Live Cell Imaging 1

Live cell imaging:the need for time-resolved microscopy in biology

Peter Lackie - III / BIU

Live Cell Imaging 2

the need for time-resolved microscopy inbiology

why?• biological processes have different time-frames• sequence and dynamics are essential parts ofthese processes• detection of rare / unpredictable events

how?• microscopy and control requirements• imaging & labelling modes• data display and analysis

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typical tissue processing( for microscopy )

cells / tissue

fix

label

localisation /morphology

lLive cells

dDead cells

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why?dynamic processes in biology

• tissue / cell morphology• localisation and compartmentalisation• cell-cell interactions• transient processes• single cell responses – vs population• rapid changes / movement e.g. muscle

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tissue / cell morphology

• development / repair

localisation and compartmentalisation

• internalisation

cell-cell interactions• transient processes e.g. stages of differentiation• single cell responses – especially with minorpopulations• infiltrating cells- neuronal contact

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rapid changes /movement• transient processes > 1 Hz - rapid calcium flux• cilia - fast movement

slow changes / movement• transient processes - unpredictable & asynchronousevents• slow motility > minutes

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live cell imaging- how is it done?

• requirements• imaging modes for live-cell imaging• slowing down• speeding up• analysis

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live cell imaging- requirements

essential• viable cells- environmental chamber – temperature,humidity & CO2 control• microscope, camera (digital) + control

useful• automated microscope for multi-mode imaging(e.g. phase + fluorescence)• motorised stage for multi-point sequencestreatment / control• minimal light exposure• autofocus

Live Cell Imaging 9FASTCAM inverted phase contrast microscope

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imaging modes for live-cell imaging

unlabelled cells• phase contrast• differential interference contrast• relief contrast / Hoffman modulationcontrast - especially for cells on plastic

labelled cells / molecules• bright field / reflected light• fluorescence, epi-reflection• confocal

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visualisation/ labelling

• unstained cells- altered morphology / autofluorescence• viable dyes e.g. Hoechst 33258• labelled probes / beads e.g. phycoerythrin• tagged molecules e.g. GFP

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live cell imaging - some cautions

• demonstrate that cell viability is maintained• light exposure (especially fluorescence)• cell behaviour can be altered by labelling e.g.antibodies / lectins

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time- shift

fast process

slow process

real time

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image analysis & archiving

a typical time-lapse experiment

• phase contrast + single fluorescence• 3 treatments• 2 locations in each• 15 minute intervals• 24 hours

2 x 3 x 2 x 4 x 24 = 1152 images2.5 Mb / image [1280*1024, 16 bit]1152 x 2.5 = 2.8 Gb … up to 50 Gb

data storage & analysis implications

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analysis

cell / border tracking• speed, direction, area covered• comparison with controls

counting• cell interactions• cell divisions• viability• cell infection

image analysis• areas• pattern

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quantifying repair

control treated - AAL

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analysis : repair area

The effect of AAL on rate of repair compared to control

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control

3 ug/ml AAL

change in area of damage per hour, measured at 60 minute intervals from time-lapse images

Effect of fucose pre-incubated with AAL on rate of repair

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control fucose pre-incubated with AAL

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conclusion

time-resolved live cell imaging provides information notavailable by other means

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tracing the progress of GFP transfected cellsin repair (time lapse sequence)

1 h 3 h

4 h 6 h

Area of damage

GFP labelledcell