Figure 7 - uploaded by Carsten M. Philipp
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Squamous cell carcinoma. (A) HE stain (magnifi cation, 40 × ; scan area, 1.5 mm × 3.0 mm), (B) overview as false-colour representation green-red, i.e., TPEF & SHG separated (digital resolution, 5 μ m × 5 μ m; scan area, 3.5 mm × 3.0 mm), (C) close-up as false-colour representation green-red, i.e., TPEF & SHG separated (digital resolution, 1 μ m × 1 μ m; scan area, 0.6 mm × 1.8 mm), and (D) overview as false-colour representation black-white, i.e., TPEF & SHG in sum (digital resolution, 5 μ m × 5 μ m; scan area, 3.5 mm × 3.0 mm).
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Non-invasive two-photon microscopy (TPM) provides a new technique which could become in the future a substitute for hematoxylin and eosin (HE) staining of tissue sections of the epidermis and upper and middle dermis as well. High-resolution imaging, in combination with false-colour representation, allows an accurate reproduction of standard microsc...
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... shown in Figure 7 A -D, histological key parameters of nodular BCC, such as cells of various shape, order and size, could clearly be identifi ed. In particular, the SHG signal marks pathologically dense and altered stroma which helps with the differentiation of tumour tissue from the adjacent normal skin as well as highlighting the tumour borders. ...
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Citations
... With a new 2PM system using a fiber guided objective, a vertical image exceeding 7 mm can be generated. Encouraged by the recent promising results of human skin ex vivo [14,15], first in vivo results are shown and the optimal measurement conditions, such as contact fluid and the influence of shaving and tape stripping of the skin, are evaluated in the presented study. ...
The optical biopsy could be a quick and painless support or alternative to a punch biopsy. In this letter the first in vivo vertical wide field two photon microscopy (2PM) images of healthy volunteers are shown. The 2PM images are fused images of two photon excited auto fluorescence (AF) and second harmonic generation (SHG) signals given as false-color images of 200 μm × 7 mm in size. By using these two nonlinear effects, the epidermis can be easily distinguished from the dermis at a glance. The auto fluorescence provides cellular resolution of the epidermal cells, and elastin fibers are partly visible in the dermis. Collagen, visible by SHG signal, is the dominant structure in the dermis. As contact agent water was evaluated to increase the AF signal, especially in the deeper layers of epidermis and dermis. For further improvement any terminal hairs should be removed by shaving and by taking tape strips of the first five layers of the stratum corneum. The first images illustrated that young skin compared to aged skin shows remarkably different dermal elastin and collagen signals in the dermis.
We experimentally investigate the two‐photon absorption cross sections and spectra of Eosin and Hematoxylin for applications in nonlinear microscopy. The experiments are carried out on pure samples of the two dyes in DI‐water solvent with different concentrations, in the typical range employed in standard staining procedures. Nonlinear fluorescence is excited by a line‐shaped beam emitted by a Ti:Sapphire mode‐locked laser in the wavelength range from 740 to 880 nm and is detected through a microscope setup. The two‐photon absorption spectral response is systematically analyzed and discussed. Finally, the staining is applied on biological tissue samples, which are imaged by two‐photon microscopy. Our results show that the employed dyes are fully suitable for applications in nonlinear imaging.
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In hellhäutigen Populationen aller Kontinente werden in ansteigender Zahl Aktinische Keratosen, auch solare Keratosen genannt, festgestellt. Diese Hautveränderungen, die einer In-situ-Karzinombildung entsprechen, finden sich vorwiegend in einer chronisch dem UV-Licht exponierten Haut. Woran Sie diese Hautveränderungen erkennen können, erfahren Sie hier.
“Photodynamic therapy” (PDT) is a term encompassing a collection of both curative and palliative modalities, traditionally described as treating precancerous lesions and superficial tumors using light. The list of medical fields in which PDT has managed to find a place as an accepted option for specific problems includes gastroenterology, dermatology, gynaecology, ophthalmology, and ENT. It is increasing importance underlined by a comparison with both traditional chemotherapy and radiotherapy, which can often significantly compromise patient’s health. The therapeutic use of these well-established therapies is accordingly limited by their toxicity. In contrast, PDT cannot only show a distinct degree of tumor specificity, but can be repeatedly applied without apparently damaging the health of the patient.
Skin is the largest organ of the human body. Usually, it is easy accessible for topical application of drugs and light dosimetry is easy due to the rather flat appearance in a first approximation. On the other hand a larger number of skin diseases cover a larger area or show multifocal appearance, which favors regional or large field therapies and impedes surgical interventions. As scar formation is another and today highly unwanted result of surgery, the widespread use of PDT in dermatology is a necessary consequence.
Two Photon Microscopy (2PM) generates microscopic images out of depth of biological samples. Up to now the method is restricted by narrow limitations of the field of view, the imaging depth and the orientation of the image field. A new approach overcomes these boundaries and delivers high resolution images revealing very specific information on clinically and biologically relevant tissue and cell structures. The status of the 2PM technology is critically reviewed and the options are discussed. The advantages of the new approach demonstrated by excellent tissue images.
Nonlinear optical microscopy (NLOM) relies on nonlinear light–matter interactions to provide images from larger depths within biological structures compared to conventional confocal fluorescence microscopy. These nonlinear light–matter interactions include multiphoton excitation fluorescence (MPEF), second-harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS). This review discusses the theories of and instrumentation for various NLOM techniques, with a particular focus on endogenous signals and exogenous probes. These signals and probes expand the breadth of information that optical imaging can provide. We also discuss the application of NLOM in biomedical research, including tissue engineering, drug delivery and clinical diagnostics. Current technological limitations are also discussed.
Most applications of multi-photon microscopy (MPM) in biology and medicine focus on high-resolution imaging of physiology, morphology and cell–cell interactions in intact tissues or live animals. In this paper, our aim is to assess whether MPM has the ability to provide comparable information for stained histology slides and to identify inflammatory fibroid polyp (IFP) tissue sections without H&E staining.
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In routine clinical work, classical histology is regarded as the gold standard for the discrimination of healthy and pathological tissue. However, the whole process can be tediously long and has some restraints. As a result, under most conditions, doctors have to wait for the histological results until decisions can be made. In this paper, a practical procedure of tissue preprocess, serial section, label-free imaging and image analysis is demonstrated, which costs less time and could give sufficiently good contrast to obtain information about cellular structure and organization of biological tissues.
