Live Cell Imaging
Quantitative Microscopy 2012, CBA Uppsala
Göran Månsson
CLICK – Center for Live Imaging of Cells at Karolinska Institutet
Dept. of Medical Biochemistry & Biophysics, KI Solna campus
[email protected] 070-748 3708
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 2
Live cell imaging - Trinity of imaging
Resolution
Speed Light
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 3
Live cell imaging - Contents of talk
! Demands on the imaging system for live cell studies
! Illumination
! Detection
! Acquisition
! Minimize bleaching and photo toxicity
! Sampling theory
! Calculate resolution and pixel size
! Post processing (deconvolution, 3D,..)
• Light sensitivity
• Cell movement
• Environmental demands
• ”Container”
• Resolution
• Restriction in dyes
• Cell volume
- Avoid ”dying cell imaging” => imaging restrictions
- Demand for speed, tracking capability
- Temperature, CO2, buffers, humidity
- Flasks " inverted microscope, dishes " coverslip bottom/dipping lens
- Water objective – NA < 1.3 Oil objective – NA < 1.5
- Uptake/production of fluorochrome must not harm cells significantly
- Bigger stacks for volume rendering/3D modeling
October 23, 2012 4 Göran Månsson Quantitative Microscopy 2012, CBA
Live cell imaging - some characteristics
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 5
Live cell imaging - Challenges
Live cells are very sensitive!
We must therefore optimize the medium, temperature, gas
atmosphere, light exposure etc, to keep the cells “happy”.
Light bleaches fluorescent proteins and can be photo-toxic.
Always check the cells when the imaging is done: Are they
affected by the imaging as such? check blebbing and for
example proliferation or differentiation afterwards. Use
non-imaged cells as control.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 6
To minimize the light exposure we must:
• Optimize the illumination
• Optimize the detection
• Optimize the acquisition parameters
• Improve images after acquisition (post-acquistion modif.)
Live cell imaging - Minimizing the light exposure
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 7
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Do you need fluorescence at all?
# Consider using transmitted light or a non-fluorescence contrast method, such as DIC, for your experiment.
# Locate your cells and find focus plane using other
contrast method than epifluorescence. Less harmful.
# If you lack a contrast method for transmitted light, you may still get enough contrast by closing the condenser aperture.
# Transmitted light imaging are dependent on a correctly adjusted light path (i.e. Köhler setting).
Live cell imaging - Obstacles to overcome
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 8
Priority one for fluorescence imaging: Lower the light intensity to the minimum!
# Remove unneccessary items from the light path (DIC prisms etc) and let detector receive 100% light.
# Use a Neutral Density (ND) filter for the fluorescence lamp (you don’t need strong signal to localize the cells). Restrict your eyepiece viewing to the minimum.
# Decrease laser power output as much as feasible.
# Avoid useless scans!
# Use filters with very high transmission.
# You don´t have to see the cells on the screen, to gather acquire useful data! (It can look black).
Live cell imaging - Obstacles to overcome
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 9
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Make sure your detector is very sensitive!
# Choose a sensitive detector at time of purchase: - EM-CCD or sCMOS camera (WF or SDC). - GaAsP/APD Detectors (LSC).
- NDD for multi-photon imaging.
# For cameras: - Consider binning pixels for higher sensitivity and higher S/N. Be aware that you lose resolution.
- Use B/W camera (lacks Bayer filter)
# For PMTs at confocals: - Make sure the sensitivity (=detector applied voltage) is high enough to see weak signals.
- Consider open up the pinhole a bit (sacrifize axial res.)
Live cell imaging - Obstacles to overcome
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 10
Make sure your detector is very sensitive! – cont.
# For EM-CCD cameras: Increase the amplification gain until you see the noise. Then you will detect even the weakest signals.
# Choose a detector with a large pixel depth, e.g. 16bit CCD camera instead of 12-bit.
# If you can choose pixel depth of the acquisition with PMT, go for the higher amount. After histogram stretch you still
have better intensity resolution than if you used 8bit.
# Another reason why the detector must be very sensitive, is so you can acquire images rapidly during cell movement or signal changes (e.g. Ca2+ imaging)
Live cell imaging - Obstacles to overcome
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 11
Live Cell Imaging - Using full dynamic range on live cells...
