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