Dual excitation spectral autofluorescence lifetime and reflectance imaging for fast macroscopic characterization of tissues

Abstract

Advancements in optical imaging techniques have revolutionized the field of biomedical research, allowing for the comprehensive characterization of tissues and their underlying biological processes. Yet, there is still a lack of tools to provide quantitative and objective characterization of tissues that can aid clinical assessment in vivo to enhance diagnostic and therapeutic interventions. Here, we present a clinically viable fiber-based imaging system combining time-resolved spectrofluorimetry and reflectance spectroscopy to achieve fast multiparametric macroscopic characterization of tissues. An essential feature of the setup is its ability to perform dual wavelength excitation in combination with recording time-resolved fluorescence data in several spectral intervals. Initial validation of this bimodal system was carried out in freshly resected human colorectal cancer specimens, where we demonstrated the ability of the system to differentiate normal from malignant tissues based on their autofluorescence and reflectance properties. To further highlight the complementarity of autofluorescence and reflectance measurements and demonstrate viability in a clinically relevant scenario, we also collected in vivo data from the skin of a volunteer. Altogether, integration of these modalities in a single platform can offer multidimensional characterization of tissues, thus facilitating a deeper understanding of biological processes and potentially advancing diagnostic and therapeutic approaches in various medical applications.

Fig. 1.

(A) Schematic representation of the optical and electronic layouts of the instrument. Electronic trigger signals used for synchronization of all modules are represented by dashed lines. DM425, DM495, and DM593 indicate dichroic mirrors and corresponding cutoff wavelengths. F1-F3: band-pass filters (see Table 1 for spectral bandwidth). D1-D3: hybrid detectors. (B) Timing diagram of the real-time acquisition. Cameras, LEDs, and spectrometer are triggered simultaneously at 50 Hz. Lasers are multiplexed at 50 Hz for sequential excitation at different wavelengths. TCSPC measurements are carried out at 50 Hz (25 Hz for each excitation wavelength) when the shutter is open. (C) Representative autofluorescence intensity decays and absorbance spectrum obtained in a single measurement with our instrument, from in vivo human skin tissue. Integration times were 15 ms for autofluorescence measurements at each excitation wavelength and 3 ms for reflectance measurements.

Fig. 2.

Workflow for real-time determination of the location of optical measurements. (A) Set of two consecutive frames, one captured with 445 nm excitation (visible), and another captured with 375 nm excitation (not visible). (B) Blue and green channels of each frame, where the excitation spot created by the 445 nm light is well visible. (C) Subtraction between the blue and green channels reduces the number of artefacts caused by specular reflections of the white light onto the tissue. (D) The excitation spot is segmented by subtraction of the two processed frames along followed by intensity thresholding. Centroid detection of the resulting binary mask permits determination of the center of the spot. (E) A circular region with 10 pixels of diameter defines the location of a single measurement.

Fig. 3.

Ex vivo autofluorescence lifetime and reflectance measurement of rectal cancer. (A) White light image of surgical specimen. Area limited by dashed line corresponds to analyzed region. (B) Magnified white light image of the specimen, delineating ulcerating lesions (white and red dashed lines). Black line indicates measurement boundaries. Arrows indicate areas of interest: tumor (red), normal (green), perilesional (orange), and necrosis (cyan). (C1-C5) Autofluorescence lifetime and (D1-D5) normalized autofluorescence intensity maps for each detection channel. (E) Normalized optical redox ratio. (F) Normalized absorbance spectra at locations of interest, as indicated in panel B. (G) Absorbance map at 630 nm normalized to the absorbance at 540 nm. (H-J) Integrated absorbance over three spectral ranges of interest. Scale bar = 10 mm.

Fig. 4.

Ex vivo autofluorescence lifetime and reflectance measurement of a sigmoid colon adenocarcinoma. Endogenous contrast between normal tissue and tumor is more evident in channels 4 and 5 (excitation at 445 nm) compared to the detection channels at 375 nm excitation. (A) White light image of the specimen. (B-F) Autofluorescence lifetime maps in detection channels 1-5, respectively. (G) Optical redox ratio map. (H) Average autofluorescence lifetimes and (I) absorbance spectra measured from normal and tumor regions, as delineated in panel A [ROI_normal = 484 (22 × 22) pixels, ROI_tumor = 324 (18 × 18) pixels]. In panel I, solid lines represent the average spectrum and dotted lines the corresponding standard deviation.

Fig. 5.

In vivo measurement of human skin tissue showing (A-E) autofluorescence lifetime and (F-J) absorbance maps for different spectral ranges. Panel F also shows normalized absorbance curves of skin in regions indicated by the pink and yellow arrows, the latter pointing to the location of the blood vessel underneath the epidermis. Panels (K-O) show absorbance profiles for each reflectance map (F-J, respectively) along the dashed line depicted in panel J. The autofluorescence lifetime maps show relatively homogenous signatures throughout the measured region. Blood vessels underneath the epidermis (indicated by arrows in panels J and O) are clearly visible in the orange and red absorbance maps (panels I and J, respectively). Scale bars = 10 mm.