Boosted local structured HOG-LBP for object localization
Summary (6 min read)
1. Introduction
- The assessment of nanoparticles (NPs) using Caenorhabditis elegans (C. elegans) has rapidly increased in the last years, supporting its suitability as an in vivo model to screen NPs.
- It is unlikely that all researchers will routinely use identical methodologies in initial experiments of novel materials, as they frequently have different objectives.
- To date, the majority of studies on NPs in C. elegans have focused on noble metal and metal oxide NPs.
- Finally, C. elegans also serves as an in vivo platform to validate the efficiency of a nanomaterial for a given application, such as imaging or targeting (application labeled as ‘in vivo validation’ in the figure).
2.1. Exposure media
- In the laboratory, C. elegans are typically grown on agar plates of Nematode Growth Media (NGM) with Escherichia coli (E. coli) OP50 as food source.
- Conversely, some authors reported formation of micrometric aggregates of Ag-NPs in K-medium that rapidly settled from suspension leading to an elevated effective local ‘‘dose’’ in the bottom of the wells.
- The left panel focuses on the exposure conditions.
2.3. Physicochemical properties of the test NPs
- This section describes general findings regarding the influence of NP size, surface coating and composition in the response of C. elegans to NPs.
- The conditions of Fig. 3 Transmission Electron Microscopy (TEM) of ZnO-NPs in the exposure media.
- They proposed that larger particle size may enable faster uptake rates by oral ingestion and thus higher mass doses than exposure to smaller stable particle sizes.
- 26 Surface charge also affected the oxidation state of Ce in the C. elegans tissues after uptake: greater reduction of Ce from Ce(IV) to Ce(III) was found in C. elegans when exposed to the neutral and negatively charged relative to positively charged CeO2-NPs.
- Besides metal and metal oxide NPs, quantum dots69 and carbonbased nanomaterials such as graphene oxide have been widely studied recently.
3. Techniques to investigate entrance route, uptake, biodistribution and fate
- Nanopharmacokinetics are critical for both environmental and biomedical research.
- Based on the small size of the materials under study, techniques with spatial resolution at the nanoscale are required to discern and identify particles with certainty at multiple biological levels, from the organismal down the intracellular scales, in order to shed light on single-NP localization, translocation routes and NP status in vivo.
- In C. elegans, entrance of metal and metal oxide NPs has been reported to occur mainly through the alimentary system, consistent with the fact that C. elegans does not discern between entities up to 5 mm when feeding.
- The most prevalent techniques applied to date to determine NP uptake and fate in this animal model have been fluorescent microscopy,42,51,52 hyperspectral dark-field microscopy,33,38 and to a much lesser extent, transmission electron microscopy (TEM),53,68 synchrotron-based techniques25 and other analytical techniques.
- The authors illustrate how the entrance route, uptake, biodistribution and fate of metal and metal oxide NPs have been evaluated in C. elegans, and propose to extend the toolkit of available techniques.
3.1. Fluorescence microscopy
- By using fluorescence microscopy Pluskota et al. showed that fluorescently labeled 50 nm NPs (PS and SiO2) were efficiently ingested by the worms during feeding, and translocated to primary organs such as epithelial cells of the intestine, as well as to secondary organs belonging to the reproductive tract.
- Within the intestine, NPs accumulate with decreasing concentrations from the anterior to the posterior regions of the intestine.
- Cytoplasmic uptake of 50 nm PS-NPs was observed in early embryos.52 Scharf et al. identified two entry portals of silica and PS-NPs: via the pharynx to the intestinal system, and via the vulva to the reproductive system.
- 48 However, it is important to note that fluorescence microscopy is limited to a spatial resolution of 200 nm, hence, without the use of complementary techniques, the possibility that single NPs penetrate further into C. elegans tissue or are taken up intracellularly cannot be excluded.
3.3. Transmission electron microscopy
- TEM has sufficient spatial resolution to allow single-NP detection.
- Adapted from Höss et al.27 (B) Table indicating the distribution of silica, polystyrene and carboxy-polystyrene NPs within the body of treated C. elegans.
- It can also provide further clues about NP translocation routes, i.e. by endocytosis, although this should be confirmed by chemical identification or molecular mechanistic evidence.
- Sampling at random locations along the body of the worm can limit the information obtained by TEM visualizations; moreover, the analysis of a large number of sections is very costly and laborious.
- To maximize the control of the anatomical area investigated in the cross-sections, targeted ultramicrotomy protocols can be applied using correlated light and electron microscopy (CLEM), with the aim of establishing a statistically significant and biologically meaningful link between the location in the body and the NP status in vivo.75.
