Significance: Sentinel lymph node (SLN) biopsy is an important method for metastasis staging in, e.g., patients with malignant melanoma. Tools enabling prompt histopathological analysis are expected to facilitate diagnostics; optical technologies are explored for this purpose.
Aim: The objective of this exploratory study was to investigate the potential of adopting multiphoton laser scanning microscopy (MPM) together with fluorescence lifetime analysis (FLIM) for the examination of lymph node (LN) tissue ex vivo.
Approach: Five LN tissue samples (three metastasis positive and two negative) were acquired from a biobank comprising tissues from melanoma patients. Tissues were deparaffinized and subjected to MPM-FLIM using an experimental MPM set-up equipped with a time correlated single photon counting module enabling FLIM.
Results: The data confirm that morphological features similar to conventional histology were observed. In addition, FLIM analysis revealed elevated morphological contrast, particularly for discriminating between metastatic cells, lymphocytes, and erythrocytes.
Conclusions: Taken together, the results from this investigation show promise for adopting MPM-FLIM in the context of SLN diagnostics and encourage further translational studies on fresh tissue samples.
KEYWORDS: Optical simulations, Beam shaping, Skin, Multiphoton microscopy, Point spread functions, Light scattering, Luminescence, Microscopes, Signal attenuation, Monte Carlo methods
Multiphoton fluorescence microscopy (MPM) is a method for high resolution, non-invasive investigations of biological tissue. The aim of introducing an annular shaped laser beam is to reduce the ouf-of-focus generated background signal improving imaging of light scattering tissue such as human skin. Simulations show that 50% of the beam radius can be blocked, while preserving the shape of the point spread function. Initial experiments performed on a phantom consisting of fluorescein and fluorescent beads embedded in agar by using a custom built MPM-set up show that by introducing a simple beam blocker to create an annular beam, the background signal is reduced with approximately 5%. Future work will include optimizing the set up, and creating phantoms with more light scattering properties.
This article [J. Biomed. Opt.. 18, , 061223 (2013)] was originally published online on 7 January 2013 with an error in the caption for Fig 1. The corrected caption appears below.
A specimen of a melanoma in situ with the SIAscopically indicated areas of increased dermal melanin content outlined with green and violet dots/lines. The planes of sectioning are indicated with black lines (*). The parallel horizontal reference lines are inked with red and green and the oblique reference line with blue color. Note the small proportions of the lesion with SIAscopic signal displaying dermal melanin.
Spectrophotometric intracutaneous analysis (SIAscopy) is an imaging technique developed for diagnostics of pigmented skin lesions. By image analysis, the displayed images indicate the potential distribution and position of melanin, blood, and collagen within the lesion. A topographic comparison was performed between SIAscopic findings and histopathology. In total, 60 patients with suspicious pigmented skin lesions were included. The lesions were SIAscopically imaged and documented before excision and histopathological preparation. Topographical comparisons between SIAscopy findings and histopathology were made. A sensitivity and specificity of 24% and 84%, respectively, were obtained for invasive melanomas. The positive and negative predicted values were 58% and 54%, respectively. The features indicating dermal melanin, blood displacement and collagen holes did only show “no” to “slight” agreement with histopathology, i.e., κ≤0.21 . It was concluded that (i) SIAscopy-based diagnosis has low diagnostic accuracy for melanoma, (ii) single SIAscopic features do not provide reliable diagnostic information relating to the lesions internal structure on histopathology examination and (iii) SIAscopy cannot be used as a guide for localizing the maximum tumor thickness when performing the histopathological examination. The importance of validating new optical tools for tumor diagnostics with histopathological findings was demonstrated.
