Download Biophotonics and medical imaging

Biophotonics and medical imaging
Prof. Johannes F. de Boer
Prof. Marloes Groot, Prof. Ruud Verdaasdonk,
Dr. Davide Iannuzzi, Dr. Freek Ariese
The impact of physics on
imaging in healthcare
Anna Berthe Röntgen: Hand mit Ringen
Wilhelm Röntgen's first "medical" x-ray,
of his wife's hand,
taken on 22 December 1895
X-Ray, CT, MRI, PET, Ultrasound
These techniques are a
mainstay of medical imaging
UHR-SD-OCT Fovea
6 x 6 mm
1 x 0.5 mm
6 x 1.1 mm
B. Cense et al. Opt. Express 12, 2435-2447 (2004)
Endoscope with
Micromotor
1.65 mm diameter, 6000 rpm
optical fiber
GRIN
OCT
dichroic fiber port
mirrors
motor
1.65 mm
Spinning catheter 3000rpm
52 images/sec
Future directions
• Most cancers develop at the epithelium
• Hollow organs can be accessed by
endoscopes
• Improve specificity
• Immuno-fluorescence and OCT in a single
device
This provides both structural (OCT) and
immunofluorescence information
Imaging mouse heart vasculature
Color codes
for depth
Scan free
depth
resolved
fluorescence
imaging
500 x 500 x 60 µm
15
75
Depth in tissue (µm)
AIM: To image life cells, label-free, with cellular
resolution in deep-tissue
Confocal and non-linear microscopy
Nonlinear microscopy with fluorescent labels
• Laser-induced nonlinear process provides contrast.
• Localized to the laser focus, since excitation ~I2-3.
• 3D-imaging by scanning the focus through the sample.
Two-photon fluorescence microscopy
•
•
•
Laser excitation of a nonlinear process.
Localized to the focal spot.
Scan the laser beam and map fluorescence vs. position.
High-resolution 3Dimaging!
Two-photon fluorescence microscopy
• Interneurons containing
Green Fluorescent
Protein.
• 2-photon excitation at
970 nm.
• Requires a dye or other fluorescent probe.
Third-harmonic generation imaging
THG microscopy on brain tissue
•
•
THG microscopy on mouse brain tissue.
Neurons are clearly visible as dark shadows.
Third-harmonic generation
I3
 3 2 (3) 3
 
  I
2 n c 

z2
z1
ik z
e
2 dz
(1 2i z /b)
2
where
 k  k3  3k 
Isotropic medium, tight focusing:
THG generated before
and after the focus cancel
due to Gouy phase (if
Δk≥0).

 No THG
Discontinuity (in nω or (3) ):
Asymmetry in phase
before and after focus, no
THG cancellation.
 THG signal!
Origin of the THG signal
• The main component providing a high (3) are the lipids in the
cell membrane.
• Checked by staining with the lipid-sensitive dye Nile Red:
THG signal:
Nile Red fluorescence:
Third-harmonic generation
imaging
Third-harmonic generation
imaging
Third-harmonic generation
imaging
THG microscopy setup
•
Optimal wavelength range 1200-1350 nm.
– UV generation at shorter λ
– Water absorption at longer λ
THG brain imaging
Depth scan through the prefrontal cortex of a mouse:
Image size 500 x 500 µm.
Scanned depth 360 µm.
THG brain imaging
Various brain structures can be imaged simultaneously:
White matter (axons):
Grey matter (neurons):
Blood vessels:
Label-free live brain imaging and targeted patching
with third-harmonic generation microscopy
Witte et al, PNAS 108, 15, 2011
Perspectives
• Brain tumors: essential to remove only malignant tissue,
develop THG for rapid non-invasive “optical biopsy”.
• Apply THG etc in neuromedical research: Image
neurodegeneration (Alzheimer) in-vivo (brain slices)
Fiberscope
Label-free cellular resolution
during tumor surgery
 Construct fiber-endoscope
-
Spatial temporal phase shaping at in coupling
Validate on mouse models, illumination dose
Combine with surgery
For sensitive applications: brain, nerves
2-photon fluorescence
group of Helmchen,
Optics Express 2008
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Our group has pioneered this technology and is currently
investigating its applications to the medical arena.