Laurent Gelman Facility for Advanced Imaging and ... · Live cell imaging and confocal microscopy...
Transcript of Laurent Gelman Facility for Advanced Imaging and ... · Live cell imaging and confocal microscopy...
Live cell imaging and confocal microscopy
Laurent GelmanFacility for Advanced Imaging and Microscopy
Friedrich Miescher Institut, Basel
Acknowledgments
Preparation of the lecture
- Arne Seitz (EPFL)
- Stefan Terjung (EMBL Heidelberg)
- Jens Rietdorf (FMI / Zeiss, Munich)
Data
- Rico Kunzmann (group A. Peters, FMI)
- Vincent Dion (group S. Gasser, FMI)
- Julia Kleylein-Sohn (Novartis)
- Flavio Donato (group P. Caroni, FMI)
- Gerald Moncayo (group B. Hemmigs)
- Jérôme Feige (UNIL / Novartis)
Overview
Growing cells under a microscope…– Physical integrity– Attachment, immobilization of the sample– Incubation systems (temperature, gases, humidity, pH control)
Imaging living cells– Autofluorescence– Photodamage– Microscope settings
(speed, laser power, pinhole, voxel size…)– Microscope setups: spinning-disk versus single-beam LSM and wide-field microscope.
When performing live cell microscopy, cell viability must be at the forefront of any measurement to ensure that the biological processes that are under investigation are not altered in any way.
Mounting: Physical Integrity
Attachment, ImmobilizationCells usually adhere better on plastic (treated) but can be also plated on glass.
Coating the coverglass / SubstratePoly-L-LysineFibronectinConcanavalin AOther…
0.5 - 1% LMP agaroseCyGel1.5 - 3% methyl cellulose Silicon oil, Halocarbon oilTransparent films
Some dishes are made of a special plastic with low autofluorescence where cells adhere well and allowing the use of immersion objectives. (www.ibidi.de) (www.willcowells.com)
Temperature
Small heating stages + Objective heater
Temperature control (on microscope stage)Temperature controller for slides Multiwell stage-top incubator
(OKOlab, www.oko-lab.com)
+ Relatively cheap - System temperature not constant (focus drift
possible!)
(www.embl-em.de)
Focus Drift
Constant temperature of the microscope is also important for time-lapse imaging:
Focus drift due to materialextension/shrinkage due to temperature changes
Microscope incubators
Advantages• Constant conditions (10 – 40 °C)• Stabilized focus• Atmosphere controllable (CO2 …)
Disadvantages
• Expensive
• Microscope access impaired
(www.lis.ch)
(www.oko-lab.com)
(www.embl-em.de)
Gases, pH, Humidity, Osmolarity
Physiological pH during imaging is required!
Without CO2 control (short-term):
• HEPES buffered medium instead of carbonate e.g. 30mM HEPES, 0.5g/l Carbonate instead of 2.2g/l Carbonate.
• Seal the sample chamber, e.g. with silicon (no gas exchange)
With CO2 control (long-term):
• Use perfusion chambers• Use incubators
Gases, pH, Humidity, Osmolarity
Possible solutions:• Seal the sample chamber, e.g. silicon (gas exchange ?)• Use perfusion chambers• Use incubators with humidity control• Use silicon oil
Humidity control during long-term experiments: • Osmolarity of the medium stays constant • sample doesn´t run dry
HEPES Buffered Media
Advantages
• Open system, easy to manipulate.
• Easy to handle and control.
Disadvantages• Usable for ~ 1 hour.• Toxicity of HEPES with some cells.• Evaporation.
Sealed chamber
Advantages
• Easy to handle and control.
• No evaporation.
• Cheap (reusable).
Disadvantages• Usable for max 3 hours depending
on volume.• No manipulation.• Mounting of the chamber might be
tedious.
Gas Permeable Chamber
Advantages• High optical quality (DIC)• Gas permeable• Water impermeable• Perfusable• Usable for days.
Disadvantages• No direct access to cells
(www.ibidi.de)
Perfusion chambers
Advantages
• Constant conditions.
• Manipulation of media.
• Usable for days.
• No evaporation problems.
Disadvantages• Hard to assemble and control.• Shear stress possible.
“scratch” or “would healing assay”Mammalian cells. Wide-field automated inverted microscope.2.5x objectiveTime-lapse: 50 points, 1 stack every 20min (16.5 hours total).
