1. Biophotonics and medical imaging
Prof. Johannes F. de Boer
Prof. Marloes Groot, Prof. Ruud Verdaasdonk,
Dr. Davide Iannuzzi, Dr. Freek Ariese
SD-OCT and OFDI at 850 and 1050 nm for retinal imaging in glaucoma and AMD

2. 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
Wilhelm C. Roentgen, Born on March 27, 1845 in Lennep. Died on February 10, In 1895, Roentgen discovered X-rays.
These techniques are a mainstay of medical imaging

3. UHR-SD-OCT Fovea 6 x 6 mm 6 x 1.1 mm 1 x 0.5 mm
B. Cense et al. Opt. Express 12, (2004)

4. Endoscope with Micromotor
1.65 mm diameter, 6000 rpm
optical fiber
GRIN
motor
1.65 mm
OCT
dichroic
mirrors
fiber port

5. Spinning catheter 3000rpm
52 images/sec

6. 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

7. Imaging mouse heart vasculature
Color codes for depth
Scan free depth resolved fluorescence imaging
As a last example I would like to show you…
Microvasculature in a mouse heart
Here filled with fluorescent beads and imaged fresh
500 x 500 x 60 µm 15 75
Depth in tissue (µm)

8. AIM: To image life cells, label-free, with cellular resolution in deep-tissue

9. Confocal and non-linear microscopy

10. 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.

11. 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 3D-imaging!

12. Two-photon fluorescence microscopy
Interneurons containing Green Fluorescent Protein.
2-photon excitation at nm.
Requires a dye or other fluorescent probe.

13. Third-harmonic generation imaging

14. THG microscopy on brain tissue
THG microscopy on mouse brain tissue.
Neurons are clearly visible as dark shadows.

15. Third-harmonic generation
where
Isotropic medium, tight focusing:
THG generated before
and after the focus cancel
due to Gouy phase (if Δk≥0).
 No THG
refractive index nω or the nonlinear susceptibility (3
Discontinuity (in nω or (3) ):
Asymmetry in phase
before and after focus, no
THG cancellation.
 THG signal!

16. 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:
refractive index nω or the nonlinear susceptibility (3

17. Third-harmonic generation imaging

18. Third-harmonic generation imaging

19. Third-harmonic generation imaging
Scatter length shorter than abs length

20. THG microscopy setup Optimal wavelength range 1200-1350 nm.
UV generation at shorter λ
Water absorption at longer λ

21. THG brain imaging Depth scan through the prefrontal cortex of a mouse:
Image size 500 x 500 µm.
Scanned depth 360 µm.

22. THG brain imaging
Various brain structures can be imaged simultaneously:
Grey matter (neurons):
White matter (axons):
Blood vessels:
Label-free live brain imaging and targeted patching
with third-harmonic generation microscopy
Witte et al, PNAS 108, 15, 2011

23. 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)

24. Label-free cellular resolution during tumor surgery
Fiberscope
2-photon fluorescence
group of Helmchen,
Optics Express 2008
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

25. …the diving board can be used for atomic force microscope purposes.
…and thus any event that has caused that movement.
Furthermore, the tip can be used to shine light onto the sample…
…or to collect light coming from the sample…
…opening the way to combined atomic force microscopy and optical analysis of surfaces.
At the heart of a ferrule-top probe is a 3mm x 3mm x 7mm glass ferrule…
Because of the presence of the tip on the free hanging end, …
…that has two central holes.
On the two holes hosts an optical fiber equipped with a sharp tip.
The other hole hosts a standard optical fiber
Light coupled from the opposite end of the standard fiber is then used to detect any tiny movement of the diving board…
The end of this double-fiber ferrule is machined in the form of a miniaturized diving board.
Our group has pioneered this technology and is currently investigating its applications to the medical arena.