Melanoma Imaging Research Articles

Abstract 4382: Intravital imaging of endogenous BRAFV600E melanoma reveals plasticity of tumor response and resistance to trametinib over time

Abstract Although the number of therapies entering clinical trials continues to increase, the success rate for targeted therapy studies is quite low due, in part, to a lack of relevant preclinical models. The use of targeted therapy has become a popular method for cancer treatment against mutant BRAF melanoma, as studies show that clinical use of selective inhibitors against MEK and BRAF extend life expectancy. However, melanoma becomes refractory to treatment. Since the tumor and stromal factors that contribute to drug resistance remain poorly understood, our ability to treat patients with advanced disease remains limited. Current genetically engineered murine (GEM) models in melanoma research have enhanced our understanding of tumor biology and therapeutic response, but fail to mimic tumor progression in clinical settings as most lack spatial and temporal control of endogenous tumor initiation. To better mimic human disease, we have developed a novel tamoxifen application method that reproducibly induces local tumor formation on the mouse ear, and we have incorporated a tdTomato fluorescent reporter allele (tdTomatoLSL) into an existing tamoxifen-inducible GEM model of BRAFV600E/PTEN-null melanoma. The tdTomato allele serves as a visual marker of Cre recombination in endogenous melanocytes and allows disease progression to be followed through macroscopic and multiphoton intravital imaging. To investigate therapeutic effects at the cell level, we have performed imaging studies of primary tumors before and after treatment with a selective MEK1/2 inhibitor (MEKi), Trametinib, which is a FDA approved therapy for malignant melanoma. Our melanoma induction method enables longitudinal studies of drug response that ultimately give rise to fully resistant tumors (up to 90 days on MEKi). Our studies show that this treatment causes a striking morphological change of the tumor cells as early as 3 days post-treatment. After several weeks of continuous treatment, the remaining tumor cells are highly spatially correlated with bundled collagen structures detected by second harmonic signal, suggesting that cellular milieu strongly influences drug response. Interestingly, our current studies with MEKi suggest that association of tumor cells with collagen fibrils initially provides a protective effect against this drug, however once true resistance emerges, this dependency on extracellular matrix in the tumor stroma diminishes. Furthermore, in parallel with our imaging studies, we have performed molecular analysis of tumor response using Mib/MS and mRNA-seq. By coupling our imaging studies with transcriptome and kinome reprogramming analysis, we hope to identify properties that promote resistance. Using these novel approaches, we have developed a model that enables direct observation of endogenous tumor development, plasticity, and resistance to targeted therapy at the cell level in situ for the first time. Citation Format: Hailey E. Brighton, Steven Angus, Tao Bo, David Darr, Norman E. Sharpless, Gary L. Johnson, James E. Bear. Intravital imaging of endogenous BRAFV600E melanoma reveals plasticity of tumor response and resistance to trametinib over time. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4382.

T2 Fluid-Attenuated Inversion Recovery Imaging of Uveal Melanomas and Other Ocular Pathology

Background/Aims: This study describes patterns of intraocular lesions on T2 fluid-attenuated inversion recovery (FLAIR) imaging, exploring a prospective role of FLAIR imaging sequence in diagnosis and treatment. Methods: A retrospective study of orbital magnetic resonance imaging (MRI) studies from the years 2000 to 2015 was performed. MRI sequences included: pre-contrast T1-weighted, T2-weighted, T2 FLAIR, and postcontrast T1 and T2 imaging gadolinium, which were evaluated by a neuroradiologist. Two cases of melanoma were correlated to their pathology. Results: Twenty-four patients with intraocular pathology were evaluated. All lesions, regardless of pigmentation, revealed previously described melanotic patterns on T1- and T2-weighted images; 80% of 10 melanomas localized were hyperintense on T2 FLAIR, which better delineated lesion margins. All of the four inflammatory pathologies on T2 FLAIR were hyperintense, as were 80% of the amelanotic neoplasms. Pathology of two large uveal melanomas paralleled the findings seen on T2 FLAIR. Conclusions: T2 FLAIR appears beneficial in the demarcation of pigmented ocular lesions and may aid in determining protein content or previous treatment. Data also promote previous assertions that blood flow impacts intensity of lesions on T2 FLAIR. Further research is warranted.