Courtesy of Gerald Moncayo (Hemmings lab, FMI)
Overview
Growing cells under a microscope…– Physical integrity– Holders, attachment– Incubation systems
(Temperature, Gases, Humidity, pH…)
Imaging living cells– Autofluorescence– Photodamage– Microscope settings: speed, laser power, pinhole, voxel size…– Microscope setups: Spinning-disk versus single-beam LSM and wide-field
microscope.
Choice of the imaging setup
Which microscope is the most appropriate for a live cell experiment?
Go more for sensitivity than resolution!
Autofluorescence
Specific sources of autofluorescence– Aromatic amino acid residues (UV)– Reduced pyridine nucleotides (UV)– Flavins (UV, blue)– Chitin (broad)– Yolk– Chlorophyll (blue, green)– Phenol red indicator use ‘Imaging medium’ !
General sources of autofluorescence– Dead cells (broad)
Cures – Longer wavelengths– Avoid stress– Use imaging medium (no phenol red)– Choose excitation and detection windows properly
Photodamage and photobleaching
With live-cell microscopy, there must be a compromise between acquiring beautiful images and collecting data that provide a high enough signal-to-noise ratio (S/N) to make meaningful quantitative measurements of a living specimen.
Triplett state:• chemical reactive• radicals• bleaching / phototoxicity
A lot of light, not absorbed by the fluorophore, can also potentially trigger chemical reactions in the sample by bringing directly energy to molecules or heating up locally the sample.
Fluorescent molecules used for the labeling of the sample can themselves cause deleterious effects.
Example: Be very careful when using DNA intercalating agents, such as DAPI or Hoechst!
Recognize damaged cells
- Cells detach and round up- Bulges form (“Blebbing”)- Mitochondria swell and/or get isolated- Large vacuoles appear- Cells do not make it through mitosis- Any sign of necrosis or apoptosis
Monitoring of cellular metabolic activity can be monitored for example with AlamarBlue (Invitrogen).
Avoid Photodamage
- Minimize illumination power, tolerate more noise and less S/N ratio
- Optimise detection:FiltersetsDetectorsResolution (x, y, z, t, intensity value, channels)
- Use decent dyes (high quantum efficiency).
- Add antioxidants (Trolox, ascorbic acid 2mg/ml)
Is max resolution needed for my live cell experiment?
Relative Brightness of objective lenses NA4/M2
Magnification (M)
The brightness of an image varies directly as the fourth power of the NA but inversely as the square of the magnification.
Increasing the number of pixels or increasing magnification decreases the intensity in each pixel Resolution is increased only as long as the signal-to-noise ratio is good
40x/1.3
20x/0.8 63x/1.4
100x/1.35x/0.15
10x/0.45 25x/0.8
Detector
Sample
Resolution, pixel size, binningIncreasing the number of pixels decreases the intensity in each pixel, so resolution is increased only as long as the signal-to-noise ratio is good (and optical resolution not limiting).
N.B.: Decreasing pixel size by two decreases signal-to-noise ratio by 2 also! (see also later section on noise).
Binning 3xNo binning
Binning pixels increases intensity but decreases resolution. Exposure time is also decreased (as well as file size) Interesting for live cell experiments.
Binning, contrast and resolutionWhat are my object size and shape? I can’t be sure… Unless I do binning !NB: “binning” is achieved on a CLSM by reducing the number of pixels while keeping the field of view constant (“Frame size” 512X512 256x256).
No binning Binning 2x2
Zeiss Z1, 100x/1.4
Binning, contrast, speed and photodamage
Mouse brain section, fixed section, Alexa488 staining, Zeiss Z1, 20x/0.8Binning 2x2, exposure time 10ms No binning, exposure time 320ms
Courtesy of Flavio Donato (group P. Caroni, FMI)
Playing on pixel size (binning) and contrast (do not use full dynamic range/well capacity) allowed here to reduce by 32 the exposure time more time for Z-stacks and less photodamage.
Is max resolution needed for my live cell experiment?
