Unité / Unit : Génétique et Biologie du Développement – Institut Curie / UMR 3215 CNRS / U934 INSERM
Équipe / Group : Imagerie Cellulaire et Tissulaire (PICT@BDD, Paris) Chef d’équipe / Group leader: Olivier Renaud
Group members :
Olivier Renaud, Research Engineer - CNRS
Olivier Leroy, Engineer - Institut Curie (from 1st april 2009)
1. Introduction
1.1. Microscopy and developmental biology
Fluorescence microscopy imaging has become an indispensable method for research in biology and particularly in the field of developmental biology. Indeed, recent technological advances have enabled the development of microscopes with higher resolution and allowing fast three-dimensional image acquisition of living biological samples. Meanwhile, significant progress has been made in the development of light stable fluorescent probes, which has revolutionized the ability to track individual cells in vivo. Combining these technologies allows us to image cells, tissues and living embryos in a non-invasive way and without disrupting their development. Live Imaging techniques can enhance our understanding of biological processes during development and thus in diseases such as cancer.
1.2. History
The platform for Cell and Tissue Imaging within the Genetics and Developmental Biology Unit (UMR3215 CNRS / INSERM U934) is an optical microscopy and image analysis facility, located in a brand new building entitled « Pole for Cancer and Developmental Biology » on the campus of the Institut Curie in Paris. This facility is part of the Institute’s Platform for Cell and Tissue Imaging (PICT-IBiSA) which includes three other sites for optical microscopy: Lhomond (Paris), Pavillon Pasteur (Paris) and Orsay. The PICT has been given IBiSA status by the GIS « IBiSA » (french infrastruture in Agronomy Biology and Health) which coordinates the national policy of on Platforms and Infrastructures for the Life Sciences.
This facility was made possible thanks to the desire of Prof. D. Louvard, S. Artavanis-Tsakonas to make high-end microscopy technologies available to research groups of this new Unit which is devoted to research in developmental biology. The technological choices were initially made by Dr. E. Heard and Y. Bellaïche.
1.3. Missions
The imaging platform should fulfills the following tasks::
• provide technology and expertise in optical microscopy and image analysis, to researchers from the Institut Curie and external,
• train, assist and advise users, • take care of the systems we have,
• make technical and software developments, • participate in scientific projects,
2. Imaging facility of the Genetics and Developmental Biology Unit 2.1. Installation
Installation of the Imaging facility within the Developmental Biology and Cancer pole took place during 2009. The facility space covers more than 120 m2 spread over 4 levels. This installation took place in several phases:
- Work to prepare microscopy rooms : air conditioning, construction of individual boxes, connection to compressed air and CO2, installing safety devices (badge access, laser safety, security CO2), office development. This phase was conducted in close collaboration with the general and technical services of the Institut Curie.
- Order microscopes.
- Installation and testing of microscopes.
- Establishment of working procedures to operate the facility: online booking system, online application request for training, online tracking and reporting incidents, online facility tutorials (wiki), web site, safety procedures, connection to the image server of the Institute (managed by the IT department), establishment of a terms of use, integration into the platform of cellular imaging.
The management board is made up of Olivier Renaud (Manager, IR CNRS) and Olivier Leroy (IE, Institut Curie), who joined the team on 1st April 2009.
The installation of the facility within the Developmental Biology and Cancer building took several months, with an official opening on 1st September 2009. Since that date all members of the Institut Curie have access to the facility. It should be noted that some microscopes were made available to members of the unit of Genetics and Developmental Biology gradually from the end of 2008.
2.2. Procedures set-up
Managing facility requires the establishment of a number of tools and procedures for managing equipment and users.
We have used a number of tools that were developed by the PICT and also at the institutional level: the online equipment booking, the PICT Terms of Use and equipement pricing; we have also developed:
- The use of wiki pages & web pages describing the resources of the platform, - The creation of an online form to request training
- The creation of an online form for reporting incidents,
All of these improvements has been made in a spirit of quality control in order to meet future certification of our quality procedures.
Website Resource Platform
In a spirit of transparency with respect to potential users of the imaging facility, we have created wiki pages accessible to all staff of the Institute Curie. You can visit this wiki site at:
http://wiki.curie.fr/u934/Accueil
This site is available in English and French. It gathers all information necessary for use of our technology platform:
- How to Access the platform (eg, application training, application to use assisted rates, etc..)