3.4. Scanning electron microscopy
- Scanning electron microscopy (SEM) allows investigation of the morphology of the C. elegans external surface, the cuticle, in detail.
- After a 24 h exposure to citrate coated 10 nm Ag-NPs in NGM agar, they observed severe epidemic edema and bursting of the cuticle of C. elegans (Fig. 7A–C), suggesting that Ag-NPs can induce adverse physical effects via the dermal route.
- Given that previous studies in liquid33 did not reveal such effects, the authors proposed they were induced by the movement of C. elegans in the agar plates where the Ag-NPs were distributed.
- More recently, the authors evaluated the external surface of worms treated with Fe2O3 and Au-NPs in liquid by SEM coupled to energy-dispersive X-ray spectroscopy (EDX), an elemental analysis technique that enables the study of chemical composition.
- The authors could not visualize NPs on the cuticle of C. elegans by SEM, nor did they detect the presence of iron or gold elements on this structure by EDX (Fig. 7D–F) after thorough rinsing of the treated worms.
3.5. Synchrotron and microprobe techniques
- Among the synchrotron techniques, synchrotron radiation X-ray fluorescence (m-SRXRF) has been used to map the metal distribution in C. elegans, while synchrotron X-ray absorption near-edge spectroscopy (m-XANES) has provided information regarding the oxidation state and coordination environment of metals.
- Cu2+ exposure resulted in a much higher absorption and accumulation.
- 25 Regarding the use of nuclear microprobe techniques, Le Trequesser combined scanning transmission ion microscopy (STIM) and micro-proton-induced X-ray emission (m-PIXE) to detect and quantify 30 nm TiO2-NPs in C. elegans.
- After 4 h exposure, NPs were visible only in the lumen of the alimentary system extending from the pharynx to the anal region, and were retained there even 24 h after feeding.
- 43 Given that alterations in the distribution of trace metal such as Fe, Cu, Zn or Mn are sometimes related to certain pathological states, the use of these techniques is of value in the study of alterations in metal homeostasis.
3.6. Analytical chemistry techniques
- Among the analytical techniques applied to investigate NPs in C. elegans, different micro-spectroscopy modalities have been used to characterize NP status, while quantitation of NP uptake Fig. 5 Use of fluorescence and hyperspectral imaging to characterize NP pharmacokinetics.
- Upper panels show epifluorescence images of carboxy 50 nm PS-NPs in the intestine (left) and cytoplasm of early embryos .
- This journal is©The Royal Society of Chemistry 2017 Mater. Horiz. has been mainly addressed by chemical elemental analysis (ICP-MS).
- Fe concentrations after a 6 h exposure (2 mg mg 1 worm), however Fe uptake decreased after 2 h under normal conditions due to defecation of the NPs contained in the intestinal lumen (disposal of 50% Fe), and it was further reduced by the disposal of the surface-attached Fe during molting (additional 80% reduction of nematodeassociated Fe) (Fig. 9B).
- 27 More recently, Johnson et al. applied ICP-MS to quantify Au-NP uptake by C. elegans and, operating in single particle ICP-MS mode, to characterize Au-NP status inside the animals.
3.7. Other experimental techniques with potential to characterize nano/bio interactions
- Use of m-PIXE to characterize NP toxicokinetics in C. elegans.
- Magnetometry can also be applied to investigate NP fate by monitoring how the magnetic moment of the sample changes over temperature at a determined applied magnetic field.
- These results provide indirect evidence of NP size evolution, and have been successfully corroborated with much more laborious analysis of C. elegans cross-sections by TEM (Fig. 12F and G).34,68 Both ZFC-FC measurements and TEM visualization supports the notion that NPs remain individual inside C. elegans.
4. Biological responses triggered by metal and metal oxide NPs: approaches and common outcomes
- In the evaluation of metal and metal oxide NPs, it is important to note that some metals, known as essential metals, have a biological role in animals and plants.
- Fig. 13 summarizes the different dissolution rates for different NP compositions at pH B 4.5.
- Genetic approaches involve manipulating the genome of an organism to probe how this alteration affects the biological response to a stressor.
- R am an m- sp ec tr os co py B io m ol ec u la r ph en ot yp e — Q u an ti ta ti ve . – Fat and carbohydrate metabolism – Oxidative stress protection (SOD) – Neurotransmitter synthesis and metabolism.
4.1. Pathways by which metal and metal oxide NPs cross biological barriers
- Given the technical difficulty of studying NP fate with nanometric resolution inside living organisms at present, there is limited evidence of the internalization and translocation mechanisms of NPs in C. elegans.