Preparation and characterization of a novel Al18F–NOTA–BZA conjugate for melanin-targeted imaging of malignant melanoma

Melanin is an attractive target for the diagnosis and treatment of malignant melanoma. Previous studies have demonstrated the specific binding ability of benzamide moiety to melanin. In this study, we developed a novel 18F-labeled NOTA–benzamide conjugate, Al18F–NOTA–BZA, which can be synthesized in 30min with a radiochemical yield of 20–35% and a radiochemical purity of >95%. Al18F–NOTA–BZA is highly hydrophilic (logP=−1.96) and shows good in vitro stability. Intravenous administration of Al18F–NOTA–BZA in two melanoma-bearing mouse models revealed highly specific uptake in B16F0 melanotic melanoma (6.67±0.91 and 1.50±0.26%ID/g at 15 and 120min p.i., respectively), but not in A375 amelanotic melanoma (0.87±0.21 and 0.24±0.09%ID/g at 15 and 120min p.i., respectively). The clearance from most normal tissues was fast. A microPET scan of Al18F–NOTA–BZA-injected mice also displayed high-contrast tumor images as compared with normal organs. Owing to the favorable in vivo distribution of Al18F–NOTA–BZA after intravenous administration, the estimated absorption dose was low in all normal organs and tissues. The melanin-specific binding ability, sustained tumor retention, fast normal tissues clearance and thelow projected human dosimetry supported that Al18F–NOTA–BZA is a very promising melanin-specific PET probe for melanin-positive melanoma.

Skin lesion image segmentation using Delaunay Triangulation for melanoma detection

Developing automatic diagnostic tools for the early detection of skin cancer lesions in dermoscopic images can help to reduce melanoma-induced mortality. Image segmentation is a key step in the automated skin lesion diagnosis pipeline. In this paper, a fast and fully-automatic algorithm for skin lesion segmentation in dermoscopic images is presented. Delaunay Triangulation is used to extract a binary mask of the lesion region, without the need of any training stage. A quantitative experimental evaluation has been conducted on a publicly available database, by taking into account six well-known state-of-the-art segmentation methods for comparison. The results of the experimental analysis demonstrate that the proposed approach is highly accurate when dealing with benign lesions, while the segmentation accuracy significantly decreases when melanoma images are processed. This behavior led us to consider geometrical and color features extracted from the binary masks generated by our algorithm for classification, achieving promising results for melanoma detection.

Melanoma-Nevus Discrimination Based on Image Statistics in Few Spectral Channels

The purpose of this paper is to offer a method for discrimination of cutaneous melanoma from benign nevus, founded on analysis of skin lesion image. At the core of method is calculation of mean and standard deviation of pixel optical density values for a few narrow spectral bands. Calculated values are compared with discriminating thresholds derived from a set of images of benign nevi and melanomas with known diagnosis. Classification is done applying weighted majority rule to results of thresholding. Verification against the available multispectral images of 32 melanomas and 94 benign nevi has shown that the method using three spectral bands provided zero false negative and four false positive melanoma detections. The proposed classifier is characterized by high sensitivity and specificity concerning statistical point estimates whereas its possible technical implementation is fairly simple. The proposed method may be instrumental for designing low cost diagnostic equipment to be used in primary care that is important for early detection of cutaneous melanoma. DOI: http://dx.doi.org/10.5755/j01.eie.22.2.12173

On the geometric modulation of skin lesion growth: a mathematical model for melanoma

Introduction Early detection of suspicious skin lesions is critical to prevent skin malignancies, particularly the melanoma, which is the most dangerous form of human skin cancer. In the last decade, image processing techniques have been an increasingly important tool for early detection and mathematical models play a relevant role in mapping the progression of lesions. Methods This work presents an algorithm to describe the evolution of the border of the skin lesion based on two main measurable markers: the symmetry and the geometric growth path of the lesion. The proposed methodology involves two dermoscopic images of the same melanocytic lesion obtained at different moments in time. By applying a mathematical model based on planar linear transformations, measurable parameters related to symmetry and growth are extracted. Results With this information one may compare the actual evolution in the lesion with the outcomes from the geometric model. First, this method was tested on predefined images whose growth was controlled and the symmetry known which were used for validation. Then the methodology was tested in real dermoscopic melanoma images in which the parameters of the mathematical model revealed symmetry and growth rates consistent with a typical melanoma behavior. Conclusions The method developed proved to show very accurate information about the target growth markers (variation on the growth along the border, the deformation and the symmetry of the lesion trough the time). All the results, validated by the expected phantom outputs, were similar to the ones on the real images.

