Histolab 5.0 GB - Fisher UK Extranet
Contents
1. Fichier Edition Image Mesure R sultats Pr sentation Curtace totale v Je Longueur Largeur Surface Emission champ Structure Oblet ium um um 136 25 Objets 415 55 3 47 2 19351 6181 25 Objets 416 214 13 7 3138 86 56 25 Objets 417 24 0 20 5 3661 78 83 25 Objets 418 11 4 11 4 87 2 84 63 25 oObets 419 47 3 430 15690 6366 25 Objets 420 433 243 819 4 84 16 NN ET 275 204 4184 mr 26 Objets 422 178 17 8 226 6 85 46 26 Objets 423 24 0 20 5 3661 76 32 26 Objets 424 41 0 22 3 679 3 73 10 26 Objets 425 45 4 356 12727 aon 26 Objets 426 178 17 8 225 5 5410 26 Objets 427 245 17 7 338 6251 26 Objets 426 153 3 103 7 132436 51 92 26 Obits 423 378 277 7645 7342 26 Objets 430 335 235 592 7 81 95 2 Ohets 41 253 136 3035 6342 26 Objets 432 30 4 23 2 662 5 51 40 notes nf 755 300 15515 65 99 27 Objets 434 72 1 352 14236 85 50 27 Objets 435 785 12 20591 01 68 a Objets 436 338 28 6 7322 8370 node Objete 437 335 28 6799 81 56 37 Objsts 438 588 323 14296 7930 2s Objs 439 494 317 12029 82 55 Ea Objete 440 125 42 349 8540 HR obeas am 154 6 66 2 74700 81 17 Loin a 275 233 4707 7541 ee DD DEEE ee 42 4 2 17A 84 31 25 GObss aaa s45 73 14819 8247 BS Soin ne 178 178 2266 BF 12 Cilia ii 1 1 C QT IL Si 4 Histolab manuel d utilisation page 33 43 The displayed measurements can be select2. item in the Results menu The first tab proposes you three types of headers e a standard header not requiring to be set which automatically includes the name of the software followed by the study reference e a customisable header for which you can define the height the title and a logo that you will have already placed in the clipboard e a blank header for printing the report onto pre printed paper You then only set the height of the space to be left blank The footer may include the software reference and page numbering or a given height may be left blank for use with pre printed paper Propri t s du rapport et Rice sta Miseenpage Documents imprim s En t te Personnalis Hauteur 1 5 cm Ele Titre Rapport de contr lel p M xa Pied de page Standard ka o 2 0 cm le EE ES Definition of the header and footer The second tab is used for defining the contents of the report Histolab manuel d utilisation page 41 43 Propri t s du rapport Domumet Mrt j Infos tude Vue d ensemble Graphique Valeurs histo Comptage Statistiques Mesures ns cpl aie eme les el Caract ristiques de l tude Coupe Histogramme Statistiques Table de mesures The list shown allows you to choose the documents to be included in the report and to determine the order and title The actual print out is launched via the Print report item in the File
3. o e o 7 VIDEO o PHASE CONTOURS F7 Mesurer les objets inf rieurs ane zz Rs NE Annuler You should indicate a min and or max size for the detected objects The effects of the sequence on the image can be visualized by placing the switch on the right in the Phase or Outlines position Histolab manuel d utilisation page 20 43 Definition of scanning When the system is equipped with a motorized stage the sample analysis is completely automatic The form and the size of the sample must then be defined The upper part of the dialog box shown above Set scanning used for this item in the Measure menu is D finition de l exploration r Echantillon Fectangulaire Longueur 67 4 mm ee 44 9 mm 42 champs Circulaire Diametre 67 40 mm Exploration f Horizontale Verticale En spirale M Inclure les champs p riph riques F Parcours al atoire Choisir champs parmi 42 Disposition des champs fe Champs jointits Pas horizontal i 0 61 mm Pas vertical 7 333 mm Annuler The zone thus defined can be scanned in several different ways with the options in the two other parts You can for example define exhaustive scanning of the sample check the Include the peripheral fields and Joining fields boxes or prefer a more rapid analysis by defining the disjoint fields or by letting the system chose fields at random The z
4. A particular measurement length width diameter perimeter surface or intensity can be Histolab manuel d utilisation page 37 43 analysed individually with a histogram you must define size classes for the considered measurement and choose which structure falls within the scope of the histogram The histogram is presented in the form of a bar graph the number of objects in each class can also be displayed Histogramme toutes structures Valeurs de l histo nee Longueur Hm Histogram of the lengths of all structures combined and the corresponding values It is possible to display the normal law corresponding to the distribution of the measurement in the defined classes by checking the corresponding option in the lower part of this window Other plotting options colour of the bars and the normal law graph gridline curve thickness can also be chosen under the histogram The choice of the structure that of the measurement as well as limits of size classes are defined by clicking on this button or via the Histogram settings button in the Measure menu The following dialog box appears Propri t s de l histogramme ae xl Structures Toutes les structures do EH Axe horizontal Longueur T Minin 0 000 mm Auto W Wlasimurm 0 000 mm Auto M Intervalle o 000 mm Auto M Axe vertical Hombre SR ASET Minimum jo uo M Dre nnuler Maximum jo Auo M SPREE ES B