- Equipment available and tutorials - Software for image analysis - Reminder of health and safety - Information on our activities - FAQs
- Links
This site is updated frequently by the imaging team.
In addition, information is also available on the institutional website and on the PICT-IBiSA website (http://pict-ibisa.curie.fr/).
Request form for training / assisted use
The training activity is an important part of service activities. It is therefore important to respond to users training requests as soon as possible and have a follow-up of conducted training. To this end, we created an online form to request training that is accessible from any computer connected to the intranet:
http://sondage.curie.fr/index.php?sid=56992&lang=en
Upon receipt of this request, we check with the user that his request is compatible with the available technologies. If so, an appointment will be scheduled with the user. The user and his team leader must also validate online acceptance of terms and conditions of use of imaging facility of Genetics and Developmental Biology Unit (PICT@BDD). For that team leaders receive a specific email inviting them to log onto a secure page. The terms and condition of use is very similar to that found on other PICT sites.
Users can request training to be autonomous to use a microscope (called "autonomy"), or requiring an assisted use in the case of a specific need or for preliminary tests.
Form of incident reports
We have also created an online form allowing users to report a problem would occur on a microscope (eg breakdown, system left in a poor condition, etc...). This form is available at:
http://sondage.curie.fr/index.php?sid=39856&lang=en
All of these procedures allows us to have a comprehensive monitoring of problems on the systems and interventions made by our suppliers.
3. Available equipement
A significant investment was made by the Institut Curie, as well as the CNRS, and other funding agencies such as FRM, INCA and ERC to equip the PICT@BDD with high-end microscopes. Indeed, more than 1.6 million were invested only in purchasing microscopes systems. This amount does not cover staff costs and equipment maintenance. Moreover, to remain at the forefront of technology, significant investments must be made in time.
3.1. Microscopy systems
Epifluorescence upright microscope (Leica / Universal Imaging)
This motorized microscope allows a routine observation of samples either using transmitted or epifluorescence light. With its highly sensitive CCD camera, this system is also capable of acquiring multi 3D images of fixed samples in fluorescence. The images must then be processed by deconvolution to eliminate image blur.
Upright microscope with epifluorescence Module ApoTome (Zeiss)
This motorized microscope allows three-dimensional observation of thick samples, multi-labeled with fluorescence probes. This system is equipped with a module "ApoTome" to generate optical slices of the sample. This module operates on structured illumination and uses a mathematical algorithm for reconstruction of data in real time.
Inverted videomicroscope with deconvolution (Delta Vision - Applied Precision)
Inverted microscope allows three-dimensional observation of multi-labeled live cell with high spatial and temporal resolution. This system is equipped with a dual excitation source (Xenon lamp) for fast image acquisition of dual labelling sample. Images acquired with this system are then deconvolved thereby improving considerably the image quality. This system is also equipped with an incubator to keep cells under physiological conditions (temperature and CO2). Inverted confocal microscope with rotating disc (Roper / Nikon)
Microscope dedicated for the observation of live samples. This system allows the rapid acquisition of multidimensional images (xyz, time, different wavelengths) with very good spatial resolution xyz. This system is equipped with a thermostatic chamber to keep samples under physiological conditions (temperature and CO2). This microscope has a CCD camera CoolSnap HQ2 and a laser bench with the lines 405, 491, 561 and 635nm.
Inverted confocal microscope disc rotary head FRAP (Roper / Nikon)
This microscope has the same technical characteristics as the previous system but is also equipped with a module to perform FRAP experiments photobleaching or photoactivation. The laser sources can be controlled with precision to expose the sample to high light intensity in time and space. The microscope is installed since December 2009.
Inverted laser scanning confocal microscope with spectral detection (LSM710 - Zeiss) Inverted microscope dedicated to the observation of specimens (live or fixed) multi-labelled and thick (tissue sections, explants, etc..). This complex system is equipped with a confocal head with spectral capabilities. Images are acquired with an excellent spatial resolution (xy theoretical resolution of 250nm and 500nm in z). Furthermore, the spectral capabilities of the device allow the separation of fluorophores with similar emission spectra. This system has a laser bench with lines 405, 458, 488, 514, 561, 594 and 633nm. This system is equipped with a température controlled chamber (temperature and CO2) to keep the samples under physiological conditions. Inverted laser scanning confocal microscope with spectral detection and multi-photon laser (Mai Tai LSM710NLO/Laser - Zeiss / Spectra Physics)
This microscope has the same technical characteristics as the previous system but is also equipped with a multi-photon powerful laser. This system is dedicated to the observation and ablation of sub-cellular structure in live samples with this additional laser.
