live cell imaging - southampton.ac.uk · the need for time-resolved microscopy in biology ......
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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
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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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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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analysis : repair area
The effect of AAL on rate of repair compared to control
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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