Photodynamic therapy (PDT) using topical application of aminolevulinic acid (ALA) and methylaminolevulinate (MAL)
has become a popular therapeutic method for the treatment of non-melanoma skin cancers such as basal cell carcinomas
(BCCs); however, the treatment response varies. An important question is if BCCs which respond poorly to PDT lack
accumulation of protoporhyrin IX (PpIX) after ALA/MAL application. In connection to PDT, fluorescence diagnostics
(FD) can be performed to detect PpIX within human skin. We investigated fluorescence images from 22 patients with 35
BCCs. They were evaluated with respect to the fluorescence contrast based on image analysis, which was considered to
be a tool to non-invasively measure the PpIX-concentration. As expected the fluorescence contrast between tumor and
normal skin was elevated after MAL-application; although no correlation between low fluorescence contrast and lack of
treatment response could be observed. In a former study, we have also investigated the transdermal penetration of ALA
and MAL in 27 BCCs in vivo using a microdialysis technique. In 15 of 16 BCCs in which the microdialysis catheter was
located superficially (i.e. at a depth of less than 1 mm), therapeutic drug concentrations were detected;.however, in the
11 lesions with a deeper catheter location (below 1 mm) drug concentrations above the detection limit of the system were
only obtained in 6 lesions (p=0.026). No difference between the transdermal penetration of MAL and ALA could be
seen. Conclusions: Lack of PpIX fluorescence cannot entirely explain why some BCCs don't respond to PDT, but
inadecuate concentrations within the full thickness of the tumor may play a role as microdialysis has shown.
PDT is an effective method when treating multiple actinic keratoses (field cancerization). The major side effect is pain.
Our objectives were to investigate the pain-relieving effect of transcutaneous electrical nerve stimulation (TENS) and
peripheral nerve blocks during PDT of field cancerization (FC) of the face and scalp. Patients with field cancerization
were included in three studies. In the first study, we examined TENS with an application site on the adjacent dermatome
from the PDT area in order to allow the use of water spray during PDT for FC of the scalp and face. In the second study,
patients with FC in the facial area received unilateral supraorbital, infraorbital and/or mental nerve blocks. The non-anaesthetised
side of the treatment area served as control. In the third study, with similar methodology as in the second
study, occipital and supraorbital nerve blocks were combined for FC of the forehead and scalp. The results of the studies
strongly support the use of nerve blocks as pain relief during PDT. The use of TENS provided a limited pain reduction,
but TENS might be an alternative if the patient disapproves of the use of nerve blocks or is afraid of injections.
Multiphoton microscopy is an interesting optical technique, which allows for non-invasive imaging of
highly light scattering media such as human skin. Recent reports have showed the potential of
applying this technique for 3D visualisation of cell structures of biological tissue without previous
sectioning of the tissue samples. In this study, we have applied two-photon microscopy on excised
lesions of human non-melanoma skin cancer ex vivo in order to find diagnostic criteria using this
technique. The skin samples have been investigated by a multiphoton microscopy system based on a
fs-pulsed Ti:sapphire laser connected to a confocal microscope. The autofluorescence of the skin was
detected using excitation at 780 nm. The cell nuclei distribution turned out to be one important
parameter, which can be used for discriminating between tumour and normal tissue. We are now
developing a technique for automatic detection and characterisation of tissue, based on an image
analysis algorithm. The detection of cell nuclei has been found crucial for this purpose. The goal is to
develop a fast characterisation algorithm that can be used on line in connection to in vivo
investigations. This would allow for a true non-invasive biopsy technique in the future.
Photodynamic therapy (PDT) is an attractive alternative treatment for patients with acne because of its efficiency and few side effects. Propionibacterium acnes (P.acnes) are bacteria present in the skin, which produce endogenous porphyrins that act as photosensitisers. In addition, application of aminolaevulinic acid or its methyl ester (mALA) results in increased accumulation of porphyrins in the pilosebaceous units. This makes it possible to treat acne with PDT. This initial study investigates the possibility of fluorescence imaging as assessment tool in adjunct to PDT of patients with acne. Twenty-four patients with acne on the cheeks have been treated with PDT with and without mALA. Fluorescence images have been obtained before and after treatment. The clinical acne score was assessed as base line before PDT, and at every follow up visit. Additionally the amount of P.acnes was determined. The clinical evaluation showed a general improvement of acne, even though no difference between treatment with and without mALA was observed. By performing texture analysis and multivariate data analsysis on the fluorescence images, the extracted texture features were found to correlate with the corresponding clinical assessment (67%) and amount of P.acnes (72%). The analysis showed that features describing the highly fluorescent pores could be related to the clinical assessment. This result suggests that fluorescence imaging can be used as an objective assessment of acne, but further improvement of the technique is possible, for example by including colour images.
