Fluorescence Microscopy: Machines and Dyes Gert-Jan Kremers - - PDF document

1 Fluorescence Microscopy: Machines and Dyes

Gert-Jan Kremers Erasmus Optical Imaging Centre Erasmus MC

BRT course, 10 November 2017

Why fluorescence microscopy?

Nucleus Peroxisomes ER Mitochondria Focal adhesion Differential interference contrast (DIC)

Overview

• 1. Principle of fluorescence
• 2. Confocal microscopy
• 3. Fluorescent proteins

Principle of fluorescence

UV Infra- red energy high low

Energy

Excitation Emission S0 S1

CHO cells Hoechst

Focus problem Principle of the confocal microscope

2

Pinhole at 1 airy unit (confocal)

Optical sectioning

Pinhole open (widefield) HeLa mCherry-tubulin Pinhole at 1 airy unit (confocal)

Optical sectioning

Pinhole open (widefield) HeLa mCherry-tubulin

XY and Z laser scanning

X Y

3D scanning

Hela mTurquoise1_CAAX

1 2 3 4

3D scanning

Hela mTurquoise1_CAAX

3D scanning

MossyFiberYFP, Z. Gao, Neuroscience

3

3D scanning

MossyFiberYFP, Z. Gao, Neuroscience

Blue - DAPI nucleus (direct) Green - α-tubulin (anti-body) Red - phalloidin actin (direct)

Fluorescent probes Fluorescent probes

• Organic Dyes

– DAPI, Hoechst – FITC, TRITC, Texas Red – AlexaFluor, Cy, ATTO – Silicon rhodamine (SiR)

• Quantum dots
• Fluorescent proteins

Fluorescent proteins

– GFP gene isolated from Aq Victoria jellyfish – GFP color variants: blue, cyan, yellow – First red FP from coral: DsRed – mFruit basket of color variants

27 kDa

Why was GFP such a breakthrough?

• GFP is a protein
• No cofactor required
• Genetic targeting:

– Organism, cell, organelle – Fusion proteins – Biosensors

• Low toxicity

!! Live cell imaging !!

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html

Genetic encoded labeling

4

Genetic labeling Live Cell Imaging Optical highlighters

Change in color of fluorescence after illumination with near-UV light

• 3 classes

– Photoactivatable FPs: dark to green – Photoswitchable FPs: dark to green (and reverse) – Photoconvertible FPs: green to red

Photoconvertible FPs

Shaner et al, JCS 2007

Photoconvertible FPs

Kremers et al (2009) Nature Methods

U2OS mMaple3-H2B

5

Summary

• Fluorescence microscopy

– Seeing is believing! – High contrast multi-color imaging

• Confocal microscopy

– No more blurry images > everything is in focus – Optical sectioning for 3D imaging

• Fluorescent proteins

– Genetic targeting – Live cell imaging – Functional imaging Erasmus OIC

Optical Imaging Centre Rotterdam

Fluorescence Microscopy: Machines and Dyes

Gert-Jan Kremers Erasmus Optical Imaging Centre Erasmus MC

BRT course, 10 November 2017