Live cell imaging and confocal microscopy

Laurent Gelman Facility 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, Immobilization Cells usually adhere better on plastic (treated) but can be also plated on glass.

Coating the coverglass / Substrate Poly-L-Lysine Fibronectin Concanavalin A Other…

0.5 - 1% LMP agarose CyGel 1.5 - 3% methyl cellulose Silicon oil, Halocarbon oil Transparent 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 material extension/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 objective Time-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: Filtersets Detectors Resolution (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, binning Increasing 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 resolution What 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/0.8 Binning 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 experiment GFP-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 immers