5. active field the data of which is displayed in the Video page In order to change the active field use the Previous field and Next field options in the Measure menu or click on the desired field while pressing simultaneously on the lt Shift gt and lt Ctrl gt keys on the keyboard The overview can be displayed in a separate window and thus remain visible even when you are working on the other pages of Histolab for that you use the Display in window option in the Results menu or the corresponding keyboard shortcut the lt F12 gt key Yue d ensemble Display of the overview in a palette Histolab manuel d utilisation page 31 43 Exportation e Transformation of the overview into a single image activate the Frame tool in the Edit menu and then frame the required zone The Copy option allows you to transfer the framed part to another software application e Saving the measured images individually in the File menu use the Export results option then the Measured images sub menu Histolab manuel d utilisation page 32 43 6 Measurements table While the fields are being analysed Histolab creates a table that groups together the values measured Each object occupies a line and is described by the analysis field number the name of the structure to which it belongs and by the result of the measurements taken The table is displayed by selecting the corresponding tab jl Histolab
6. menu Histolab manuel d utilisation page 42 43 9 Secure operating modes For the use of Histolab on slides with very constant shades it may be interesting to freeze the settings to prevent any accidental changes Histolab provides the following three operating modes e The Administration mode is the normal working mode in which all software functions can be accessed e The Operations mode is a secure mode in which all routine operations are possible However the settings are no longer accessible they remain identical to those made in the Administration mode e the Supervision is a secure mode for which the conditions of access to the software s functions are defined by a distinct software component specifically developed for certain companies 1 S curit active b w Administration men _ Param tres de s curit Op rations O Supervision The operating modes shown in the menu The Security settings item allows the administrator to define the software operating mode and to protect access to the Administration mode with a password Histolab manuel d utilisation page 43 43
7. selecting the structure There are three methods for selecting the structure e In the Measure menu the Structures item opens a sub menu containing the names of the defined structures you just have to select the required structure which is then checked e The name of the structure currently selected is displayed in the toolbar This is a pull down menu clicking on it displays the other structures e During the measurements there is a quick means of switching from one structure to the next the Next structure item in the Measure menu and in particular the corresponding keyboard shortcut the lt F4 gt key A colour used for plotting the readings is associated to each structure The colour appears in particular in the toolbar and in the pull down menu In order to change the colour of a structure you should use the Drawings colors item in the Layout menu Using the measurement tools Manual or automatic measurement tools are provided depending on the type of structures chosen The manual tools can be used directly whereas the automatic tools must be at least set at the start of a section analysis e The Tools item in the Measure menu opens a sub menu that contains the tools list The selected tool is checked e If the toolbar is displayed each tool is represented by a button The button corresponding to the selected tool appears depressed sometimes none of the buttons may be depressed at the start of a study Mo
8. contents Using the measurement tools Measuring the intensity Checking the measurements Correcting the errors Validating a measured field Automatic sample measuring with a motorized stage 5 Overview of the section Exportation 6 Measurements table Exportation 7 Statistics and Graphs The statistics The counting table The histogram of a measurement Exportation 8 Page layout and Report print out 9 Secure operating modes Histolab manuel d utilisation 25 27 27 28 28 29 30 32 33 35 36 37 37 37 39 43 page 3 43 1 Overview of Histolab Histolab has been devised in order to take several kinds of measurements on various types of histological tissue It thus provides rich functionalities for processing images printing images and numerous interactive and powerful measurement tools We have taken particular care with regards to simplicity and comfort of use and we wanted the documents produced to be usable straight away annotation of the images print out of the images and the results storage in files that can be manipulated with other applications Histolab provides for the accumulation of measurements in an unlimited number of fields It allows the simultaneous analysis of several types of structures Statistics concerning each type of structure are provided at all times The measurements provided are as follows X axis and Y axis of the centre length width perimeter surface and light int
9. is possible to individually modify the upper and lower levels for each of the red green and blue channels by clicking on one of the coloured bars until the threshold reaches the desired value Setting adaptative thresholding This method is set with the following dialog box Seuillage adaptatif Objets sombres 2 a a 53 e eerie eee VIDEO PHASE CONTOURS Taille 5 ae Delta 20 Annuler TA Setting adaptative thresholding This dialog box indicates e the general shade of the objects e a size parameter that corresponds to the radius in pixels of the largest objects to be detected e the minimum shade variation to be taken into account between the objects and the image background As in the previous case the result of the thresholding operation appears if you click on the Phase button superimposition of a mask on the thresholded parts of the image or on the Outlines button the thresholded parts are bordered Setting smoothing The smoothing dialog box is presented as follows R glage du lissage VIDEO Pie T CONTOURS Annuler Ok Taille du lissage Setting smoothing Histolab manuel d utilisation page 18 43 If small insignificant objects interfere with the measurement increase the smoothing if various elements of a structure are assimilated reduce the smoothing The result of the thresholding operation appears if yo