Advances in noninvasive imaging of melanoma.

Melanoma is the most dangerous type of skin cancer and its incidence has risen sharply in recent decades. Early detection of disease is critical for improving patient outcomes. Any pigmented lesion that is clinically concerning must be removed by biopsy for morphologic investigation on histology. However, biopsies are invasive and can cause significant morbidity, and their accuracy in detecting melanoma may be limited by sampling error. The advent of noninvasive imaging devices has allowed for assessment of intact skin, thereby minimizing the need for biopsy; and these technologies are increasingly being used in the diagnosis and management of melanoma. Reflectance confocal microscopy, optical coherence tomography, ultrasonography, and multispectral imaging are noninvasive imaging techniques that have emerged as diagnostic aids to physical exam and/or conventional dermoscopy. This review summarizes the current knowledge about these techniques and discusses their practical applications and limitations.

A pilot study to image the vascular network of small melanocytic choroidal tumors with speckle noise-free 1050-nm swept source optical coherence tomography (OCT choroidal angiography).

To visualize and measure the vascular network of melanocytic choroidal tumors with speckle noise-free swept source optical coherence tomography (SS-OCT choroidal angiography). Melanocytic choroidal tumors from 24 eyes were imaged with 1050-nm optical coherence tomography (Topcon DRI OCT-1 Atlantis). A semi-automated algorithm was developed to remove speckle noise and to extract and measure the volume of the choroidal vessels from the obtained OCT data. In all cases, analysis of the choroidal vessels could be performed with SS-OCT without the need for pupillary dilation. The proposed method allows speckle noise-free, structure-guided visualization and measurement of the larger choroidal vessels in three dimensions. The obtained data suggest that speckle noise-free OCT may be more effective at identifying choroidal structures than traditional OCT methods. The measured volume of the extracted choroidal vessels of Haller's layer and Sattler's layer in the examined tumorous eyes was on average 0.982463955mm(3) /982463956μm(3) (range of 0.209764406mm(3) /209764405.9μm(3)to 1.78105544mm(3) /1781055440μm(3)). Full thickness obstruction of the choroidal vasculature by the tumor was found in 18 cases (72%). In seven cases (18%), choroidal vessel architecture did not show pronounced morphological abnormalities (18%). Speckle noise-free OCT may serve as a new illustrative imaging technology and enhance visualization of the choroidal vessels without the need for dye injection. OCT can be used to identify and evaluate the choroidal vessels of melanocytic choroidal tumors, and may represent a potentially useful tool for imaging and monitoring of choroidal nevi and melanoma.

ニボルマブによりpseudoprogressionを起こしたと思われた悪性黒色腫の当科での検討

ニボルマブによりpseudoprogressionを起こしたと思われた悪性黒色腫の当科での検討

Automatic Triple-A Segmentation of Skin Cancer Images based on Histogram Classification

Skin cancer has been the most common and represents 50% of all new cancers detected each year. If detected at an early stage, simple and economic treatment can cure it mostly. Accurate skin lesion segmentation is critical in automated early diagnosis system. This paper present a triple segmentation procedure based on the pixels distribution Bell-shaped (Normal), J-shaped, Reverse J-shaped and U-shaped peaks that is bimodal. According to the nature of dermoscopy images distributions, three segmentation methods are used to identify the normal skin cancer from malignant skin and to extract the tumor region. First, active contours are used for bell distribution shape. Second segmentation is done using adjusted ant colony optimization when the U-shaped peaks distribution was classify. Third segmentation strategies apply adaptive threshold for two J-shapes. Experiments on synthetic and real dermoscopy images demonstrate the advantages of the proposed methods that is able to produce ant colony optimization accurate segmentation when applied to a large number of skin cancer (melanoma) images.

Radiopharmacological characterization of ⁶⁴Cu-labeled α-MSH analogs for potential use in imaging of malignant melanoma.

