Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and noncentrosymmetric-structured proteins is rapidly gaining interest in biomedical applications. The advantage of this technique includes high imaging penetration depth and minimal phototoxic effects on tissues. Because fluorescent dyes are not used, discrimination between different components within the tissue is challenging. We have developed a nonlinear spectral imaging microscope based on a home-built multiphoton microscope, a prism spectrograph, and a high-sensitivity CCD camera for detection. The sensitivity of the microscope was optimized for autofluorescence and second harmonic imaging over a broad wavelength range. Importantly, the spectrograph lacks an entrance aperture; this improves the detection efficiency at deeper lying layers in the specimen. Application to the imaging of ex vivo and in vivo mouse skin tissues showed clear differences in spectral emission between skin tissue layers as well as biochemically different tissue components. Acceptable spectral images could be recorded up to an imaging depth of ~100 µm.
The deep-tissue penetration and submicron spatial resolution of multi-photon microscopy and the high-detection
efficiency and nanometer spectral resolution capability of a spectrograph were combined to study the intrinsic emission
of mouse skin post mortem biopsy and section, and in vivo tissue samples. The different layers of skin could be clearly
distinguished based on both their spectral signature and morphology. Auto fluorescence could be detected from both
cellular and extra cellular structures. In addition SHG from collagen and a narrowband spectral emission band related to
collagen were observed. Visualization of the spectral images in RGB color allowed us to identify tissue structures such
as epidermal cells, lipid-rich keratinocytes and intercellular structures, hair follicles, collagen, elastin, and dermal
fibroblasts. The results also showed morphological and spectral differences between the mouse skin post mortem biopsy
and in vivo samples which explained by biochemical differences, specifically of NAD(P)H. Overall, spectral imaging
provided a wealth of information not easily obtainable with present conventional multi-photon imaging methods.
We combined a homebuilt multiphoton microscope and a prism-CCD based spectrograph to develop a spectral imaging system capable of imaging deep into live tissues. The spectral images originate from the two-photon autofluorescence of the tissue and second harmonic signal from the collagen fibers. A highly penetrating near-infrared light is used to excite the endogenous fluorophores via multiphoton excitation enabling us to produce high quality images deep into the tissue. We were able to produce 100-channel (330 nm to 600 nm) autofluorescence spectral images of live skin tissues in less than 2 minutes for each xy-section. The spectral images rendered in RGB (real) colors showed green hair shafts, blue cells, and purple collagen. Analysis on the optical signal degradation with increasing depth of the collagen second-harmonic signal showed 1) exponential decay behavior of the intensity and 2) linear broadening of the spectrum. This spectral imaging system is a promising tool for both in biological applications and biomedical applications such as optical biopsy.
Two-photon microscopy revolutionized deep and live tissue imaging. It uses near-infrared femtosecond-pulsed laser sources, usually Ti:Sa lasers, to excite fluorescence. Several endogenous and synthetic fluorophores are, however, excited with wavelengths shorter than 360 nm, including NADH, tryptophan and the ratiometric Ca<sup>2+</sup> indicators. Efficient two-photon excitation imaging of these endogenous fluorophores is difficult at present due to the lack of suitable laser sources. To address these concerns, we investigated the use of photonic crystal fibers as a laser source for visible wavelength two-photon microscopy. The high nonlinearity of the photonic crystal fibers leads to supercontinuum generation that can span the visible to the near-infrared spectral regions. We investigated the spectral and temporal properties of photonic crystal fibers excited by a near-infrared femtosecond Ti:Sa laser. Our results show that the fiber emission can be tuned by variation of laser excitation wavelength and laser intensity. Our autocorrelation measurements show that the pulse duration of the PCF nonsolitonic radiation is in the order of a few picoseconds. We also demonstrate the application of the photonic crystal fiber output to two-photon microscopy of tryptophan.
Interest in the development of optical technologies that have the capability of performing in situ tissue diagnosis without the need for surgical biopsy and processing has been growing. In general, optical diagnostic techniques can be classified into two categories: (1) spectroscopic diagnostics and (2) optical imaging. Spectroscopic diagnostic techniques are used to obtain an entire spectrum of a single tissue site (point-measurement method). On the other hand, optical imaging methods are aimed at recording a two- or three-dimensional image of a sample region. A third category, which combines the two modalities, is currently in an early development phase. This category, referred to as spectral imaging, has been applied to cytomics, fluorescence resonance energy transfer (FRET) analysis, histology, fluorescence microscopy and autofluorescence microscopy. In this study, we combined a multi-photon microscope with a sensitive prism-based spectrograph and employed it for intrinsic emission spectral imaging microscopy of in vivo mouse skin tissues. We show results on: (1) spectral image RGB real-color visualization; (2) tissue layer discrimination using spectral signatures; (3) depth-resolved skin tissue spectral imaging; and (4) tissue component determination by spectral (linear) unmixing.
The last two decades saw the emergence of spectroscopy and microscopic imaging as techniques for tissue diagnostics. The biochemical state of the tissue is revealed by spectroscopy, while the morphological information is visualized by microscopic imaging. Little research has been carried out to diagnose tissues based on the combination of spectroscopy and microscopic imaging. Here, we report on tissue spectroscopy and microscopic imaging employing two-photon excitation of tissue autofluorescence and second harmonic generation. We designed and constructed a prism-based spectral imaging system coupled to a two-photon microscope. Full emission spectra with a 1-7 nm spectral resolution covering 330nm to 600nm can be recorded at a maximum rate of 500 spectra per second equivalent to about 0.5 frames/min (224x224 pixels). We present results on spectral imaging of human skin sections and in-depth imaging of pig skin tissue. Different skin layers show clear differences in their intrinsic emission spectral signature that can be used for diagnosis.
We combined a non-linear microscope with a sensitive prism-based spectrograph and employed it for the imaging of the auto fluorescence of skin tissues. The system has a sub-micron spatial resolution and a spectral resolution of better than 5 nm. The spectral images contain signals arising from two-photon excited fluorescence (TPEF) of endogenous fluorophores in the skin and from second harmonic generation (SHG) produced by the collagen fibers, which have non-centrosymmetric structure. Non-linear microscopy has the potential to image deep into optically thick specimens because it uses near-infrared (NIR) laser excitation. In addition, the phototoxicity of the technique is comparatively low. Here, the technique is used for the spectral imaging of unstained skin tissue sections. We were able to image weak cellular autofluorescence as well as strong collagen SHG. The images were analyzed by spectral unmixing and the results exhibit a clear spectral signature for the different skin layers.