Exp.time = 851ms
-Much data, high S/N
-High photo toxicity
and bleaching!
-“Dying cell imaging”
Exp.time = 11ms
-Postprocessed using
5x5 Median filter, to
reduce noise
Exp.time = 11ms
-Less data, lower S/N
-77x less exposure!
Faster
-Live cell imaging
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 12
Live Cell Imaging - Using full dynamic range on live cells...
Exp.time = 11ms
-Less data, lower S/N
-77x less exposure! Faster
-Live cell imaging
Best image is often the worst “quality” image – that still gives you the information you look for.
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 13
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Items to choose and use wisely
# Objective (magnification, corr ring, NA, immersion, Ph?, WD)
# Immersion (glycerol for many mounting media, temperature)
# Cover slip (Refractive index, thickness). (Live imaging?)
# Sample container (bottom thickness, plastic/glass, coating)
Choices of above affects the Spherical Abberation.
Also, remember to clean the objectives beforehand!!
Live cell imaging - Reduce spherical abberations (SA)
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 14
Live cell imaging - Decrease/eliminate spherical abberation
Widefield microscope, 40X/0.6 air
objective with correction collar
Bad setting of collar Collar set for 0.17mm cover slip
Double exposure time vs correct Half the exposure time vs bad
- Note! Mismatch of Refractive Index gives similar result to the images.
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 15
Live cell imaging - Decrease/eliminate spherical abberation
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Material Refractive Index
Air 1
Water 1.33
Quartz glass 1.45
Glycerol 1.47
Immersion oil 1.52
D 263M glass 1.52
Polystyrene (flasks, dishes) 1.56
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 16
Live cell imaging - Decrease/eliminate spherical abberation
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
FITC TRed FITC TRed
Confocal
1AU
Confocal
Max pinhole
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 17
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Adjust the pixel time (or exposure time) and use averaging filter if applicable.
This so you can minimize the light intensity on the cells, but still keep good S/N ratio.
The cells are happier – but the experiment takes longer time. You lose temporal resolution.
Live cell imaging - Obstacles to overcome
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 18
Avoid photo toxicity and bleaching
- Minimize light exposure intensity
Laser 40%
Gain 447V
Laser 1%
Gain 750V
Laser 0.2%
Gain 700V
High laser
Low sensitivity
40x less intensity!
Still not much noise
200x less intensity
Low contrast
Laser 0.2%
Gain 700V
Histogram stretch
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 19
Avoid photo toxicity and bleaching - Avoid photo-toxicity and bleaching
Laser power 4% Detector gain 548V Pixel time 10µs Averaging 1
Laser power 0.2% Detector gain 700V Pixel time 2µs Averaging 1
Laser power 0.2% Detector gain 700V Pixel time 2µs Averaging 1 – histogram stretch
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Save the cells and decrease the noise!
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 20
Live Cell Imaging - what is histogram stretch?
Image is displayed with its full dynamic range – here 12 bit (212=4096 grey levels)
Before After
Image is displayed with a reduced dynamic range – here from 0 to 670 (instead of 4095)
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 21
Avoid photo toxicity and bleaching - Save the cells and decrease the noise
Laser power 4% Detector gain 548V Pixel time 10µs Averaging 1
Laser power 0.2% Detector gain 700V Pixel time 2µs Averaging 1
Laser power 0.2% Detector gain 700V Pixel time 2µs Averaging 1 – histogram stretch
Laser power 0.2% Detector gain 700V Pixel time 10µs Averaging 1
Laser power 0.2% Detector gain 700V Pixel time 10µs Averaging 5
Laser power 0.2% Detector gain 700V Pixel time 10µs Averaging 5 – histogram stretch
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 22
Live cell imaging - Minimize light exposure intensity
Intensity Averaging Pixel time Rating
High No Short
Low No Long
Low Yes Short
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Bad
Better
Best
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 23
Live Cell Imaging - Proper sampling
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Structure pattern of specimen
Pixel size twice as big as
object structures
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 24
Live Cell Imaging - Proper sampling
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Structure pattern of specimen
Pixel size as big as object
structures
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 25
Live Cell Imaging - Proper sampling
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Structure pattern of specimen
Pixel size as big as object
structures
Pixel size as big as object
structures…unlucky sampling
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 26
Live Cell Imaging - Proper sampling
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Structure pattern of specimen
Pixel size as big as object
structures…unlucky sampling
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 27
Live Cell Imaging - Proper sampling
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Structure pattern of specimen
Pixel size half the size of
the object structures
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 28
Live Cell Imaging - Nyqvist-Shannon sampling theorem
Lateral:
Axial:
Temporal:
Spectral:
Pixel size at specimen plane ≤ ½ resolution in x/y-plane.
