Light & fluorescence microscopy and its application to biofilms

Biophotonic Imaging Group Lab. General Biochemistry and Physical Pharmacy

Ghent University Belgium

Kevin Braeckmans

Red blood cells (~7-8 mm)

Things Natural

Fly ash ~ 10-20 mm Human hair

~ 60-120 mm wide

Ant ~ 5 mm

Dust mite

200 mm

ATP synthase

~10 nm diameter

Mic

row

orl

d

0.1 nm

1 nanometer (nm)

0.01 mm

10 nm

0.1 mm

100 nm

1 micrometer (mm)

0.01 mm

10 mm

0.1 mm

100 mm

1 millimeter (mm)

1 cm

10 mm 10-2 m

10-3 m

10-4 m

10-5 m

10-6 m

10-7 m

10-8 m

10-9 m

10-10 m

Vis

ible

Nan

ow

orl

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1,000 nanometers = In

frar

ed

U

ltra

vio

let

M

icro

wav

e S

oft

x-r

ay

1,000,000 nanometers =

The Scale of Things – Nanometers and More

Atoms of silicon spacing 0.078 nm

DNA ~2-1/2 nm diameter http://science.energy.gov/bes/news-and-resources/scale-of-things-chart/

Light microscope

Resolution

• Resolution = min. distance to resolve neighboring structures • Human eye: ~ 0.2 mm • Since 17th century: development of microscopes → see (resolve) smaller things

Antonie van Leeuwenhoek (1632-1723) Robert Hooke (1635-1703)

Resolution – continuous improvement

•

Fluorescence nanoscopy

Atomic force microscopy

Resolution

• Point Spread Function, PSF = 3-D diffraction pattern of a point source of light imaged through a lens

• In focal plane: ‘Airy disk’ surrounded by low intensity diffraction rings

• Resolution limit pptical microscope: r l /2 r 250nm for visible light

Image plane Optical axis

Resolution

Original = sum of point sources of light

Image = sum of PSFs

lens

Resolution & objective lenses

𝑟 =𝜆

2𝑛 sin𝜃

𝑛 sin𝜃 = 𝑁𝐴 (Numerical Aperture)

𝑟 = 0.5𝜆

𝑁𝐴

(radius of airy disk)

(r = 290 nm, if l=550 nm & NA=0.95)

1840 - 1905

Resolution & objective lenses

Low NA Medium NA High NA

Immersion medium: • Air: n = 1 → NA = n sinq < 1 • Water: n = 1.33 → NA < 1.33 • Oil: n = 1.52 → NA < 1.52

× 1.5!

(r = 230 nm, if l=550 nm & NA=1.2) (r = 200 nm, if l=550 nm & NA=1.4)

Cover slip

Water / oil

Digital imaging & sampling

Image formed by lens

CCD camera CCD chip

Matching sampling to resolution

2×r0

0 0.5rNA

l

Nyquist theorem: pix size = r/3

-2 0 20

0.1

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-2 -1 0 1 20

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-3 0 30

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00

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Incr

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pixsize = 5 µm NA 1.2 r = 230 nm M = 60x

R = 15 µm

R = 13.8 µm

Aberrations and lens types

Spherical aberrations

Good objective lens

Correction for • Particular cover slip thickness • Particular n of sample only, immersion medium e.g. air, water, oil!

Aberrations and lens types

Spherical aberrations

Spherical aberrations

Corrected for spherical aberrations

Use WI lens for high-resolution live cell imaging

Aberrations and lens types

Chromatic aberrations

Chromatic aberration

Chromatic correction

Aberrations and lens types

Lens types

Chromatic correction Spheric correction

Achromat R+B R+B

Fluorite R+B+G R+B

Apochromat R+B+G+UV R+B+G

Aberrations and lens types

Lens types

Chromatic correction Spheric correction

Achromat R+B R+B

Fluorite R+B+G R+B

Apochromat R+B+G+UV R+B+G

Aberrations and lens types

Lens types

Chromatic correction Spheric correction

Achromat R+B R+B

Fluorite R+B+G R+B

Apochromat R+B+G+UV R+B+G

Aberrations and lens types

Lens types

Chromatic correction Spheric correction

Achromat R+B R+B

Fluorite R+B+G R+B

Apochromat R+B+G+UV R+B+G cost

Light microscopy of bacteria

Streptococci & Fusobacteria

Low contrast

Burkholderia biofilm

Staining of bacteria

Paul Ehrlich (1854 – 1915)

“Magic bullet”

(1940)

• Demonstrated specific bacterial staining • Cure for syphilis • Founder of chemotherapy • Nobel Prize in Physiology or Medicine 1908

Staining of bacteria

Fluorescence

Jablonski-diagram Excitation and emission spectrum

Selective labeling of cells and subcellular components

Selective visualisation with high contrast

Fluorescence microscopy

Microscopy sample holders for biofilms

Glass bottom dishes / chambers / plates → inverted microscope!

