light & fluorescence microscopy and its …tcoenye/iuap/braeckmans.pdflight & fluorescence...
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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
d
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
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
-2 -1 0 1 20
0.1
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1
-3 0 30
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00
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Incr
easi
ng
mag
nif
icat
ion
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
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