Region bleached for FRAP
Ex: FRAP experimentGFP-labeled protein in the paternal pronucleus of a mouse zygote.LSM710 Zeiss microscope, 40x/1.3 objective.Time-lapse: 250 points, 1 frame every 134msec.Pinhole ~1.5 AU
Courtesy of Rico Kunzmann (Group A. Peters, FMI)
Refraction around the coverslip
NA and immersion medium
The higher the refractive index of the immersion medium, the higher the NA…
The higher the NA, the brighter the sample and the higher the resolution…
air, n = 1 oil, n = 1.51
Immersion medium and mounting medium refractive indexesThe refractive index of the immersion medium must fit that of the mounting mediumwhen objects are located far away from the coverslip.
oil, n = 1.51
Thick tissue slicen < 1.51
Objective numerical aperture - definition
Apochromat 63x/1.3 glycerol immersion Apochromat 63x/1.2 water immersion
Moss in water
(Courtesy of Nathalie Garin, Leica)
Working distanceThe working distance is defined as the distance (in millimeters) from the front lens element of the objective to the closest surface of the coverslip when the specimen is in sharp focus.
In most instances, the working distance of an objective decreases as magnification increases.
Principle of Confocal Laser Scanning Microscopy (CLSM)
PMT
Objective
Specimenfocal plane
Dichroic
Laser
Emission Filter
Pinhole
Laser beam shape and photobleaching
confocal microscopy multi-photon microscopy
focal plane
coverslide
focal plane
Photo
bleac
hing
patte
rnLa
ser b
eam
shap
e
Scanned area
Reduced phototoxicity but heating problems and higher photobleaching may occur due to high laser power.
Scanned area
Principle of Confocal Laser Scanning Microscopy (CLSM)
PMT
Objective
Specimenfocal plane
Dichroic
Laser
Emission Filter
Pinhole
Principle of Confocal Laser Scanning Microscopy (CLSM)
The acquisition speed depends on - pixel dwell-time (time spent per pixel)- number of pixels
tone frame > number of pixel * pixel dwell time
Example 1: x*y format = 512 * 512pixel dwell time = 2.16 μst > 512 * 512 * 2.16 = 0.57 s
Example 2:with x*y format = 1024*1024pixel dwell time = 3.5 μsaverage = 4t > 1024 * 1024 * 305 * 4 = 14.7 s
Scanning is a slow process !! To go faster, parallel acquisition of several pixels is required (spinning-disk microscope)
PMT
Objective
Specimenfocal plane
Dichroic
Laser
Emission Filter
Pinhole
Live Cell Confocal Settings
• Avoid averaging, but if it is necessary then use line average instead frame average .
• Use line-by-line sequential instead of frame-by-frame sequential
• Try to acquire channels in parallel and not sequentially
• Minimize laser power, compensate by:– Increased sensitivity (higher gain) more noise, process
the image after acquisition (see later)– Opening pinhole increased optical thickness– Less pixels larger voxel size (x, y, z) or scan every
second line.
Lineaverage
Frameaverage
Linesequential
Framesequential
Parallel versus single point acquisition
Nipkow Disk
Optical Path Configuration in CSU22
Comparison CLSM, SD and wide-field systems
Camera chip
Hg Bulb
Pinhole
PMT
.…
Laser
Camera chip
Laser
Higher sensitivity in SD/WF due to (EM-)CCD cameras
- NB: Confocal microscopes become more and more sensitive though, owing to a new generation of detectors: the GaAsP detectors (“BIG” for Zeiss, “HyD” for Leica).
- Read-out noise can be lowered when using lower read speeds of the camera.
