Functional cellular imaging by light microscopy

MICROSCOPIES

Why use Light?

Good (enough) resolution:Spatial – classically a few hundred nanometers; now tens of nmTemporal - <millisecond

Compatible with live cells, tissues, organisms

Many probes available for imaging:Fluorescent antibodies, GFP, indicators for Ca2+

membrane potential, etc.

Relatively inexpensive and simple (vs. E.M., PET, FMRI etc)

Three decades of building microscope systems

1977 2008

On the importance of looking

“You can observe a lot just by watching.”

Yogi Berra

Former Yankee Catcher andGreat American Sage

And sometimes you can see beautiful things!

Chaotic and spiral calcium waves in Xenopus oocyte: Jim Lechleiter, U. Texas

Major types of light microscopy;

1. Transmitted (reflected) light. Poor contrast; paucity of specific labels/functional probes; poor depth resolution.

2. Fluorescence. High contrast (black background); numerous fluorescent dyes, proteins and functional probes; permits 3D

imaging (confocal, 2-photon)

A fluorescence microscope

But – conventional fluorescence imaging provides little depth discrimination, so images are terrible because of out-of-focus fluorescence

e.g. Pollen grain imaged by conventional epifluorescence microscopy

One solution – Confocal microscopyOut-of-focus light is rejected by blocking it with a pinhole

aperture

Confocal sections through pollen grain at 1 um intervals

3-D reconstruction of pollen grain

Another way to avoid out-of-focus fluorescence and achieve 3D imaging –

Two-photon microscopy

Especially good for looking deep into tissues (e.g. brain) without damaging cells

Practical theory of 2-photon microscopy

1. Near simultaneous absorption of the energy of two infrared photons results in excitation of a fluorochrome that would normally be excited by a single photon of twice the energy.

2. The probability of excitation depends on the square of the infrared intensity and decreases rapidly with distance from the focal volume.

Advantages of 2-photon microscopy

1. Increased penetration of infrared light allows deeper imaging.

2. No out-of-focus fluorescence.3. Photo-damage and bleaching are confined to diffraction-

limited spot.4. Multiple fluorochrome excitation allows simultaneous,

diffraction-limited, co-localization.5. Imaging of UV-excited compounds with conventional optics.

Two-photon imaging of exocytosis in pancreatic acinar cells

Exocytic events evoked by addition of acetylcholine

C25 m

5 m15 m

DC

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PF

TZ

100 m

Single-cell imaging in intact lymph node

Miller et al., 2002. Science 296: 1869-1873

Another solution – Total Internal Reflection (TIRF) Microscopy

Excite fluorescence in only a very thin layer right next to a coverglass

Good for looking at things happening in or very near the plasma membrane of a cell

Total internal reflection microscopy

glass

air

glass

Total internal reflection microscopy

glass

air

Evanescent wave

Through-the-lens total internal reflection fluorescence microscopy

(TIRFM)

© Molecular Expressions Microscopy Primer

Cultured cells expressing GFP-tagged membrane protein imaged by conventional epifluorescence

The same cells viewed by TIRFM

TIRFM imaging of single-channel Ca2+ fluorescence signals (SCCaFTs): Ca2+ entry through plasma membrane channels

expressed in Xenopus oocytes

Imaging single channel events with high time resolution: SCCaFTs recorded at 500 frames s-1

© Molecular Expressions Microscopy Primer

The diffraction limit

The position of a single point source (e.g. a fluorescent molecule) can be localized with much higher precision, limited only by the number of

photons that can be collected.

What we then need is to have only sparse sources at any given time, so as to avoid unresolved overlap

Sidling around the diffraction limit

Photoactivation Localization Microscopy (PALM)

(Betzig et al., Science 2006)• Express protein of interest tagged with a photoactivatable fluorescent

protein (eg.g. EOS) in cell

• Stochastically photoactivate a low density of molecules per frame and localize using Gaussian function

active state

Fluorescence emission

Bleached state

Activating laser405 nm

Excitation laser532 nm

Repeat thousands of times

Non-fluorescent state

Photoactivation Localization Microscopy (PALM)

• Express protein of interest tagged with a photoactivatable fluorescent protein (eg.g. EOS) in cell

• Stochastically photoactivate a low density of molecules per frame and localize using Gaussian function

Imaging actin tagged with EosFP (photoactivatable protein)

Eos-actin TIRFEos-actin PALM

Fibroblasts expressing DsRed

“Greening” of DsRed

“greening” results from enhancement of green fluorescence and reduction of red fluorescence

Clustered T cells after activation

Evanescent field excites Ca2+-dependent fluorescence only in a thin layer next to cell membrane

Ca2+indicator (fluo-4)in cytosol