Live Cell Imaging Resolution Light Speed - Image...

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


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


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.


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


!  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


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).


!  Illumination

!  Detection




!  Illumination

!  Detection



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.)



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)


!  Illumination

!  Detection




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


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




!  Illumination

!  Detection



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)


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





!  Illumination

!  Detection



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



!  Illumination

!  Detection



FITC TRed FITC TRed

Confocal

1AU

Confocal

Max pinhole


!  Illumination

!  Detection



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.



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




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



Save the cells and decrease the noise!


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




Avoid photo toxicity and bleaching - Save the cells and decrease the noise

Laser power 4% Detector gain 548V Pixel time 10µs Averaging 1







Live cell imaging - Minimize light exposure intensity

Intensity Averaging Pixel time Rating

High No Short

Low No Long

Low Yes Short

!  Illumination

!  Detection



Bad

Better

Best


Live Cell Imaging - Proper sampling

!  Illumination

!  Detection



Structure pattern of specimen

Pixel size twice as big as

object structures



!  Illumination

!  Detection




Pixel size as big as object

structures



!  Illumination

!  Detection





structures


structures…unlucky sampling



!  Illumination

!  Detection





structures…unlucky sampling



!  Illumination

!  Detection




Pixel size half the size of

the object structures


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





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




Correct size of pixels (<1/2 of resolution)

Too big pixels (undersampling)


!  Illumination

!  Detection




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



Determine the lateral resolution

Calculate the pixel size

Size at specimen = Pixel size of CCD

Total magnification



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




Live Cell Imaging - Deconvolution

!  Illumination

!  Detection



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



!  Illumination

!  Detection



Widefield

40X/0.6

CLSM confocal

40X/1.2

Before 3D Blind

Deconvolution

After 3D Blind

Deconvolution



!  Illumination

!  Detection



Confocal deconvolved

Widefield raw

Widefield

deconvolved



!  Illumination

!  Detection



Deconvolution increases S/N " lower your signal and maybe

Nyqvist sampling is feasible.


Live Cell Imaging - Background subtraction

!  Illumination

!  Detection



image with

bad vignetting

background

image

background

subtracted


Live Cell Imaging - Volume rendering

!  Illumination

!  Detection


!  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

October 23, 2012 39 Göran Månsson Quantitative Microscopy 2012, CBA October 23, 2012 Göran Månsson Quantitative Microscopy 2012, CBA 40

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

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