Microscopy sample holders for biofilms

Perfusion chambers / flow cells / flow chambers: → Study biofilm growth & structure under laminar flow conditions → Optical window needed! (correct thickness → aberrations!)

Custom made Commercially available

Fluorescence microscopy

Staining bacteria - examples

Live / dead stain (BacLight, Invitrogen) → 2 nucleic acid stains: • Syto9: live cells • Propidium Iodide: inactive/dead cells

Streptococcus Gordonii

Staining bacteria - examples

Fluorescent Proteins → mutant strains expressing FP → fusion proteins (subcellular)

GFP: P. Putida Syto 62: Acinetobacter

GFP: E. Coli P.I.: dead cells (after AB treatment)

Staining bacteria - examples

Fluorescently labeled lectins → labeling EPS components (glycoconjugates)

AlexaFluor 488-Solanum lectin SYTO 60: nucleic acid stain

Cy5-Solanum Tuberosum TRITC-Arachis hypogaea Syto 9: nucleic acid stain

Confocal Laser Scanning Microscopy (CLSM)

Eliminates out-of-focus fluorescence light optical sections

Improved contrast & 3-D imaging

3-D confocal images - examples

Confocal resolution

𝑟𝑧 = 0.64𝜆

𝑛 − (𝑛2 −𝑁𝐴2)

For very small pinhole size (< 25% Airy disk)

Lateral: 𝑟 = 0.5𝜆

2𝑁𝐴 = 0.35

𝜆

𝑁𝐴

Axial:

However, small pinhole → few photons per pixel → noisy images Trade-off: pinhole ~ Airy disk

WI lens: NA=1.2, n=1.33, l=550 nm → 𝑟 = 160 nm

𝑟𝑧 = 460 nm

𝑟𝑧 = 0.88𝜆

𝑛 − (𝑛2 −𝑁𝐴2)

Lateral: 𝑟 = 0.45𝜆

𝑁𝐴

Axial:

Sampling

Remember Nyquist: match pix size to resolution

Fixed # pixels (e.g. 512×512) Change zoom setting (field of view)

Fixed zoom setting (field of view) Adjust # pixels (e.g. 1024×1024)

Sampling – also in 3-D

Nyquist criterion also applies to z-direction

𝑟𝑧 = 0.88𝜆

𝑛 − (𝑛2 −𝑁𝐴2)

Δ𝑧

Δ𝑧 =𝑟𝑧3

NA=1.2, n=1.33, l=550 nm → 𝑟𝑧 = 460 nm Δ𝑧 = 150 nm

Z-step (focus motor)

2-photon microscopy

Excitation by 2 coinciding photons of halve energy (double l)

→ Pulsed laser (pico/femto second pulses)

intrinsic optical sections (no confocal pinhole needed)

Only happens at focus point

2-photon microscopy

Excitation by long (NIR) wavelengths Less scattering, better penetration depth

Confocal Confocal Confocal

2-photon 2-photon 2-photon

Advanced fluorescence microscopy methods for measuring molecular dynamics

application to transport and diffusion in biofilms

1. FRAP

Fluorescence Recovery After Photobleaching (FRAP)

From FRAP-curve:

• diffusion coefficient D

• (im)mobile fraction k

a, t<0 b, t=0 c, t>0 d, t>>0

e f g ha, t<0 b, t=0 c, t>0 d, t>>0

e f g h

0

50

100

150

200

250

N

FRAP – old … but not worn out!