3.2. Processing and image analysis software Imaris (Bitplane)
Imaris software allows real time visualization of sets of images acquired with different microscopes. This software allows visual evaluation of 3D and 4D datasets. Imaris has a very good rendering quality, speed of reconstruction and interactivity with the data. This software also provides efficient tools for segmentation to separate and visualize individual objects to quantify.
Zen2009 (Zeiss)
Zen2009 software tallows to view and analyze data acquired with the Zeiss confocal system. Metamorph (Universal Imaging)
Metamorph is a complete processing and image analysis software. ImageJ (NIH)
ImageJ is a free complete processing and image analysis software. This software allows users to create custom macros or add new features to the software through the sharing of applications developed by the Java community.
MATLAB (MathWorks)
The software MATLAB is both a programming language and development environment. This software allows to perform numerical calculations driven and is used in the construction stages of projects.
Softworx (Applied Precision)
Software for processing and analyzing images acquired on the Delta Vision microscope. This software allows especially the deconvolution of data acquired on the microscope.
Denoising software (J. Boulanger, Institut Curie/INRIA & J. Sedat, UCSF)
This software allows to denoise images. It provides good contrast in areas of high frequencies while it removes noise of homogenous areas. We are using this software on low contrast images coming from the group of E. Heard. The algorithm we used has been optimized by the group of J. Sedat (UCSF).
4. Usage statistics
In 2009, the imaging facility has been used by 68 users, which on average have been trained over two microscopes. We count 146 autonomous users means they are able to use a given microscope without external supervision.
We received 146 applications for training, 2 requests for additional training and 3 applications for assisted use. This represents more than 450 hours of training. Requests for assisted training relate only to formal requests made by users. Indeed, this figure does not represent the daily activity of user support by the team of the platform (advice, help with settings, etc..). An average of 10 days elapses between the demand for training by the user and effective training.
Over 75% of training requests (see Fig. 1) were carried out by the Unit of Genetics and Developmental Biology (UMR3215 / U934). This figure is not surprising since we are part of this unit and the platform is located in the same building. We have also received requests for
training from six other units whose UMR144-Compartmentation and cellular dynamics (7%) and UMR168 - Physical chemistry Curie (6%).
Fig. 1: Distribution of training requests per unit (2009)
Regarding the distribution of training requests per team (see Fig. 2) the largest number of request come from the teams of E. Heard (27% of total applications), Y. Bellaïche (29.2%) and JR Huynh (15.3%) for Unit UMR3215, and from the team of M. Piel (4.4%) for other units.
Fig. 2: Distribution of training applications per research group (2009).
Applications for training also vary depending on the type of microscope. For example, the laser scanning confocal microscope with 2-photon source is a specific system allowing laser ablation experiments. On this system 45% of the autonomous users come from teams outside our unit (see Fig. 3).
Fig. 3: Distribution of the number of autonomous users on the confocal laser scanning microscope with 2-photon laser (LSM710 NLO) in 2009.
The number of requests for training from Units other than the UMR3215 has increased since the platform opened last September (see Fig. 4). Indeed, the vast majority of requests from the UMR3215 were dealt with in the first part of 2009.
Fig. 4: Evolution of training requests between September and December 2009.
The time-use of the microscopes is high because we have over 10,300 hours of use from March to December 2009 (for 6 microscopes). For comparison, use of 9 hours daily for 218 days per year represents 11,772 hours. The average use per month is 1030 hours. It may be noted that the activity was sharply reduced in June because of work in the microscopy main room. Microscopes LSM710NLO confocal, spinning disk confocal and Delta Vision were not available for 1 month. These systems are the most used in the platform.