√ (read noise)2 + (dark noise)2 + (Shot noise)2
Signal to noise ratio (S/N)
S/N = Signal
Number of photons
S/N
Andor catalogue (2006)
noise dominated byphotonic component
noise dominated byread and dark components
Duplication of supernumerary centrosomes
Multi-beam scanSingle-beam scan
mitotic DdCP224-GFP/GFP-histone2B cells
Comparison of signal to noise ratio between LSM and SD
Single-beam LSM scan Multi-beam SD scan
SD have weak illumination powers
Imaging limitations: available signal
(Laser) excitation is not the only limiting factor, fluorophore saturation decreases overall signal
0
0.2
0.4
0.6
0.8
1
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
Excitation photons x1026
Satur
ation
Extended Jablonski diagram
Saturation and triplet state in single- and multi-beam scanning
0.4
0.2
0.6
0.8
1.0
1E20 1E21 1E22 1E23 1E24 1E25Intensity (photons cm-2s-1)
Single-beam scanMulti-beam scan
1mW 488nm, 40x 1.25 NAFlu
orop
hore
satur
ation
Single-beam scanMulti-beam scan
0.4
0.2
0.6
0.8
1.0
1E20 1E21 1E22 1E23 1E24 1E25Intensity (photons cm-2s-1)
Tripl
et po
pulat
ion a
t stea
dy st
ate
Intensity levels in single and multi-beam scanning
Beams 1Illumination 1 mWEmission rate 1.26 x 108 photons/secFluorophore saturation 63%Detection efficiency 0.25Overall efficiency 1
Beams > 1000Illumination 0.4-0.6 µWEmission rate 1.72 x 105 photons/secFluorophore saturation 0.09%Detection efficiency 0.9Overall efficiency 4
Model calculations based on scanning fluorescein at 1 mW 488 nm excitation with a 40x/1.25NA objective, from Live Cell Spinning Disk Microscopy, Ralph Gräf , Jens Rietdorf & Timo Zimmermann, Adv Biochem Engin/Biotechnol (2005) 95: 57–75
Light-dependent photobleaching?
“Photochemical damage is one of the most important yet least understood aspects of the use of fluorescence in biology; in this discussion we can do little more than define our ignorance.”Roger Tsien (Nobel Prize for Chemistry 2008)
Triplett state:• chemical reactive• radicals• bleaching / phototoxicity
Consequences of a not adjustable pinhole size in a SD
N.A. → ↘ → MAG → → ↘λ ↘ → →Resolution ↗ ↘ ~ ↘
Comparison LSM vs. Spinning-Disk confocal
Property Single-beam scanning confocals
Multi-beam scanning confocals
Wide-field setup
Acquisition point by pointslow
Numerous pointsfast
All frame at a timevery fast
Detection Photomultiplier Tubelow sensitivity
CCD/EM-CCD Camerahigh sensitivity
CCD/EM-CCD Camerahigh sensitivity
Multichannel imaging
Parallel/Sequential= fast
Sequential/(Parallel)slow (time x nb of channels)
Sequential/(Parallel)slow (time x nb of channels)
Region bleaching Integrated Add-on Add-on
Optical sectioning Adjustable pinholesFlexible
a lot of light is lostNo crosstalk between
scan beams
Fixed pinhole sizeinflexible/suboptimala lot of light is lost
Crosstalk between scan beams
No sectioningall light collected
Possibility to do TIRF
Image processing in live cell imaging
Very often, images are noisy, as low laser powers are used, and/or short exposure times, and/or fluorescent proteins with low quantum yields.
Deconvolution or denoising can often improves dramatically image quality.
Image processing in live cell imaging: PureDenoise pluginPureDenoise is a plugin in ImageJ, Florian Luisier at the Biomedical Imaging Group (BIG), EPFL, Switzerland, http://bigwww.epfl.ch/algorithms/denoise/
Mammalian cells, GFP-labeled microtubule-organizing centre (MTOC)SD-microscope, 40x/1.3 objective, exposure time: 100ms per frame.Z-stack: 81 planes, dist=500nm.Time-lapse: 210 points, 1 stack every 2 min. After acquisition, images are denoised with PureDenoise over time and MIPs over Z-dimension are generated.
After denoising Before denoising
Courtesy of Julia Kleylein-Sohn (Novartis)
17’010 frames within 7 hours
Image processing in live cell imaging: Deconvolution
YFP-labeled locus in Yeast nucleus.SD-microscope, 100x/1.45 objective, exposure time: 30ms per frame.Z-stack: 14 planes, dist=200nm.Time-lapse: 201 points, 1 stack every 1.5 sec.
After acquisition, images are deconvolved with Huygens professional (www.svi.nl) and MIPs over Z-dimension are generated.
Courtesy of Vincent Dion (Gasser lab, FMI)
After deconvolution Before deconvolution
2’814 frames within 5 minutes
Working with fluorescent fusion proteins: be careful!- Check expression level sand degradation
- Check the functionality of the fusion protein… Adjusting expression levels to those of the endogenous protein!
ng of vector transfected 10 20 30 40 10 20 10 20A-ECFP B-EYFP EYFP-B
Working with fluorescent fusion proteins: be careful!
- Look for artificial clustering of the over-expressed proteins
YFP-PPARα
End