10. only box Histolab manuel d utilisation page 22 43 4 The slide analysis The characteristics of the study have been defined the system calibration has been checked the image of the section s is correct on the screen everything is ready for the measurements They are taken field by field and then in principle structure by structure Pre viewing the slide with a motorized stage If the system is equipped with a motorized displacement stage it is possible to pre view the slide before the actual analysis in order to locate the areas of interest in the overview In order to do this e Choose an objective with weak magnification on the microscope and select the corresponding calibration in the list of calibrations given in the Measurement menu and then e Position the scanning area around the tissue section to be analysed by moving and redimensioning its representation in the Overview page and e Select the Acquire preview images item in the Measure menu The system then produces an image of the entire scanning area e Return to the desired objective for the analysis and select the corresponding calibration in the list Plotting a reference area If when defining the study a structure has been defined as the reference area it is generally Histolab manuel d utilisation page 23 43 plotted before any other measurement Select this structure in the local menu in the toolbar or in the list of Structures
11. stage 3 Name of the structure being measured This is a local menu click on it to display the list of the defined structures and it allows you to change the structure 4 Setting the detection of the selected structure 5 Switching from field to field With a motorized stage all of the buttons are active The displacement buttons pilot the stage the button on the left allows you to define an initial position With an indexed displacement stage the buttons are inactive moving the stage changes the field With a manual system the field displacement buttons are active and allow you to display the measurements already taken 6 Measurement of a structure Measurement of all of the structures Measurement of the sample motorized systems 7 Interactive measurement tools from left to right selection of objects measurement in a manually plotted area counting plotting a contour plotting a segment measurement of the reference light intensity The tools are validated according to the type of structure selected Histolab manuel d utilisation page 7 43 8 Setting the properties for the displayed documents measurements displayed in the tables definition of the graph report page layout The tabs pages and documents The tabs located on the left of the window allow you to display five pages that are representative of the current study e The real time video image is of course the most important page as it allows yo
12. system is likely to miss the correct position reduce the analysis pitch Histolab manuel d utilisation page 13 43 3 Preparing the study Defining the study During this first stage the user defines the structures that he wishes to analyse and documents the study that is starting reference of the section name of the operator comment s This is done using the New study item in the File menu Histolab manuel d utilisation page 14 43 Nouvelle tude ne Structures Intitul Figures one de mesure Membrane Za Suraces E l Mitochondries Ga Obits MS Membrane w Epaisseur IJI Lignes gt I Nouaux Points fl et Membrane v Ajouter if tL Supprimer fafa aun R f rence Lame F468 PG i D p rateur EL Notes Coloration 1710 Analyse de l chantillon Choisir les champs manuellement a Suivre les positions des platines fe Suivre une exploration pr d finie Annuler M Conserver les images mesur es The structures are used for coherently grouping together the measurements especially for the statistical calculations The name of each structure is indicated in the left column It can be modified once it has been partially or totally selected Each structure will contain a certain number of figures whose type determines the measurements taken This is selected in the local menu situated in the centre of the list which features
13. Champ 23 Hm Hrm Hi l E Champ 30 Nombre 2a oe 445 bas 445 445 Champ 31 Champ 32 Total Histogramme toutes structures Moyenne Int de Confiance Longueur um 0 2 4 somr2002 1420 if SE CTY x4 Histolab manuel d utilisation page 36 43 The statistics The statistics table contains the measurements taken for each structure It may remain visible at all times if the Statistics item of the Display in window sub menu in the Results menu is checked The counting table The counting table displays field by field the results of the counts for each structure during a study Statistics for these counts are displayed at the end of the table This table is updated during the analysis of a tissue section It may also be displayed at all times if the Statistics item of the Display in window sub menu in the Results menu is checked Table de comptage Champ Objets ur Champ 1 Champ 2 Champ 3 Champ 4 Champ 5 Champ 6 Champ 7 Champ 8 Champ 9 Champ 10 Champ 11 Champ 12 Champ 13 Champ 14 Champ 15 Champ 16 Champ 17 Champ 18 Champ 19 Champ 20 Champ 21 Champ 22 Champ 23 Champ 24 Champ 25 Champ 26 Champ 27 Champ 28 Champ 29 Champ 30 Champ 31 Champ 32 Total Moyenne Int de confiance The histogram of a measurement ti as j as bt 0 1 5 6 1 0 4 6 6 7 8 3 3 6 2 2 3 g 8 1 1 5 5 1 0 5 0 1
