Prof. Johannes F. de Boer

Prof. Marloes Groot, Prof. Ruud Verdaasdonk,

Dr. Davide Iannuzzi, Dr. Freek Ariese

Biophotonics and medical imaging

The impact of physics on imaging in healthcare

X-Ray, CT, MRI, PET, Ultrasound

Anna Berthe Röntgen: Hand mit RingenWilhelm Röntgen's first "medical" x-ray,

of his wife's hand, taken on 22 December 1895

These techniques are a mainstay of medical imaging

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, 2435-2447 (2004)

Endoscope with Micromotor

1.65 mm diameter, 6000 rpm

dichroic mirrors

GRIN motorOCT

fiber port

1.65 mm

optical fiber

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

500 x 500 x 60 µm

15 75Depth in tissue (µm)

Color codes for depth

Scan free depth resolved fluorescence imaging

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 3D-imaging!

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

THG generated before and after the focus cancel due to Gouy phase (if

Δk≥0).

No THG

Isotropic medium, tight focusing:

Asymmetry in phasebefore and after focus, no THG cancellation.

Discontinuity (in nω or (3) ):

THG signal!

€

I3ω =3ω

2nω c

⎛

⎝ ⎜

⎞

⎠ ⎟

2

χ (3) Iω3 e iΔk z

(1+ 2 i z /b)2z1

z2∫ dz

2

€

Δ k = k3ω − 3kω( )where

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:

Grey matter (neurons): White matter (axons): Blood vessels:

Label-free live brain imaging and targeted patchingwith third-harmonic generation microscopyWitte et al, PNAS 108, 15, 2011

• 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)

Perspectives

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 fluorescencegroup of Helmchen,Optics Express 2008

At the heart of a ferrule-top probe is a 3mm x 3mm x 7mm glass

ferrule…

…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 fiberThe end of this double-fiber

ferrule is machined in the form of a miniaturized diving board.

Light coupled from the opposite end of the standard fiber is then

used to detect any tiny movement of the diving board…

Because of the presence of the tip on the free hanging end, …

…and thus any event that has caused that movement.

…the diving board can be used for atomic force microscope

purposes.

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.

Our group has pioneered this technology and is currently investigating its applications to the medical arena.