The melanocortin-1 receptor (MC1R) plays an important role in melanoma growth, angiogenesis and metastasis, and is overexpressed in melanoma cells. α-Melanocyte stimulating hormone (α-MSH) and derivatives are known to bind with high affinity at this receptor that provides the potential for selective targeting of melanoma. In this study, one linear α-MSH-derived peptide Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) without linker and with εAhx-β-Ala linker, and a cyclic α-MSH derivative, [Lys-Glu-His-D-Phe-Arg-Trp-Glu]-Arg-Pro-Val-NH2 (NAP-NS2) with εAhx-β-Ala linker were conjugated with p-SCN-Bn-NOTA and labeled with (64)Cu. Radiochemical and radiopharmacological investigations were performed with regard to transchelation, stability, lipophilicity and in vitro binding assays as well as biodistribution in healthy rats. No transchelation reactions, but high metabolic stability and water solubility were demonstrated. The linear derivatives showed higher affinity than the cyclic one. [(64)Cu]Cu-NOTA-εAhx-β-Ala-NAP-NS1 ([(64)Cu]Cu-2) displayed rapid cellular association and dissociation in murine B16F10 cell homogenate. All [(64)Cu]Cu-labeled conjugates exhibited affinities in the low nanomolar range in B16F10. [(64)Cu]Cu-2 showed also high affinity in human MeWo and TXM13 cell homogenate. In vivo studies suggested that [(64)Cu]Cu-2 was stable, with about 85 % of intact peptide in rat plasma at 2 h p.i. Biodistribution confirmed the renal pathway as the major elimination route. The uptake of [(64)Cu]Cu-2 in the kidney was 5.9 % ID/g at 5 min p.i. and decreased to 2.0 % ID/g at 60 min p.i. Due to the prospective radiochemical and radiopharmacological properties of the linear α-MSH derivative [(64)Cu]Cu-2, this conjugate is a promising candidate for tracer development in human melanoma imaging.

Reducing perceived pain levels during nonbreast lymphoscintigraphy.

The aim of this study was to quantify the reduction of perceived pain levels during lymphoscintigraphy for melanoma by altering the pH of the Tc-sulfur colloid to near the physiologic value of 7.40. This is an institutional review board- and Food and Drug Administration-approved randomized, double-blinded, prospective crossover trial, registered with clinicaltrials.org. Before beginning the procedure and after signing informed consent, 60 serial enrollees presenting for sentinel lymph node imaging of melanoma of the thorax and appendicular structures completed a questionnaire addressing background information, administered by a research support nurse.An investigator (N.H.) prepared the injections to be used and labeled them such that no one else could discover which injections contained standard-of-care solution and which contained the pH-altered solution (buffered to near pH 7.40 using sodium bicarbonate).After each injection, the enrollee was asked by a research support nurse to quantify the pain of each injection using a scale of 0 to 10. The injection site location (head, thorax, appendicular structures, and other) was also recorded. Sixty subjects were enrolled in the study, of which 57 had complete data. On average, there was a significant mean (SD) decrease of 1.42 (2.17) points (95% confidence interval, 0.85-2.00) on a pain scale of 0 to 10 when the buffered injections were used as compared with the standard-of-care injections (P < 0.0001). Ordering of injections did not significantly affect perceived pain scores. The use of sodium bicarbonate to buffer the pH of Tc-sulfur colloid to near the physiologic value of 7.40 significantly reduced perceived pain levels during nonbreast lymphoscintigraphy.

Micellar formulation of indocyanine green for phototherapy of melanoma

Phototherapy (PT), a light activated treatment modality, is a potential therapeutic option for the treatment of melanoma. In spite of the excellent safety profile and absorption in the near infrared (NIR) range, clinical potential of indocyanine green (ICG) as PT is limited by its short half-life and inefficient tumor accumulation. In this study, we have covalently conjugated ICG-NH2 to the pendant carboxyl groups of poly (ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG-PCC) copolymer using carbodiimide coupling, which self-assembled into micelles with a particle size of 30–50nm and high ICG loading. These ICG conjugated micelles exhibited significant in vitro photodynamic cytotoxicity. Use of sodium azide and NIR radiation at 4°C revealed photodynamic and photothermal as mechanism of cytotoxicity of ICG solution and ICG conjugated micelles, respectively. In vivo NIR imaging demonstrated that ICG conjugated micelles prolonged its circulation and increased tumor accumulation through enhanced permeability and retention (EPR) effect. Enhanced tumor accumulation improved therapeutic efficacy with complete tumor regression in NIR irradiated ICG conjugated micelles compared to ICG solution and control in a A375 human melanoma tumor model in athymic nude mice. These results suggest that ICG conjugated micelles can be potentially utilized for PT and imaging of melanoma.