Fluorescence imaging has been shown to be a potential complement to visual inspection for demarcation of basal cell carcinoma (BCC), which is the most common type of skin cancer. Earlier studies have shown promising results when combining autofluorescence with protoporphyrin IX (Pp IX) fluorescence, induced by application of -5-aminolaevulinic acid (ALA). In this work, we have tried to further improve the ability of this technique to discriminate between areas of tumor and normal skin by implementing texture analysis and Fisher linear discrimination (FLD) on bispectral fluorescence data of BCCs located on the face. Classification maps of the lesions have been obtained from histopathologic mapping of the excised tumors. The contrast feature obtained from co-occurrence matrices was found to provide useful information, particularly for the ALA-induced Pp IX fluorescence data. Moreover, the neighborhood average features of both autofluorescence and Pp IX fluorescence were preferentially included in the analysis. The algorithm was trained by using a training set of images with good agreement with histopathology, which improved the discriminability of the validation set. In addition, cross validation of the training set showed good discriminability. Our results imply that FLD and texture analysis are preferential for correlation between bispectral fluorescence images and the histopathologic extension of the tumors.
The demand for fast and effective tools for diagnosis of skin
cancer is increasing due to the escalating incidence of skin
cancer in recent years. Fluorescence imaging has gained rising
interest for detection and delineation of basal cell carcinoma
(BCC), which is the most common type of skin cancer. By applying
delta-5-aminolaevulinic acid (ALA) protoporphyrin IX (Pp IX) is
accumulated in the tumour; hence, the Pp IX can be used as a
fluorescent marker for tumours. More information may be obtained
by combining the ALA induced fluorescence with fluorescence
without externally applied fluorophore, so called
autofluorescence. In this work we present a method for demarcation
of BCC, using a non-expensive multispectral imaging set up
assisted by image warping for image alignment. To calibrate the
method, histopathological mapping has been carried out by excising
the tumours with Mohs micrographic surgery. The Z-images,
combining information of autofluorescence and ALA induced
fluorescence, showed good agreement with the histopathological
mapping of BCCs located on the face in 5 out of 12 patients and
partial agreement in 7 patients. Since the face is an area where
demarcation of tumours usually is considered difficult, this
result shows a potential for the method as a pre-operative guiding
tool for this type of skin lesions.
Fluorescence imaging has shown a potential for demarcation of basal cell carcinoma (BCC), which is the most common type of skin cancer. The technique is based on imaging the fluorescence from protoporphyrin IX (Pp IX), after application of α-5-aminolevulinic acid (ALA). One limitation with the technique is that it is sensitive for undesired local intensity variations. But it has been shown that by combining autofluorescence, i.e. fluorescence without any externally applied photosensitiser, and the Pp IX fluorescence, higher contrast between tumour and normal skin can be obtained. This has earlier been reported using a laser-based technique allowing for simultaneous recording of autofluorescence
and Pp IX fluorescence. In this work we present a method, using a simple set up for multispectral imaging assisted by computerised image warping. The set up was evaluated investigating 9 patients with histologically verified BCC located in the face. Z-images, defined as the ratio between the autofluorescence and the Pp IX fluorescence images, were obtained and compared to the clinically marked tumour borders. Agreement with the tumour border was found in 8 out
of the 9 patients. These results imply that multispectral fluorescence imaging is a potential diagnostic tool for
demarcation of BCC.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.