The analysis of the distribution of the number of hours used per system (see Fig. 5) shows that the spinning disk confocal microscope is the most used sytem, then the confocal LSM710, then the Delta Vision Core, then the ApoTome microscope and finally the widefield microscope. The number of hours used per system depends heavily on the type of technology of the microscope. For example, the spinning disk confocal microscope is used for the acquisition of live experiments (lasting several hours). The average time of a session is 8 hours. On the other hand the widefield Leica microscope is mainly used for observations on fixed samples and therefore the average duration of a session on this system is 2 hours (the user must be present during the acquisition).
Fig. 5: Distribution of the number of hours used per system for 2009.
The confocal microscopes (either spinning disk or laser scanning), are under great demand from users. To avoid any overload of these microscopes and allow more users to have access to these technologies, we have doubled the spinning disk confocal microscope. The second system was installed in December 2009. We also bought another laser scanning microscope using funding from CNRS and Institut Curie. This microscope will be installed during 2010. This system is also equipped with a UV laser to produce DNA damage.
Furthermore, since November 2009 we established a system to track incidents that occur on the microscopes. Incidents are classified into different categories according to whether the incident involves a partial or complete stop of the device. We have recorded 36 incidents (see Fig. 6) between November and December 2009 with 2 crashes and 14 partial failures. Other incidents correspond to misuse, unexplained crashes, suggestions, maintenance.
Fig. 6: Number of incidents per system from November to December 2009.
There is a higher number of incidents on the most used and more complex (confocal scanning with multi-photon source) microscopes. In 2009, all systems were still under warranty.
5. Other activities
5.1. Software development Adaptive filtering
We have developed a tool based on the local variance of the image to enhance the contrast in order to facilitate the segmentation of cells. Initially designed to address problems encountered by the team of Y. Bellaïche, the algorithm can also be used for other types of applications.
5.2. Practical workshops, demonstrations and tests
The platform of the imaging unit of Genetics and Developmental Biology regularly organizes practical workshops in partnership with suppliers of materials. These workshops allow users to have demonstrations and to test microscopes that are not available at the Institut Curie. In general, the workshop is organized around a theoretical presentation made by the supplier, a demonstration of the device and then tested in real conditions with users samples. The systems are often available for several days after the demonstration to allow scientists / engineers to conduct more comprehensive testing. Five practical workshops were organized in 2009:
« The spinning disk microscopy by Zeiss », organised by PICT@BDD & Zeiss company, 16 June to 13 July 2009.
« New Olympus microscope: the FV10i and FSX100», organised by PICT@BDD & Olympus France company, 3 July 2009.
« 3D & 4D reconstruction software by Volocity», organised by PICT@BDD & Perkin Elmer company, 16 September to 6 October 2009.
« Digital virtual microscopy », organized by PICT@BDD & Hamamatsu company, 5 to 9 October 2009.
« System Incucyte : microscopy under controlled atmosphere », organized by PICT@BDD & Essen Instruments company, 28 October to 16 November 2009.
We also tested from 8 - 12 June 2009 the confocal macroscope from Nikon (Nikon Imaging Center) with four teams (Teams Heard, Bourc'his, Bellaïche and Huynh), as well as the SPIM (Single Plane Illumination Microscope) and DSLM (Digital Scanned Laser Light Sheet
Red : Substraction of the 2 images (effect of the filter) Green : Result
Junctions (high variance) are not changed, only the center of cells (low variance) is smoothed making segmentation easier.
After treatment Before treatment
Microscope) microscopes on 14 & 15 October 2009. This evaluation was carried out with the team of Y. Bellaïche (Institut Curie / UMR 3215 CNRS / Inserm U934 - Genetics and Developmental Biology, Polarity, division and morphogenesis) at the EMBL (European Mocular Biology Laboratory, Heidelberg, Germany) in the laboratory of Dr. Stelzer.
5.3. The activity of training and education
The training and education are important activities of our service. Indeed, one of our main tasks is to transfer knowledge in the field of microscopy and image processing and analysis.
To be autonomous on a microscope, users have to follow a training program that involves four parts:
- Training which includes theoretical description of the system and recall the basics of microscopy,
- A theoretical safety regulations training specific to our platform and in particular the lasers risks and procedures for evacuations,
- Practical training including, microscope start and stop procedures, acquisition of 2D to 4D images if necessary, data backup, data transfer, cleaning procedures, procedure to book a microscope.
- Individual validation of knowledge. The user is supervised during his first microscopy session.