14. France Belgique Belgi Fisher t l 038867 1414 t l 056 260 260 Bioblock Selentifie fax 0388671168 fax 056260270 email infos bioblock fr email belgium bioblock com Parc d innovation BP 50111 F67403 illkirch cedex www bioblock com www bioblock be Mode d emploi Espa a tfno 9151592 34 fax 91 515 92 35 email ventas bioblock com www es fishersci com User Manual Histolab Analysis of histological tissue edition 8 Histolab 8 July 2003 Table of contents 1 Overview of Histolab 4 Starting up Histolab 4 The Histolab work sheet D The menus 6 The toolbar 7 The tabs pages and documents 8 The status bar 8 The structure of a work session 9 Preparing the equipment 9 Preparing the study 9 The slide analysis 10 Using the results 10 Protecting the settings 10 2 Preparing the equipment 11 Image acquisition 11 Access to the stage controller 12 System calibration 12 Setting the autofocus 12 3 Preparing the study 14 Defining the study 14 Setting the detection of structures 16 Setting fixed thresholding 17 Setting adaptative thresholding 18 Setting smoothing 18 Defining a macro 19 Choice of separation 20 Setting the criteria 20 Definition of scanning 20 Saving a study 21 Saved templates and files 22 4 The slide analysis 23 Pre viewing the slide with a motorized stage 23 Plotting a reference area 23 Repositioning a sample 24 Selecting the structure 25 Histolab manuel d utilisation page 2 43 Table of
15. HASE CONTOURS SP RE Dees ff Setting threshold levels for a colour image This box shows the shades present in the image depending on the type of image in shades of grey or three red green and blue channels the lower and upper detection thresholds currently defined for each of the red green and blue channels or if need be only in black and white under the histogram During automatic analysis of the video field lt F5 gt the points considered in the image are those with each of their red green and blue components inside the defined range calculates the mean value and the standard deviation for each of the components of the image points in the designated area and the lower and upper detection thresholds modulated by the defined tolerance click on this button and box the shade to be extracted in the video image Histolab then The result of the thresholding operation appears if you click on the Phase button superimposition of a mask on the thresholded parts of the image or on the Outlines button the thresholded parts are bordered Histolab manuel d utilisation page 1 7 43 If the detection carried out is not satisfactory there are several ways of adjusting it e If the field measurement has missed elements in the structure being analysed increase the tolerance if elements have been unduly allocated to the structure reduce the tolerance e If the setting is still not satisfactory it
16. an be displayed individually in separate windows included in the report or copied to an external software application e The Report is a true copy of the document that is likely to be printed It always follows the progress of the work in hand The report page layout can be personalised size and contents of the header and footer title and order of the printed documents Histolab manuel d utilisation page 8 43 The status bar The status bar is situated at the very bottom of the sheet It contains information relating to the work in hand 0 0 Oo Q 6 6 Q The Histolab status bar 1 System date System time Scale in which the video image or overview is displayed Name of the currently applied calibration Name of the image file if loaded oo Or OS amp N Reference of the sample being analysed or name of the study file in which the measurements are recorded 7 Position of the crosshairs when you click on one of the graduated rules When you use the framing tool the size of the selected area is displayed here 8 Indicator of memory occupied on the microcomputer The structure of a work session Preparing the equipment During this stage you essentially set e the acquisition and framing of the video image using the options in the Image menu you try to display the image on the screen with a sufficient contrast e the calibration of the system operation that consist in establishing the equivale
17. documents between several different software applications You can also cancel an action and access the image annotation module the Image menu is dedicated to video management acquisition storage and improvement of images the Measure menu contains the functions used during a study system calibration properties of the study choice of structures and measurement tools the Results menu concerns the documents presented on the Table Results and Report pages It also provides access to dialog boxes for defining the contents of these Histolab manuel d utilisation page 6 43 documents and items allowing you to display certain of them in windows that remain visible at all times e the Layout menu contains various more general options personalising the environment and options for plotting measurements e the menu information about the Histolab software and secure operating modes The functionalities provided in these menus are described in detail in the Reference Manual The toolbar The toolbar provides quick access to certain frequently used functions and information 100000 22 90900 OOOO O OOO ae ee E a 0 g The Histolab toolbar 1 Commonly used video functions from left to right Acquisition Freeze image Automatic contrast Image integration 2 Activation Deactivation of position tracking when the system is equipped with an indexed or motorized displacement