Development of a (99m)Tc-labeled lactam bridge-cyclized alpha-MSH derivative peptide as a possible single photon imaging agent for melanoma tumors.

Melanocortin-1 (MC1) receptor is an attractive melanoma-specific target which has been used for melanoma imaging and therapy. In this work, a new lactam bridge α-MSH analog was labeled with (99m)Tc via HYNIC and EDDA/tricine as coligands including gamma aminobutyric acid (GABA) as a three carbon chain spacer between HYNIC and the N-terminus of the cyclic peptide. Also, stability in human serum, receptor bound internalization, in vivo tumor uptake, and tissue biodistribution were thoroughly investigated. HYNIC-GABA-Nle-CycMSHhept was synthesized using a standard Fmoc strategy. Labeling was performed at 95 °C and analysis involved instant thin layer chromatography and high performance liquid chromatography methods. The receptor bound internalization rate was studied in MC1 receptor expressing B16/F10 cells. Biodistribution of radiopeptide was studied in nude mice bearing B16/F10 tumor. Labeling yield of >98 % (n = 3) was obtained corresponding to a specific activity of 81 MBq/nmol. Peptide conjugate showed efficient stability in the presence of human serum. The radioligand showed specific internalization into B16/F10 cells (12.45 ± 1.1 % at 4 h). In biodistribution studies, a receptor-specific uptake was observed in MC1 receptor-positive organs so that after 2 h the uptake in mouse tumor was 5.10 ± 0.08 % ID/g, while low accumulation in the kidney uptake was observed (4.58 ± 0.68 % ID/g at 2 h after injection). The obtained results show that the presented new designed labeled peptide conjugate may be a suitable candidate for diagnosis of malignant tumors.

Iterative Threshoding and Morphology Operation based Melanoma Image Segmentation

is a suitable diagnostic technique for biology observation of pigmented skin lesions used in dermatology. Nowdays there is great interest in the prospects for methods of automatic image analysis for dermoscopy image, it Provide quantitative information about the lesion, which can be of link the doctor, and as a standalone early warning tool. This paper presents a good method of melanoma images segmentation. It based on threshoding as segmentation and mathematical morphology used to remove unwanted part in order to obtain a better segmentation. The proposed method is compared with the famous method of segmentation of skin lesions in images dermoscopic such adaptive thresholding and fuzzy K-means clustering for the segmentation and evaluated with two metrics, False Positive Rate (FPR) and the False Negtive Rate (FNR) , using the segmentation results obtained by a dermatologist experienced and ground truth. KeywordsOperation ,images segmentation, skin lesions.

Evaluation of (68)Ga- and (177)Lu-DOTA-PEG4-LLP2A for VLA-4-Targeted PET Imaging and Treatment of Metastatic Melanoma.

Malignant melanoma is a highly aggressive cancer, and the incidence of this disease is increasing worldwide at an alarming rate. Despite advances in the treatment of melanoma, patients with metastatic disease still have a poor prognosis and low survival rate. New strategies, including targeted radiotherapy, would provide options for patients who become resistant to therapies such as BRAF inhibitors. Very late antigen-4 (VLA-4) is expressed on melanoma tumor cells in higher levels in more aggressive and metastatic disease and may provide an ideal target for drug delivery and targeted radiotherapy. In this study, we evaluated (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A as a VLA-4-targeted radiotherapeutic with a companion PET agent for diagnosis and monitoring metastatic melanoma treatment. DOTA-PEG4-LLP2A was synthesized by solid-phase synthesis. The affinity of (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A to VLA-4 was determined in B16F10 melanoma cells by saturation binding and competitive binding assays, respectively. Biodistribution of the LLP2A conjugates was determined in C57BL/6 mice bearing B16F10 subcutaneous tumors, while PET/CT imaging was performed in subcutaneous and metastatic models. (177)Lu-DOTA-PEG4-LLP2A showed high affinity to VLA-4 with a Kd of 4.1 ± 1.5 nM and demonstrated significant accumulation in the B16F10 melanoma tumor after 4 h (31.5 ± 7.8%ID/g). The tumor/blood ratio of (177)Lu-DOTA-PEG4-LLP2A was highest at 24 h (185 ± 26). PET imaging of metastatic melanoma with (68)Ga-DOTA-PEG4-LLP2A showed high uptake in sites of metastases and correlated with bioluminescence imaging of the tumors. These data demonstrate that (177)Lu-DOTA-PEG4-LLP2A has potential as a targeted therapeutic for treating melanoma as well as other VLA-4-expressing tumors. In addition, (68)Ga-DOTA-PEG4-LLP2A is a readily translatable companion PET tracer for imaging of metastatic melanoma.