- Moreover, the negative membrane potential of most cells interacts differently with particles with a positive or negative surface charge.
- The electrostatic interaction of NPs with the negatively charged bilayer of a membrane mediates their binding and their toxicity.
- For biodegradable particles such as ZnO or Fe2O3, NP metabolism inside C. elegans has been demonstrated using a range of techniques, including magnetometry or spectroscopy.
- 37 Gonzalez-Moragas et al. reported intracellular uptake by clathrin-mediated endocytosis of 6 nm Fe2O3-NPs, and down-regulation of the early endosome formation gene dyn-1, among other intestinal-related genes, however internalization of 11 nm Au-NPs were not detected either by electron microscopy or by gene expression analysis.
4.2. Toxicological mechanisms of metal nanoparticles
- In an attempt to study particle-specific effects of manufactured nanomaterials, Tsyusko et al. chose gold NPs as a model since they are resistant to oxidative dissolution.
- In good agreement, Contreras et al. showed that Ag-NP pre-exposed nematodes suffered cumulative damage.
- In a later study, Roh et al. reported the formation of ROS and analyzed the expression of genes in MAPK signaling pathways.
- This study highlighted a critical role for dissolved silver in the toxicity of all tested Ag-NPs, and also proposed a specific nano-Ag effect via oxidative stress typically for the less soluble Ag-NPs, hence encompassing the two most prevalent Ag-NPs toxicity mechanisms reported previously.
- Overall, the data is consistent with multiple mechanisms of action playing larger and smaller roles in different contexts.
4.3. Toxicological mechanisms of metal oxide nanoparticles
- Ma et al. investigated the toxicity of ZnO-NPs and did not find differences from Zn2+ at the same molar (Zn) concentrations.
- Cyp35a2 is a highly stress-responsive gene, and these results may highlight the principle that induction of a gene does not prove that the mechanism of toxicity of the stressor is closely related to the biological function of the gene induced, since genes are often inducible by multiple stressors.
- Many metal ions can cause indirect oxidative stress via inhibition of antioxidant and other enzymes, depletion of glutathione and other antioxidants, or disruption of the electron transport chain.
- Arnold et al. could not attribute CeO2NP-induced growth inhibition to oxidative or metal stress, but rather proposed a non-specific inhibition of feeding caused by NPs aggregating in the test media and/or inside the gut tract.
- Only after long exposures and high doses (25 mg L 1 for 24 h), which suggest the relatively safe properties of TiO2-NPs.
4.4. Toxicological mechanisms of other nanomaterials
- The cellular and molecular mechanisms of nanotoxicity of other nanomaterials including quantum dots (QD) and carbon nanomaterials such as graphene oxide (GO) have also been addressed recently in C. elegans.
- In a study of QD-exposed nematodes, the intestinal barrier played a crucial role in transgenerational toxicity.
- 116 Wu et al. found a key role of innate immunity in regulating in vivo GO toxicity.
- 112 Recently, Subramani et al. investigated the shielding efficacy of BSA and poly ethylene imine (PEI) on graphene oxide (GO) in C. elegans and reported reduced toxicity of BSA-coated GO NPs compared to PEI-coated NPs, confirming the importance of surface properties in particle toxicity in vivo.
- Hence, these studies offer a perspective complementary to metal-based NP evaluation, and open new avenues for future research on these materials.
5. Proposed integrated workflow for biological and materials science-based
- The data is heterogeneous and still limited, hampering metadata analysis.
- Therefore, to further their understanding of nano/bio interactions and facilitate data integration, the authors propose adoption of a minimal set of materials science and toxicological assessments that should be included in all studies.
- First, it is of vital importance that NPs be very wellcharacterized after their synthesis or their purchase.
- In addition, colloidal stability of NPs should be investigated in different exposure media well-tolerated by C. elegans.
6. Conclusions
- The authors have presented how toxicology and materials science experts are contributing to better understand nano/bio interactions in the model organism C. elegans, although they have often worked separately, missing joint opportunities between their research fields.
- A major recurrent shortcoming identified is that most of the toxicological studies to date have focused on identifying toxicity endpoints, while very few also investigate NP status inside C. elegans.
- From the materials science viewpoint, magnetometry can find a novel use in the study of worms exposed to magnetic NPs; absorbance micro-spectroscopy can be applied to characterize noble metal NPs inside C. elegans; and FT-IR micro-spectroscopy might determine the level of tissue oxidation in NP-treated animals.
