short
term observations with operator present
Perfect focus
• To overcome drift due to mechanical
and thermal changes over time
Other features of microsocopes
useful for cell imaging
• Keep the exposure constant
• Motorised stage to follow multiple cells (also
need appropriate software)
• Shutter on illuminators so that the cells don’t
bleach
Photodamage
• Live cells poorly tolerate high exposure to lighttrue for transillumination and epifluorescence:
cell death, compromised cell function and stress
• Targets: the cell, the medium, the fluorophore
• Generation of reactive oxygen species
• Blue light is very toxic to cells
• The longer the wavelength, the better
• You have to compromise!
Light flux at specimen
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Illumination system:
75W Xenon arc
490/10nm exciter filter (60%T)
505nm dichromatic mirror (85% reflectance)
Flux at specimen: 380W/cm2
• 2500 times the flux of sunlight on the
brightest day!
Minimize the exposure to the necessary for
your experiment, not to make a pretty movie
Kinetochore tracking in 3D
20 z-sections
Every 7.5s seconds
5 minutes
That’s a lot of exposure!
Minimum exposure to reduce
photodamage
Use a minimal exposure to maximize your data collection. Kinetochores are still there after 4min!
Deconvolution (1cycle) can help restore your signal for presentation purposes.
Correcting for photobleaching
Type of live-cell imaging
experiments one might do
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Time-lapse imaging (BF or TIRF)
Photoactivated localized microscopy-PALM
Fluorescence Recovery After Photobleaching-FRAP
Fluorescence Correlation Spectroscopy-FCS
Fluorescence Speckle Microscopy-FSM
• Fluorescence Resonance Energy Transfer-FRET
TIRF imaging of cells to image processes
close to the membrane and focal adhesion
TIRF resolution in live-cell imaging
• 100-250nm in z-axis
• The evanescent field, resulting from total internal
reflection of the beam excites fluorophores in a
SMALL volume, close to the coverslip. Therefore
sample photobleaching is very low
Fluorescence Recovery After PhotobleachingFRAP to look at 2D diffusion
Very good for
membrane dynamics
Photoactivation to determine
movement of molecules and lifetime
of subcellular structures
Fluorophore Photoconversion
• EosFP is a green fluorescent protein (emits at
516nm) from stony coral
• Near-UV radiation induces a conformational
change in the protein
• Protein emission at 581nm
• Especially good for cell tracking in organisms
The birth of speckle microscopy
Fluorescence speckle microscopy to look
at motion and turnover of macromoleulcar
assemblies
Courtesy of M. Mendosa/S. Besson
FSM gives information on flux and
movement of actin during migration
Courtesy of M. Mendosa/S. Besson
Quantitative analysis of FSM imaging gives
information on actin movement during cell migration
Courtesy of M. Mendosa/S. Besson
Fluorescence resonance energy transfer (FRET)
• FRET involves non-radiative energy transfer between donor and
acceptor fluorophores
• Occurs over distances of 1-10 nm
• Emission and excitation spectrum must significantly overlap
• Can be used to measure close interaction between fluorophores and
as a ‘spectroscopic ruler’ to measure intermolecular distance
Donor molecule
Excitation
Acceptor molecule
Emission Excitation
FRET
Wavelength
Emission
Fluorescence resonance energy transfer (FRET)
Example: the emission and absorption spectra of cyan fluorescent protein (CFP, the
donor) and yellow fluorescent protein (YFP, the acceptor), respectively.
CFP & YFP pair is currently the ‘best’ for FP-based FRET.
When to use FRET?
An Aurora B FRET probe as a tool to
monitor differential phosphorylation
Donor
Acceptor
FRET occurs when it is not phosphorylated
Violin et al. 2003
Fuller et al. 2008
We;burn rt al, 2010
Decreasing phosphorylation
Aurora B phosphorylation varies with
substrate position
Michael Lampson, Dan Liu
Fluorescence resonance energy transfer (FRET)
Donor molecule
No FRET
Excitation
Acceptor molecule
Emission
Wavelength
Donor molecule
Excitation
Acceptor molecule
Emission
Emission
FRET
Wavelength
An important control in FRET studies is to photobleach the
acceptor and demonstrate that donor emission does NOT
decrease