18. e points intended for carrying out counts e lines intended for measuring lengths and thicknesses e objects intended for carrying out individual geometric measurements on closed contours length width equivalent diameter perimeter surface and light intensity e areas intended for taking global measurements All of the figures can be plotted manually All of them except lines can be acquired automatically Histolab manuel d utilisation page 15 43 The structures taken can be restricted inside a reference area by using the local menu situated to the right to the list unless otherwise stated measurements are taken for the entire video field The local menu features the structures that can be considered as the reference area they are defined by a closed contour Setting the detection of structures displayed by pressing this button or via the Detection settings item in the Setting the detection of structures with automatic tools is presented in a palette that is Measure menu You first choose whether to apply automatic detection or not depending on whether the structure to be measured is in a homogeneous shade or not R glages de la d tection D tection de Marquage Trac manuel f D tection automatique R glages f Fixe couleur Seuillage Fixe monochrome Adaptatif F glages Filtrage if Lissage Macro S paratio
19. ed by pressing this button or by using the corresponding item in the Results menu Choix des mesures Abscisse Ordonn e Longueur Largeur Diam tre a P rim tre ae oe Bes ee Surface Annuler Emission EBEAEBHROERKOCO Selection of the displayed measurements It is also in this window that you can choose the precision of a measurement by clicking on the corresponding pull down menu In order to choose the intensity expressed display the following window via the Measure menu e Taux d absorption lumi re blanche FE mae aR Are Se Bu Taux d mission fluorescence Choose e Absolute intensity to display the mean value of the grey levels in an object e Absorption rate to analyse the intensity of marking on a histological section with transmission lighting white light e Emission rate to analyse the intensity of marking on a fluorescent histological section Histolab manuel d utilisation page 34 43 Exportation Exporting the results consists in formatting the results obtained by Histolab so that they can be used by external software applications in general spreadsheet programs Two techniques are available e via the clipboard select the block of cells When it is highlighted select the Copy option in the Edit menu Activate the destination software application and paste in the desired position To select entire lines click and drag in the lege
20. ensity The results are all expressed in real units either in a comprehensive table or summed up with statistical values Histolab provides the user with a whole range of measurement tools thus adapting to many kinds of histological sections Certain tools are automatic i e once they have been set they measure a structure on all or part of the field displayed in the video window Other tools are pure plotting tools particularly for counting measuring thicknesses or even for when the colorations of the section are not sufficiently homogeneous to be detected with shade criteria Finally everything is done so that the user can use the results provided by Histolab as smoothly as possible a work sheet can be saved and resumed later The results statistics and counts can be exported to common calculation applications either in compatible files or simply by using Copy Paste Finally images lists of values and statistics can be edited on a printer Histolab manuel d utilisation page 4 43 Starting up Histolab A Histolab shortcut is normally accessible in the Microvision program group situated in the Programs menu within the Windows Start menu The Histolab work sheet The work sheet is the window displayed when Histolab starts up It contains the standard elements of the Windows environment the title bar 1 which includes the system menu box on the left and the window enlargement reduction boxed on the right the me
21. h manual plotting tools and automatic measurement tools Chapter 5 presents the Overview page which displays the mapping of the section or the background history of the measurements taken Using the results This final stage consists in defining the presentation for the tables and graphs provided as well as the page layout for the measurement report These functions are described in Chapters 6 to 8 Protecting the settings The settings made during the first two stages are fundamental and determine the validity of the measurement results It is possible to restrict access to these settings by setting the software to a secure operating mode The secure operating modes are described in Chapter 9 Histolab manuel d utilisation page 10 43 2 Preparing the equipment Image acquisition The first stage consists in acquiring the image of the field to be measured It is stored in the image memory and a part of it is displayed on the work sheet Real time acquisition is activated using this button or the New image option This button automatically improves the image contrast It may be useful to freeze the image using this button or the Freeze image option Acquisition by integration is used when the light intensity is very weak the image is acquired with a special camera called an integration camera The image integration is set via the corresponding item in the Image menu E During a study image acquisi
