Jonathan A. Palero

In vivo nonlinear spectral imaging in mouse skin

We report on two-photon autofluorescence and second harmonic spectral imaging of live mouse tissues. The use of a high sensitivity detector and ultraviolet optics allowed us to record razor-sharp deep-tissue spectral images of weak autofluorescence and short-wavelength second harmonic generation by mouse skin. Real-color image representation combined with depth-resolved spectral analysis enabled us to identify tissue structures. The results show that linking nonlinear deep-tissue imaging microscopy with autofluorescence spectroscopy has the potential to provide important information for the diagnosis of skin tissues.

A simple scanless two-photon fluorescence microscope using selective plane illumination

We demonstrate a simple scanless two-photon (2p) excited fluorescence microscope based on selective plane illumination microscopy (SPIM). Optical sectioning capability is presented and depth-resolved imaging of cameleon protein in C. elegans pharyngeal muscle is implemented.

Short-wavelength two-photon excitation fluorescence microscopy of tryptophan with a photonic crystal fiber based light source

We report on a novel and simple light source for short-wavelength two-photon excitation fluorescence microscopy based on the visible nonsolitonic radiation from a photonic crystal fiber. We demonstrate tunability of the light source by varying the wavelength and intensity of the Ti:Sapphire excitation light source. The visible nonsolitonic radiation is used as an excitation light source for two-photon fluorescence microscopy of tryptophan powder.

Image formation by linear and nonlinear digital scanned light-sheet fluorescence microscopy with Gaussian and Bessel beam profiles

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivo Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy

Nonlinear spectral imaging microscopy (NSIM) allows simultaneous morphological and spectroscopic investigation of intercellular events within living animals. In this study we used NSIM for in vivo time-lapse in-depth spectral imaging and monitoring of protein-bound and free reduced nicotinamide adenine dinucleotide (NADH) in mouse keratinocytes following total acute ischemia for 3.3 h at ~3 min time intervals. The high spectral resolution of NSIM images allows discrimination between the two-photon excited fluorescence emission of protein-bound and free NAD(P)H by applying linear spectral unmixing to the spectral image data. Results reveal the difference in the dynamic response between protein-bound and free NAD(P)H to ischemia-induced hypoxia/anoxia. Our results demonstrate the capability of nonlinear spectral imaging microscopy in unraveling dynamic cellular metabolic events within living animals for long periods of time.

Fast nonlinear spectral microscopy of in vivo human skin

An optimized system for fast, high-resolution spectral imaging of in vivo human skin is developed and evaluated. The spectrograph is composed of a dispersive prism in combination with an electron multiplying CCD camera. Spectra of autofluorescence and second harmonic generation (SHG) are acquired at a rate of 8 kHz and spectral images within seconds. Image quality is significantly enhanced by the simultaneous recording of background spectra. In vivo spectral images of 224 × 224 pixels were acquired, background corrected and previewed in real RGB color in 6.5 seconds. A clear increase in melanin content in deeper epidermal layers in in vivo human skin was observed.

In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues.

We demonstrate the capability of nonlinear spectral imaging microscopy (NSIM) in investigating ultraviolet and visible light induced effects on albino Skh:HR-1 hairless mouse skin non-invasively.

Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope

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 approximately 100 microm.

Spectrally resolved multiphoton imaging of post-mortem biopsy and in-vivo mouse skin tissues

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.

Three-dimensional multiphoton autofluorescence spectral imaging of live tissues

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.