We investigated different kinds of human cutaneous ex-vivo skin samples by combined two photon intrinsic fluorescence
(TPE), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), spectral
lifetime imaging (SLIM), and multispectral two photon emission detection (MTPE). Morphological and spectroscopic
differences were found between healthy and pathological skin samples, including tumors. In particular, we examined
tissue samples from normal and pathological scar tissue (keloid), and skin tumors, including basal cell carcinoma (BCC)
and malignant melanoma (MM). By using combined TPE-SHG microscopy we investigated morphological features of
different skin regions, as BCC, tumor stroma, healthy dermis, fibroblastic proliferation, and keloids. A score, based on
the SHG to autofluorescence aging index of dermis (SAAID), was assigned to characterize each region. We found that
both BCC and surrounding dermis have a negative SAAID value, tumor stroma has a positive SAAID value, whereas
fibroblastic proliferation and keloids have a SAAID value close to the unit. Further comparative analysis of healthy skin
and neoplastic samples was performed using FLIM, SLIM, and MTPE. In particular, BCC showed a blue-shifted
fluorescence emission, a higher fluorescence response at 800 nm excitation wavelength and a slightly longer mean
fluorescence lifetime. MM showed an emission spectrum similar to the corresponding healthy skin emission spectrum,
and a mean fluorescence lifetime distribution shifted towards shorter values. Finally, the use of aminolevulinic acid as a
contrast agent has been demonstrated to increase the constrast in BCC border detection. The results obtained represent
further support for in-vivo non-invasive imaging of diseased skin.
We investigated human cutaneous basal cell carcinoma ex-vivo samples by combined time resolved two photon intrinsic
fluorescence and second harmonic generation microscopy. Morphological and spectroscopic differences were found
between malignant skin and corresponding healthy skin tissues. In comparison with normal healthy skin, cancer tissue
showed a different morphology and a mean fluorescence lifetime distribution slightly shifted towards higher values.
Topical application of delta-aminolevulinic acid to the lesion four hours before excision resulted in an enhancement of
the fluorescence signal arising from malignant tissue, due to the accumulation of protoporphyrines inside tumor cells.
Contrast enhancement was prevalent at tumor borders by both two photon fluorescence microscopy and fluorescence
lifetime imaging. Fluorescence-based images showed a good correlation with conventional histopathological analysis,
thereby supporting the diagnostic accuracy of this novel method. Combined morphological and lifetime analysis in the
study of ex-vivo skin samples discriminated benign from malignant tissues, thus offering a reliable, non-invasive tool for
the in-vivo analysis of inflammatory and neoplastic skin lesions.
We report the effects on two-photon excitation microscopy of applying
optical clearing agents to human skin tissue samples. We
demonstrate that the agents glycerol, propylene glycol and glucose in
aqueous solution are all effective in enhancing penetration depth (by
up to a factor of 2) and in increasing image contrast (by up to a factor
of 90 at 80 μm depth) in 150 μm thick sections. We analysed the
dynamics of the clearing process, by developing a simple theoretical
model based on the free diffusion of the agent into the tissue. In
experiments employing simultaneous two-photon excitation and
second harmonic generation microscopy similar contrast was
produced. A preliminary measurement of the clearing effect on a bulk
skin sample is also presented. All three agents are potentially biocompatible
and effective in reducing scattering; hence, in improving
light penetration depth and image contrast. As such, they could be
suitable for in vivo application in two-photon microscopy, as well as in
other techniques performing optical biopsy of human skin tissue.
We used combined simultaneous two-photon excitation fluorescence microscopy (TPE) and second harmonic generation microscopy (SHG) on human skin tissue slices. We studied the effect caused by topical application of optical clearing agents (OCAs). We demonstrated that hyperosmotic agents as glycerol, propylene glycol and glucose in aqueous solution, are all effective in improving excitation light penetration depth and in enhancing image contrast. The effect caused on acquired images by sample immersion in OCAs or in their aqueous dilution, was studied. We observed a similar clearing effect with TPE and SHG acquisitions, with different effectiveness and rising time for each agent. The TPE acquired data are in good agreement with a simple diffusion model developed. From the SHG acquisition some different behaviour was observed. All three agents are potentially bio-compatible and effective in reducing scattering, improving light penetration depth and image contrast. Use of OCA can be suitable for in vivo application in two-photon microscopy, as well as in other techniques performing optical biopsy of human skin tissue.
We used two-photon microscopy towards the imaging of cutaneous basal cell carcinoma (BCC). Our aim was to evaluate the morphology of BCC using two-photon fluorescence excitation and to establish a correlation with histopathology. We built a custom two-photon microscope and we measured the system capabilities. The system allowed to perform a preliminary measurement on a fresh human skin tissue sample. A human skin tissue sample of BCC excised during dermatological surgery procedures were used. The clinical diagnosis of BCC was confirmed by subsequent histopathological examination. The sample was imaged using endogenous tissue fluorescence within 2-3 hours from the excision with a two photon laser scanning fluorescence microscope. The acquired images allowed an obvious discrimination of the neoplastic areas toward normal tissue, based on morphological differences and aberrations of the intensity of the fluorescence signal. Our results showed that BCC tissue has a more homogeneous structure in comparison to normal tissue as well as a higher fluorescent response. The images obtained by two photon microscopy were further compared to the images acquired by an optical microscope after the conventional histopathological examination on one part of the respective sample. Our suggested method may represent a new diagnostic tool that improves the diagnostic accuracy of clinical examination alone, enabling the accurate discrimination of basal cell carcinoma from normal tissue.
Dynamic scattering of coherent light by moving particles causes a Doppler shift of the original frequency, depending on the velocity and the scattering angles. This phenomenon was used for the detection of abnormal spontaneous muscle activity caused by the denervation of muscles. Transmission measurements of low frequency modulated laser irradiation have been made in 110 denervated first dorsal interosseous who had previously been imposed to electromyography which detected abnormal activity. Measurements have also been made in 173 normal muscles. The laser used was a diode laser emitting at 830nm and a pulse generator modulated the laser radiation to a
low repetition frequency of 84Hz. While passing through the denervated muscles, the incoming laser beam gets a frequency shift due to the contraction of the denervated muscle fibers and mixes with the ballistic part of the beam. To analyze the inherent information the outcoming laser light was transformed into electric current by a photodiode and the signal after being selected by an A/D card was submitted to the Fast Fourrier transformation. The findings of our
suggested method have been compared to those of the normal muscles as well as to the electromyographic findings of each denervated muscle.