22. in the Measure menu Once this is done select the free plotting tool by pressing the corresponding button in the toolbar or by selecting the Outlines item in the list of Tools in the Measure menu Then scan the sample and point at as many points as necessary in order to describe the section edge with the desired precision However the greater the number of points in the plotted profile the longer the analysis of each field will take as an inclusion test for each measurement within the profile must be carried out Repositioning a sample In order to replace a sample in a reference area display the overview select the Overview page or display the palette by pressing the lt F12 gt key or by using the Results menu Yue d ensemble Overview Locate a detail that is characteristic of the profile move the section until the detail is situated in the video field i e until the white rectangle representing the video field in the overview and the detail in question are superimposed then close the overview lt F12 gt again display the Video Histolab manuel d utilisation page 24 43 page and to stop controlling the profiles at the position of the microscope stage then move the stage until the located detail is superimposed on the corresponding tissue Press this button again to return to automatic position control CO press this button or select the Disengage stage item in the Measure menu in order
23. n f Aucune Rapide Standard Fine Crit re F glages E Aucun Surface The detection setting palette Structures in an image are detected in four stages e Thresholding which extracts the phase to be measured from the image There are two methods Fixed thresholding consists in manually setting the values of shades characterising the objects This method allows greater precision of detection but requires stable lighting and uniform preparation of the samples There are two versions that take account of either the grey levels in the image or the levels of each of the red green and blue components Histolab manuel d utilisation page 16 43 Adaptative thresholding allows you to detect the objects in fields where lighting is not homogeneous It is always carried out on an image converted into grey levels The detection takes a few seconds e Filtering which corrects imperfect thresholding that leaves artefacts or does not fully detect objects Filtering can be either a simple smoothing of the contours of the detected objects or a sequence of personalised processes e possibly a separation of the detected objects often useful for points sometimes for objects e the application of a criteria to the measured values this stage is also optional useful for objects Setting fixed thresholding It is set in the following dialog box R glages de la teinte 5 N YIDED P
24. nce between a real distance and the length that is visible on the screen The preparation of the equipment is presented in Chapter 2 The reference manual deals with many aspects of this in detail video acquisition in Chapter 4 calibration in Chapter 5 Preparing the study The preparation of the study consists in providing the software with the number and the names of the structures to be measured as well as some of the properties of the sample to be analysed Histolab manuel d utilisation page 9 43 and if the system is equipped with a motorized stage the form and the size of the area to be scanned Then the detection of each structure is set i e all of the image processes that will lead the equipment to detect the structure in the video image The programmed processes are summarised in a palette that can be displayed via the Measure menu These operations are described in Chapter 3 The slide analysis The analysis can be completely automatic if the system is equipped with a motorized stage and if the slide preparation is sufficiently homogeneous so that the settings made may apply to the entire sample from one end to the other Otherwise the analysis is semi automatic after having positioned the microscope stage so that the area of interest is displayed in the video image the operator must select the structure then he wants to analyse and then a measurement tool Chapter 4 is dedicated to taking measurements with bot
25. nd columns To select entire columns click and move in the heading lines To select the entire table click in the top left corner of the table e via a text format file in the File menu use the Export results sub menu then the Measurements item IMPORTANT NOTE Exportation must not be confused with saving a study A study file contains the measurements and their environment but can only be read by Histolab An exported file only contains the value but can be read by other software applications It is always preferable to save the study as you can then extract the values in order to export them whereas the reverse is not possible Histolab manuel d utilisation page 35 43 7 Statistics and Graphs Besides the measurements table presented above Histolab also provides four other documents in the Results page the statistics table the counting table a bar histogram and the numerical values associated with the graph i Histolab Fichier Edition Image Mesure R sultats Pr sentation 7 QE Suface totae ow o Eee YS Statistiques Longueur Largeur Surface Emission pm Hm HIT 6 Champ 20 Nombre de valeurs o 32 Champ 21 59700 Champ 22 Champ 23 Surface totale Valeur minimale Valeur maximale 7520000 Somme 1 49e 08 Champ 24 Valeur moyenne 4640000 Champ 25 Ecart type 2050000 Champ 26 Intervalle de confiance 711000 Champ 27 Champ 25 Longueur Largeur Surface Emission
