Live cell imaging

Why live cell imaging?

• Live cell analysis provides direct spatial and temporal information

• Planning your experiment– 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…

• 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

• 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 light-true 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

• 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 3D20 z-sectionsEvery 7.5s seconds5 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

• 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 Photobleaching-FRAP 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 Acceptor molecule

Excitation Emission Excitation Emission

FRET

Inte

nsity

Wavelength

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.

Fluorescence resonance energy transfer (FRET)

When to use FRET?

An Aurora B FRET probe as a tool to monitor differential phosphorylation

FRET occurs when it is not phosphorylatedViolin et al. 2003Fuller et al. 2008We;burn rt al, 2010

Donor

Acceptor

Aurora B phosphorylation varies with substrate position

Decr

easi

ng p

hosp

hory

lati

on

Michael Lampson, Dan Liu

Fluorescence resonance energy transfer (FRET)

Inte

nsity

Wavelength

Donor molecule Acceptor moleculeExcitation Emission

No FRET

Inte

nsity

Wavelength

Donor molecule Acceptor moleculeExcitation Emission Emission

FRET

An important control in FRET studies is to photobleach the acceptor and demonstrate that donor emission does NOT decrease