This training course has a minimum duration of 2 hours, and the period may vary depending on the system and user knowledge. Training can be done individually or in small group of 3-4 persons. The training validation is done individually.
The PICT@BDD team is also involved in the organization of internal training sessions at the Institut Curie, such as "Training Image J" held from 7 to 9 December 2009 at the CNRS in Gif / Yvette . We also have a teaching activities at the University of Paris VI and VII. All lessons / training / praticals undertaken during the year 2009 is described below:
« Atelier de Microscopie Confocale », Continuing Education University Paris VII, 10 & 13 March,
2009, Organized by Dr. El Nemer & Prof. Le Van Kim (University of Paris VII and Institut National de Transfusion Sanguine), Course « Etude des interactions protéines-protéines par microscopie optique », Practicals « Les bases de la microscopie confocale ».
« Visite des laboratoires de l’unité de Biologie du Développement par une classe de l’école maternelle de la Rue de Turenne, Paris», 17 March 2009, Organized by Dr. Bellaïche,
Practicals « microscopie optique ».
« Les nouvelles technologies en microscopie photonique », University Paris VI, April 3, 2009,
organized by the Network for Cell Imaging (University Paris VI), Course « Tour d'horizon des techniques en développement ».
« Intervention orale », Club Français de Microscopie, 14 April 2009, Organised by Prof. Ingrand,
Course « Les nouvelles technologies en microscopie photonique ».
« Licence professionnelle en Biophotonique », University Paris VII, 2-3 June 2008, Resp.
Charlotte Py, Course « La microscopie confocale » and Practicals « Vidéomicroscopies & microscopie confocale ».
« Action thématique C: Imagerie fonctionnelle du noyau », CNRS GdR2588, 30 June 2009,
Pacticals « L’utilisation de la microscopie multi-photonique pour induire des dommages à l’ADN».
« Atelier de Microscopie Confocale », Continuing Education University Paris VII, 10 & 13 March,
2009, Organized by Dr. El Nemer & Prof. Le Van Kim (University of Paris VII and Institut National de Transfusion Sanguine), Course « Etude des interactions protéines-protéines par microscopie optique », Practicals « Les bases de la microscopie confocale ».
« Module Ecole Doctorale », Ecole doctorale GC2ID, 25 & 26 November 2009, Organised by
Prof. Le Van Kim (University Paris VII and Institut National de Transfusion Sanguine), Course « Etude des interactions protéines-protéines par microscopie optique », Practicals « Les bases de la microscopie confocale ».
« Formation Image J », Institut Curie, 7-9 December 2009, Organized by Dr. Cordelières, Le
Baccon et Renaud (Institut Curie / CNRS), Course and practicals « Traitement et analyses d’images avec Image J ».
5.4. Collaborations
J. Sedat, University of California, San Francisco, USA & E. Heard, Institut Curie / UMR 3215 CNRS / Inserm U934 - Genetics and Developmental Biology, Epigenetics and development of mammals, Institut Curie - "Development of high-resolution optical microscopy tools to study the organization of chromosomes within nuclei".
Y. Bellaïche & F. Graner, Institut Curie / UMR 3215 CNRS / Inserm U934 - Genetics and Developmental Biology, Polarity, Division and Morphogenesis, Institute Curie - "Development of new tools for imaging micro-rotation of pupae of Drosophila".
J. Sedat, University of California, San Francisco, USA & J. Boulanger, Johann Radon Institute for Computational and Applied Mathematics, Austria - "An adaptive statistical method for 4D-fluorescence image sequence denoising with spatio-temporal discontinuities preserving".
T. Boudier (IFR83, UPMC), F. Chiappini (UPMC), F. Ropes (Institut Curie), A. Dauphin (CHU Pitie-Salpetriere), C. Klein (INSERM IFR58) & O. Leroy (Institut Curie) - "Made of imaging professionals from different institutions, this new working group create an exciting thinking and scientific cooperation. The main task of this working group is to develop and disseminate tools and 3D methods for image analysis using ImageJ software.
W. El Nemer, UMRS 665 "Proteins of the erythrocyte membrane and Non-Eryhtroïdes Homologs", Institut National de Transfusion Sanguine, Paris, France - "Interaction of Lu / BCAM glycoproteins with the membrane skeleton negatively modulates cell adhesion to laminin a5: Role of the increased adhesion properties of heriditary spherocytosis red cells".