Sentinel lymph node in melanoma: present aspects and future trends

In melanoma, the sentinel lymph node (SLN) status is the most important factor determining overall survival. Lymphoscintigraphy is a current practice evolving since more than 20 years. It represents the standard practice in detecting SLN and includes dynamic imaging and SPECT/CT. This article reviews the different technical aspects of lymphoscintigraphy with their advantages. It also reviews the main other ways of SLN imaging in melanoma, including more specific techniques, some of them representing a field of research. A PUBMED (MeSH) search was performed with the following keywords: sentinel lymph node melanoma imaging and reviewed relevant articles. We excluded case reports, publications with an Impact Factor lower than 2 and older than 10 years. The use of dynamic and delayed images combined with preoperative SPECT/CT and blue dye during surgery remains the method of choice in sentinel lymph node melanoma detection. SPECT/CT provides several advantages, in particular a higher rate of node detection, better nodes localization and reduction of operative time, with significantly reducing costs. To provide longer retention in lymphatic nodes, some targeted agents are developed but their clinical use is limited. Lymphatic staging with imaging contrast agents could directly assess nodal status without surgery and could be a promising method for the future.

Label-free imaging of melanoma with nonlinear photothermal microscopy.

Nonlinear photothermal microscopy, in which the intensity of the pump heating beam is modulated at f and the photothermal signal is extracted from the probe beam with a lock-in amplifier referred to 2f, is applied to the imaging of mouse melanoma without any staining. The pump and probe pulses, with central wavelengths of 488 and 632 nm, and a pulse duration of ∼100 ps, are filtered from a compact commercial supercontinuum fiber laser source. An auto-balanced detector is applied to accumulate the signal and remove the laser noise of the probe. The spatial resolution of the nonlinear photothermal imaging is enhanced by ∼18% in both theoretical calculations and experiments, compared with a linear photothermal mechanism, and the resolution enhancement is theoretically ∼42% compared with conventional optical microscopy. This imaging technique shows possibilities for the clinical evaluation of melanoma with a high contrast and spatial resolution.

Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo.

We applied a linear-array-based photoacoustic probe to detect melanin-containing melanoma tumor depth and volume in nude mice in vivo. This system can image melanomas at five frames per second (fps), which is much faster than our previous handheld single transducer system (0.1 fps). We first theoretically show that, in addition to the higher frame rate, almost the entire boundary of the melanoma can be detected by the linear-array-based probe, while only the horizontal boundary could be detected by the previous system. Then we demonstrate the ability of this linear-array-based system in measuring both the depth and volume of melanoma through phantom, ex vivo, and in vivo experiments. The volume detection ability also enables us to accurately calculate the rate of growth of the tumor, which is an important parameter in quantifying the tumor activity. Our results show that this system can be used for clinical melanoma diagnosis and treatment in humans at the bedside. Linear-array-based PA images of melanoma acquired in vivo on day 3 (a) and day 6 (b).

Sensitivity enhancement of photothermal microscopy with radially segmented balanced detection.

A novel detection method is proposed for highly sensitive photothermal microscopic imaging. This method is based on the characteristics of an angular-dependent photothermal signal; it improves signal intensity by up to two times and rejects the intensity noise of the probe beam. The subdiffraction resolution photothermal imaging of mouse skin melanoma is demonstrated using a laser diode-based photothermal microscopy system to evaluate this method. We confirm that the signal intensity is enhanced 1.7 times compared with the conventional detection method. Moreover, the intensity noise of the laser diode used for the probe beam is effectively reduced by approximately 31 dB, even for a sample with non-uniformity of the refractive index and stationary absorption. This method is implemented by means of a commonly used balanced detector and is thus potentially useful for high-speed imaging.