Sub‐cellular tumor identification and markerless differentiation in the rat brain in vivo by multiphoton microscopy

Objective/Background Aim of the current study was to localize and differentiate between tumor (glioma) and healthy tissue in rat brains on a cellular level. Near-infrared multiphoton microscopy takes advantage of the simultaneous absorption of two or more photons to analyze various materials such as cell and tissue components via the observation of endogenous fluorophores such as NAD(P)H, FAD, porphyrins, melanin, elastin, and collagen, with a very high resolution, without inducing the problems of photo-bleaching on out-of-focus areas. Methods In vitro and in vivo studies on healthy rat brains as well as C6 glioma cell line allografts have been performed. Near-infrared laser pulses (λ = 690–1060 nm, τ ∼140 fs) generated by an ultrafast Ti:Sapphire tunable laser system (Chameleon, Coherent GmbH, Santa Clara, CA) were coupled into a laser scanning microscope (LSM 510 META, Carl Zeiss, Germany) to observe high quality images. Results Several image acquisitions have been performed by varying the zoom scale of the multiphoton microscope, image acquisition time and the wavelength (765, 840 nm) to detect various tissue components. With a penetration depth of ∼200 µm in vitro and about 30–60 µm in vivo into the brain tissue it was possible to differentiate between tumor and healthy brain tissue even through thin layers of blood. Conclusion Near-infrared multiphoton microscopy allows the observation and possibly differentiation between tumor (glioma) and healthy tissue in rat brains on a cellular level. Our findings suggest that a further miniaturization of this technology might be very useful for scientific and clinical applications in neurosurgery. Lasers Surg. Med. 44: 719–725, 2012. © 2012 Wiley Periodicals, Inc.