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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 ik 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 Because At On Furthermore, The …or …opening Light …the …and …that the The the other to coupled heart diving two end collect thus of has hole the holes the of ofany two board the this afrom presence light way hosts ferrule-top central event hosts tip double-fiber can coming the to can acombined that an standard opposite be holes. be ofoptical used probe the from has used tip ferrule end to fiber isatomic for on shine acaused of 3mm atomic the equipped is the light machined force free the optical standard x that 3mm force sample… onto hanging microscopy with movement. fiber xmicroscope the in7mm a fiber the sharp sample… end, is form glass and … then tip.of used a miniaturized optical to detect analysis purposes. ferrule… anydiving of tinysurfaces. movement board. of the diving board… Our group has pioneered this technology and is currently investigating its applications to the medical arena.