26. nt stage these buttons allow you to move from one field to another Histolab manuel d utilisation page 28 43 Automatic sample measuring with a motorized stage Move the sample to the upper left corner of the rectangle to be analysed or the summit of the circle and press this button it defines the position of the first scanning field Click on this button automatic scanning will begin Histolab manuel d utilisation page 29 43 5 Overview of the section The overview is displayed when you select the Overview tab tl Histolab Histolab manuel d utilisation page 30 43 The overview presents a background history of the analysed fields The profiles corresponding to the measurements taken in each field for all of the defined structures are represented in a scaled down view the overview magnification is displayed in the status bar at the bottom of the window The images of the fields are also displayed if the Keep measured images option was activated when the study was started When the system is equipped with a stage that transmits its position to the microcomputer indexed or motorized stage the background history is replaced by a mapping of the section If the stage is motorized the preview if acquired is displayed here with the scanning area The latter can be moved and deformed with the mouse so that it can be adjusted to the area of interest on the tissue section The bordered field is the
27. nu bar 2 provides access to all of the software s functions When the sheet does not occupy the entire screen it can be moved by clicking on the title bar or deformed by clicking within the frame The toolbar 3 contains some buttons for quickly accessing the software s most used functions On the left of the sheet some tabs 4 allow you to display either the video image or various documents that are representative of the work in hand a mapping or a histogram of the fields measured the measurements table a graph and statistical tables the printed report The video image or the document occupies most of the sheet 5 They are bordered by rules 6 graduated in real units and by scroll bars 7 that are active when the document or image is larger than the window itself In the lower part of the work sheet the status bar 8 contains information relating to the work in progress Histolab manuel d utilisation page 5 43 ds 8 5008 1742 JC xi8s if SI CTY x12 i 10 tamesas pe if JAM I The Histolab work sheet The menus They provide access to all Histolab functions Here is a quick overview of them the File menu is dedicated to the studies start of a study and definition of its main properties saving the study exporting and printing the results It also enables you to archive load and print images the Edit menu provides classic Copy Paste functions that allow you to exchange images or
28. o express the light absorption rate for each of the detected objects for example for analysing the intensity BRDU marking on a histological section Before taking the measurements in this case you must provide the system with a light reference corresponding to 100 of transmitted light i e an absorption rate of 0 In order to do that press this button or select the Measure background intensity item in the Measure menu and click in an area of the image where all the light is transmitted e In fluorescence the light source is used for stimulating fluorescent markers in specific wavelengths It is then the emission rate of each of the fluorescent objects in comparison with the dark background intensity that is characteristic of the marking intensity Before taking the measurements in this case you must provide the system with a light reference corresponding to 0 of emitted light In order to do that press this button or select the Measure background intensity item in the Measure menu and click in an area of the image where nothing fluoresces Checking the measurements The last values measured can be displayed in a small window superimposed onto the work sheet Measurement results item in the Results menu Histolab manuel d utilisation page 27 43 Mesures de l objet 181 Champ 10 Nowau Results expressed Correcting fhe errors In the case of an error during plot
29. one to be analysed is represented in the Overview page The size and position of the zone can be adjusted with the mouse Saving a study While the measurements are being taken it is recommended to save the measured values on a regular basis The Save study item in the File menu is used for that purpose The first time this item displays the standard Windows dialog box in which you should indicate a directory and a file name to identify the study the suffix hlb is automatically added This Histolab manuel d utilisation page 21 43 name then appears in the Histolab toolbar The same file is used for the following recordings unless it is changed explicitly with the Save study as item Saved templates and files It may be useful to define study templates that conserve in particular the definition of the structures and the settings for the detection thereof In order to do this the information prepared in a file is saved using the Save study item In order to make use of this template e Open the file with the Open study item File menu this restores the defined structures e Enter the characteristics specific to the new study with the corresponding item in the Measure menu e Define a new file with the Save study as item in the File menu Note the template files can be protected from any accidental changes from within the Windows Explorer display the properties of the file and check the Read