K. Marin, CNRS URA2578 "Macrophages and immunity development", Institut Pasteur, Paris, France - "Confocal microscopy study of hematopoiesis in the fish danio rerio".
5.5. Future projects
The imaging platform of the Unit of Genetics and Developmental Biology works closely with research teams of the unit, to offer dedicated tools for cellular imaging in the development of living organisms.The challenges of optical microscopy in the context of developmental biology require to increase optical resolution to see with greater precision events at sub-cellular level but also to monitor biological processes with good accuracy at the level of a whole organisms (like embryos).
High-resolution microscopy
The resolution of optical microscopy remains a limiting factor for the precise observation of subcellular structures, such as nuclear proteins. Indeed, the lateral resolution of a standard confocal microscope is about 250 nanometers (0.000000250 meters), the size of a small bacterium (eg Mycoplasma). But the structures we want to see are often inferior to that size (eg, proteins, microtubules, etc..). Substantial progress has now been made through the joint efforts of international laboratories for physics, optics, chemistry and biology. The physical limits of optical field are being outdated by the development of new technological approaches and intensive computer processing of images. These technologies can be grouped into three categories:
- Structured illumination (eg OMX microscope from Applied Precision, SIMs from Zeiss, etc..): the sample is illuminated using a specific template to reveal the interference of the sample with light source (moiré effect),
- STED microscopy ("Stimulation Emission Depletion Microscopy") uses physical characteristics of fluorescent probes to reduce the size of the impulse response of optical microscope,
- PALM microscopy (“Photo-Activated Localization Microscopy”) / STORM ( "Stochastic Optical Reconstruction Microscopy”) enables activation of a small fraction of fluorescent molecules in the sample in order to locate them individually and accurately. This process is repeated many times until a complete and accurate map of the location of all fluorescent molecules is generated.
These techniques are now central for optical resolution improvement and offer the possibility to achieve a finer level of observation. We plan to purchase a high resolution microscope for observing biological processes during development. Currently only the STED and the structured illumination microscopy are able to provide higher resolution in a three-dimensional space. However, it should be noted that these technologies are evolving rapidly both in academic laboratories and manufacturers, and new solutions can emerge quickly. We will therefore in the year 2010 assess the various technologies available in order to choose the best system for our applications.
Whole sample microscopy
Current microscopy methodologies do not allow scientists to image a small entire living organism. The team of Dr. Stelzer at EMBL (European Molecular Biology Lab) has developed a new microscope (SPIM "Single Plane Illumination" and DSLM "Digital Scanned Laser Light Sheet Microscope") capable of imaging small sample sizes (2-3mm), while increasing the spatial resolution and reducing the phototoxicity associated with the excitation of fluorophores during acquisition. We evaluated the system DSLM at EMBL and we believe that this technology would enable new experimental approaches in particular for the study of morphogenetic movements during development of Drosophila and the fish Danio rerio. We will develop collaborations in this direction.
HIV-1 Nef inhibits ruffles, induces filopodia and modulates migration of infected lymphocytes. Nobile C, Rudnicka D, Hasan M, Aulner N, Porrot F, Machu C, Renaud O, Prévost MC, Hivroz C, Schwartz O, Sol-Foulon N. J Virol. 2009 Dec 16. [Epub ahead of print]
Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells. Le Saux B, Chalmond B, Yu Y, Trouvé A, Renaud O, Shorte SL. J Microsc. 2009 Mar;233(3):404-16.
Electron-multiplying charge-coupled detector-based bioluminescence recording of single-cell Ca2+. Rogers KL, Martin JR, Renaud O, Karplus E, Nicola MA, Nguyen M, Picaud S, Shorte SL, Brûlet P. J Biomed Opt. 2008 May-Jun;13(3):031211.
A Bayesian reconstruction method for micro-rotation imaging in light microscopy. Laksameethanasan D, Brandt SS, Engelhardt P, Renaud O, Shorte SL. Microsc Res Tech. 2008 Feb;71(2):158-67.
High-resolution 3-D imaging of living cells in suspension using confocal axial tomography. Renaud O, Viña J, Yu Y, Machu C, Trouvé A, Van der Voort H, Chalmond B, Shorte SL. Biotechnol J. 2008 Jan;3(1):53-62.