- In C. elegans, to date these techniques have revealed NP-induced adverse effects in different cell organelles, among them mitochondria, lysosomes and the nucleus.
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Cites background from "Boosted local structured HOG-LBP fo..."
...This hypothesis is grounded, for example, in the wide range of papers that attempt to boost detection accuracy with work along four axes: (1) rich structured models [20, 42]; (2) multiple feature learning [38, 41]; (3) learned histogram-based features [11, 29, 32]; or (4) unsupervised feature learning [34]....
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...First, handcrafted image features such as HOG [5, 45, 44], SIFT [25], and Fisher Vector [4] are extracted at every location and scale of an image....
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...We do not use any context cues in this paper and leave it as a future work....
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...Contexts from local or global appearance have been explored to improve object detection [23, 27, 8, 5, 18, 29]....
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References
31,952 citations
"Boosted local structured HOG-LBP fo..." refers background or methods in this paper
...Following the bottom-up paradigm, we propose a boosted Local Structured HOGLBP based object detector....
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...This paper focuses on two basic problems: how to accurately describe object structure at feature level and how to fuse multiple Local Structured Descriptors for part based model at topology level....
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15,935 citations
"Boosted local structured HOG-LBP fo..." refers background or methods in this paper
...Secondly, at topology level, we present a boosted feature selection and fusion scheme for part based object detector....
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...Experimental results show that our method achieves the state-of-the-art performance....
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...This paper focuses on two basic problems: how to accurately describe object structure at feature level and how to fuse multiple Local Structured Descriptors for part based model at topology level....
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10,501 citations
"Boosted local structured HOG-LBP fo..." refers background in this paper
...During the past few years, many variants of HOG have been presented, such as Co-occurrence Histograms of Oriented Gradients(CoHOG) [26] in which the co-occurrence with various positional offsets is adopted to express complex shapes of object....
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...This paper focuses on two basic problems: how to accurately describe object structure at feature level and how to fuse multiple Local Structured Descriptors for part based model at topology level....
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...One representative feature is Histogram of Oriented Gradients (HOG) [1]....
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...Although existing part based models have achieved impressive progress in the past several years, their improvement is still limited by low-level feature representation....
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...Therefore, this paper mainly studies the description of object structure from both feature level and topology level....
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6,650 citations
"Boosted local structured HOG-LBP fo..." refers background or methods in this paper
...Experimental results show that our method achieves the state-of-the-art performance....
[...]
...This paper focuses on two basic problems: how to accurately describe object structure at feature level and how to fuse multiple Local Structured Descriptors for part based model at topology level....
[...]
6,598 citations
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Frequently Asked Questions (11)
Q2. How do the authors reduce the negative effect caused by the non-linear changes?
In order to reduce the possible negative effect caused by these non-linear changes, the authors clip the entry of uniform pattern with 0.2.
Q3. What is the purpose of this paper?
This paper focuses on two basic problems: how to accurately describe object structure at feature level and how to fuse multiple Local Structured Descriptors for part based model at topology level.
Q4. What is the purpose of the training of a part model?
In the stage of training object detector, the authors firstly train the root model using the learnt feature from the first stage, then initialize parts models from the root model.
Q5. Which version of latent SVM is used in other experiments?
The latent SVM from V4 is only adopted in the full experiments on PASCAL VOC datasets and latent SVM from V3 is used in other experiments.
Q6. What is the method for boosting multiple features?
3) Boosted multiple features fusion scheme for part based model stably improves the localization performance and performs best among these methods.
Q7. What is the way to truncate a pattern?
1) Truncation is helpful for robustness to nonlinear illumination changes; 2)Different truncation for uniform patterns and non-uniform patterns is reasonable.
Q8. How much is the maximum value of the entry of uniform pattern?
the entry of non-uniform pattern is often much larger than uniform patterns, so the authors limit its maximum value to 0.3 empirically.
Q9. How does the LSLBP score compare with traditional LBP?
LSLBP achieves 32.4% best AP score, with an improvement by 1.4% and 2.2% over LBP with trilinear interpolation and traditional LBP, respectively.
Q10. How many categories outperforms the proposed method?
The authors outperforms other methods except Oxford-MKL method(Oxford-MKL method adopted four types of multi-level features and achieved very competitive results on VOC2007 datasets) in 16 out of 20 categories.
Q11. What is the performance of the person and chair?
As shown in Figure 7, for the person category, the Boosting based method achieves the best results, by improving 4% over baseline V3, 3% over naı̈ve combination and 2.4% over MKL based method.