Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clin. cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochem. properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radiofrequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is sepd. in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calc. in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.

Endoscopic tissue fluorescence life-time imaging by frequency domain light-induced fluorescence

André Studzinski, Hubert van den Bergh, Georges Wagnières

An instrumentation is being developed to draw a fluorescence life-time map of tissue endoscopically. This fluorescence life-time of an endogenous or exogenous fluorochrome gives information about the physico-chemical environment which is thought to vary between normal and diseased tissue. The excitation light from a cw laser is modulated in amplitude at high frequencies by an electro-optic modulator and delivered to the endoscopic site through an optical fiber. The image of the tissue is spectrally split in two parts, the one being the backscattered excitation light, the other the fluorescence of the fluorochromes. Each image is focused on the photocathode of an image intensifier whose gain is modulated at the same frequency. By acquiring frames at different phases between the excitation and the emission, it is possible to calculate pixel by pixel the apparent fluorescence life-time of the corresponding tissue region.

1995

Endoscopic frequency-domain fluorescence lifetime imaging for clinical cancer photodetection: apparatus design

André Studzinski, Hubert van den Bergh, Georges Wagnières

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the image contrast is derived from the fluorescence lifetime of the fluorochrome at each point in a 2D image. Lifetime images are created from a series of images obtained from two gain-modulated image intensifiers. One of them (II-1) detects the light-induced tissue fluorescence, whereas the other (II-2) detects the backscattered fluorescence excitation light. This light is modulated at the same frequency as the detectors, resulting in homodyne phase-sensitive image. These stationary phase-sensitive images are collected using two CCD cameras, digitized and manipulated with a mathematical operator in real time. A series of such images, obtained with both image intensifiers at various phase shifts between their gain modulation and the modulation of the excitation light, is used to determine phase angle and/or the modulation of the fluorescence emission at each pixel. The reference values of these phase angles and modulations are obtained with II-2, whereas II-1 enables the measurement of the phase and modulation of the fluorescence. Phase and modulation are related to the fluorescence lifetime of the fluorochrome. An advantage of the experimental method proposed here is that pixel-to-pixel scanning is not required to obtain the fluorescence lifetime image, as the information from all pixels is obtained at the same time.

1995

Time-resolved spectrofluorometer for clinical tissue characterization during endoscopy

Hubert van den Bergh, Georges Wagnières

Time-resolved fluorescence spectroscopy has the potential to provide more information for the detection of early cancer than continuous wave spectroscopy. A new optical fiber-based spectrofluorometer for time-resolved fluorescence spectroscopy of biol. tissue during clin. endoscopy is presented. The app. is based on a nitrogen laser pumping a dye laser as excitation source and a streak camera coupled with a spectrograph as time-resolved spectrometer. The excitation and fluorescence light is carried by an optical fiber to the tissue under investigation and back to the detector, resp. This optical fiber can be inserted into the biopsy channel of a conventional endoscope. Hence, the app. can be used to perform in situ tissue characterization during endoscopy. The instrument enables the measurement of the decays of entire fluorescence spectra within 15 s with a dynamic range of the spectro-temporal images of up to three orders of magnitude. Luminescence lifetimes from the sub ns up to the ms range can be measured. Spectral and temporal resoln., sensitivity, and dynamic range of the instrumentation were detd. The accuracy of the app. was checked by the measurement of the fluorescence lifetimes of various fluorophores with known lifetimes. For the first time, two-dimensional time-resolved spectra with sub-ns temporal resoln. of tissue fluorescence of the human bladder, the bronchi, and the esophagus taken during endoscopy are presented as a demonstration of performance of the instrumentation. The excitation wavelengths were 337 nm in the case of the bladder and the esophagus and 480 nm in the case of the bronchi. Lifetime contrasts between normal and neoplastic tissue were found in all three organs. The spectral anal. of the fluorescence decays showed that the fluorescence between 370 and 490 nm, excited at 337 nm, consisted in several overlapping spectra. In the case of the esophagus, the contrast between normal and tumoral tissue was inverse in two different spectral bands proving the importance of the choice of the appropriate spectral range for time-resolved autofluorescence measurements for an optimal contrast. The in vivo fluorescence decay of the photosensitizers 5-aminolevulinic acid hexylester hydrochloride-induced protoporphyrin IX was measured in the human bladder and found to be mono-exponential with a lifetime of 15.9 (+-1.2) ns. An in vivo fluorescence lifetime of 8.5 (+-0.8) ns was found in the case of the photosensitizer 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in the esophagus.

American Institute of Physics1999

Time-resolved spectrofluorometer for clinical tissue characterization during endoscopy

Hubert van den Bergh, Georges Wagnières

American Institute of Physics1999