Early experimental days (specialized groups)

Commercialization of CLSM

Landmark papers: 1974: Peters et al., Biochim Biophys Acta 367, 282-94. → invention of FRAP 1976: Axelrod et al., Biophys J 16, 1055-1069. → first quantitative FRAP model 1983: Soumpasis, Biophys J 41, 95-97. → mathematical simplification of Axelrod’s FRAP model

As from the ’90s: FRAP with a laser scanning microscope

LSM = Laser Scanning Microscope (confocal and/or multiphoton)

Photobleaching with a scanning laser beam

a, t<0 b, t=0 c, t>0 d, t>>0

e f g h

a, t<0 b, t=0 c, t>0 d, t>>0

e f g h

FRAP model for circular bleach

• Circular bleach:

0 0 1

0

1 1 I IF t

K eF

where 2

2

2

4 eff

w

Dt r

2 22

2

d beff

r rr

Smisdom et al., J. Biomed. Opt. (2011)

Braeckmans et al., Biophys. J. 85, 2240-2252 (2003)

Average fluo a.f.o. time (spatial information lost)

FRAP model for rectangular bleach

0 2 2 2 20

, , , 1 2 2 2 21 erf erf erf erf4 4 4 4 4

y yx x

eff eff eff eff

l ll lx x y yF x y z t

KF r Dt r Dt r Dt r Dt

2 22

2

d beff

r rr

where

Deschout et al., Optics Express 18 (2010)

Full spatial profile is analyzed → improved accuracy / precision

FRAP on biofilms

Waharte et al., Appl Eniron Microbiol 76 (2010)

Stenotrophomonas maltophilia (loaded with 150 kDa FITC-dextrans)

FRAP on biofilms

Waharte et al., Appl Eniron Microbiol 76 (2010)

Lactococcus lactis

FRAP on biofilms

Waharte et al., Appl Environ Microbiol 76 (2010)

Grey: S. Maltophilia Black: L. Lactis

2. FCS

0.4 mm

2 mm

Fluorescence Correlation Spectroscopy (FCS)

Experimental setup

D, N Reference: Remaut et al., J. Controll. Release 2007

FCS autocorrelation analysis

1

211 1D xy z DG

N

For 3-D Gaussian detection volume:

1E-3 0.01 0.1 1 10 100 1000

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35(C)

Tijd (ms)

1/N

D

G(

)

• Charact. diffusion time D

• Average # molecules N

FCS application to biofilms

Diffusion of bacteriophages in Stenotrophomonas maltophilia biofilms

Briandet et al., Appl Environ Microbiol 74 (2008)

● Sytox Green in solution

▪ Bacteriophages in solution

○ & ∆ Bacteriophages in biofilm Inset: different depths in biofilm

Slowed diffusion of c2 bacteriophage attributed to its long rigid tail

FCS application to biofilms

2-Photon FCS: 28 nm latex nanospheres in Stenotrophomonas maltophilia biofilms

Guiot et al, Photochem Photobiol 75 (2002)

• Diffusion slowed down in biofilm • Cationic beads get stuck

3. SPT

Single particle tracking (SPT)

Imaging movement of individual particles

Sensitive widefield laser microscope

trajectories of single particles

quantitive analysis (mode of motion, colocalization, size …)

SPT set-up (dual color)

Nanoparticle transport in biofilms and lung sputum

Improved treatment of biofilm infections in Cystic Fibrosis patients

Nanoparticle transport in biofilms and lung sputum

1. Mucus barrier 2. Biofilm matrix 3. Biofilm resistance Problems:

• Degradation and binding • Limited diffusion • Plugged parts of the lung • …

Inhaled/intraveneous antibiotics

The dose needed to eradicate the biofilm bacteria is too high

Nanoparticles containing antibiotics

Challenge: create nanoparticles with a high mobility in both CF lung mucus and biofilms

Evidence: antibiotic-containing nanoparticles show increased activity against biofilm bacteria

Nanoparticle transport in biofilms and lung sputum

Sputum: Freshlys expectorated by CF patients

Biofilm: Burkholderia multivorans LMG 18825, clinical isolate

Nanoparticles: 100 & 200 nm

Carboxylate ≈ -45 mV

2 & 5 kDa Poly(ethyleneglycol) (PEG) ≈ -5 mV

N

+ -

Nanoparticle transport in biofilms and lung sputum

PEG Carboxylate Amine

Dw/D = 1.1 Dw/D = 22.7 Dw/D = 14.9

K. Forier et al., Nanomedicine 8 (2013)

Nanoparticle transport in biofilms and lung sputum

PEG Carboxylate Amine

Dw/D = 7.4 Dw/D = 23.8 Dw/D = 28.3

K. Forier et al., Nanomedicine 8 (2013)

Nanoparticle transport in biofilms and lung sputum

PEGylation:

• decreases interactions

• increases mobility

Mucus is tougher

barrier for drug delivery

Thank you