Z-step distance in 3D stack ≤ ½ resolution in z-plane.
Time point interval ≤ ½ wished resolution in time.
Sampling bandwidth ≤ ½ wished spectral resolution.
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 29
Live Cell Imaging - Nyqvist-Shannon sampling theorem
Consequences of undersampling (too sparse sampling)
• The finer detail information is lost
• Aliasing may occur, i.e. artifacts in your image due to the sampling
Consequences of oversampling (too frequent sampling)
• Decreased S/N due to decreased detection capacity per pixel
• Photo-toxicity/bleaching increases due to more exposure/higher intensity
• Acquisition time increases (more pixels) " decreased temporal resolution
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 30
Correct size of pixels (<1/2 of resolution)
Too big pixels (undersampling)
Live Cell Imaging - Nyqvist-Shannon sampling theorem
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 31
Live Cell Imaging - calculate lateral (x/y) resolution
Widefield Confocal
R =0.61∗λemission
N .A.R =
0.45∗(λexc + λemi) /2
N.A.
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Determine the lateral resolution
Calculate the pixel size
Size at specimen = Pixel size of CCD
Total magnification
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 32
Live Cell Imaging - Nyqvist-Shannon sampling theorem
Example how to calculate resolution
and pixel size - for widefield
fluorescence imaging
≈ 465nm
16μm
100X/1.4 oil
0.63X
Emission peak:
CCD pixel size:
Objective:
Camera adapter:
Pixel =16
100∗0.63≈ 255nmd =
0.61∗λ
N .A.≈0.61∗465nm
1.4≈ 200nm
Lateral resolution Pixel size at specimen
Pixel =16
100∗0.63∗1.6≈160nm
1.6X Magnification changer:
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 33
Live Cell Imaging - Deconvolution
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Microscopy images contains both noise and blur, i.e. out-
of-focus light.
Confocal images much less blur but usually more noise,
than widefield images.
• Deconvolution reduces or eliminates both noise and
blur.
• Deconvolution also increases the S/N ratio, resolution
and contrast.
Confocal image Image deconvolved
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 34
Live Cell Imaging - Deconvolution
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Widefield
40X/0.6
CLSM confocal
40X/1.2
Before 3D Blind
Deconvolution
After 3D Blind
Deconvolution
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 35
Live Cell Imaging - Deconvolution
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Confocal deconvolved
Widefield raw
Widefield
deconvolved
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 36
Live Cell Imaging - Deconvolution
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
Deconvolution increases S/N " lower your signal and maybe
Nyqvist sampling is feasible.
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 37
Live Cell Imaging - Background subtraction
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif.
image with
bad vignetting
background
image
background
subtracted
October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 38
Live Cell Imaging - Volume rendering
! Illumination
! Detection
! Acquisition parameters
! Post-acquisition modif. Zebra fish after 8 iterations
Summary
• Minimize/optimize light exposure
• Maximize/optimize detector sensitivity
• Avoid spherical abberations
• Aim for Nyqvist sampling
• Always deconvolve your acquisitions
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Sources of information
! Book
Handbook of biological confocal microscopy. Edited by James Pawley.
! User Forum
Confocal microscopy list serv at http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy.
! Articles - Frigault MM, Lacoste J, Swift JL, Brown CM. Live-cell microscopy - tips and tools. J Cell
Sci. 2009 Mar 15;122(Pt 6):753-67.
- www.nature.com/milestones/light-microscopy. Highlights excellent new microscopy applications/inventions