30. st of the tools are used with the mouse by carried out a sequence of actions of the type lt press gt lt move gt lt release gt each action is used for reading a particular point either in the selected area in the video image or in the plotted figure The mouse buttons can be used indifferently No special keys on the keyboard are used with one exception the lt Shift gt lt F5 gt keys for automatic analysis of the entire video field and the lt F5 gt key for automatically detecting a structure in the entire video field Histolab manuel d utilisation page 25 43 The uses of each tool are presented below There is also a reminder of the measurements taken by each tool ae Double butterfly net automatic measurement of the video field e Measurements taken coordinates length width equivalent diameter perimeter surface e Principle successively applies the defining settings for each of the automatically detected structures to the video field Butterfly net shortcut lt F5 gt automatic measurement of the selected structure in the video field e Measurements taken coordinates length equivalent diameter perimeter surface e Principle applies the settings defined in the Detection settings box for the selected structure to the video field Wizard automatic measurement of an area in the video field Measurements taken coordinates length width equivalent diameter perimeter surface light intensit
31. tem you could make do with a routine inspection place a ruler or a micrometer under the camera and check that the graduations match those displayed by Histolab For more precisions refer to Chapter 5 of the Reference Manual Setting the autofocus If the observations are made with a microscope equipped with a motorized focusing axis three axis motorized system it is possible to carry out automatic focusing during the analyses This operation requires a setting that depends on the objective chosen The set autofocus item is available in the Measure menu It displays the following dialog box Histolab manuel d utilisation page 12 43 R glages de l autofocus t Activer l autofocus Pendant l exploration ajuster le focus tous les 2 champs Lors d un autofocus mesurer le contraste tous les 50 pm Essai Annuler Setting the autofocus In order to set the autofocus e enter in the second text box a distance with the same order of magnitude as the field depth of the objective used it defines the analysis pitch e click on the Test button the system moves the focusing axis and takes a measurement at each stage A curve is plotted in red and the system moves to the position that corresponds to the maximum level of this curve e if the curve is large and flattened the correct position is likely to be outwith the analysed zone increase the analysis pitch e if the curve is too narrow the
32. the objects that are larger than the indicated parameter while preserving the contours The list on the right is the sequence formed by some of these transformations Proceed in the following way to include a transformation e click in the left hand area on the desired transformation and then click on the button or simply double click in the left hand area on the desired transformation the transformation is Histolab manuel d utilisation page 19 43 then placed at the end of the list e click on the button marked with an arrow in order to move the transformation to the desired location e if required by the transformation indicate the size then validate with the lt Enter gt key The effects of the sequence on the image can be visualized by placing the switch on the right in the Phase or Outlines position Choice of separation Separation is useful for structures for which an object count has been carried out object count or measurement It allows you to consider to a certain extent objects that appear to be agglomerated as separate objects Three automatic separation methods are available e rapid for only separating large objects e standard for separating medium sized objects e fine for separating small objects Setting the criteria It is possible to terminate detection with classification according to one of the measurements taken using the following dialog box M Mesurer les objets sup rieurs
33. ting or automatic measuring there are several solutions e The last profile or the last measurement can be cancelled with the corresponding function in the Edit menu This tool allows you to select a profile The Delete item in the Edit menu deletes the selected profile If the profile contains handles it is possible to correct it by clicking on one of the handles and with the mouse button depressed bringing it to the desired location e A measurement of the section that is being analysed can be deleted by removing the corresponding line from the results table It is recommended to save the current study on a regular basis In the event of any wrong manipulations that are not seen at once and that can not be cancelled afterwards re opening the file with the corresponding item in the File menu still remains possible Validating a measured field Press this button or select the Next field item in the Measure menu to validate the measurements already taken and to analyse a new field Then move the section on its base until the field to be analysed is fully displayed in the video image This button allows you to return to a previously measured field Note these buttons are invalidated when the microscope is equipped with an indexed displacement stage connected to the computer you just have to move the stage to measure a new field or to return to a field already measured When it is equipped with a motorized displaceme
34. tion is repeated for each analysed field The techniques mentioned here are described in detail in Chapter 4 of the Reference Manual Histolab manuel d utilisation page 11 43 Access to the stage controller If an indexed or motorized displacement stage is installed it is controlled by an independent software application The necessary settings are set at the factory or during commissioning of the equipment This software can be displayed via the Show stage driver item in the gt menu the streaked button on the bottom right of the display provides access to the menu and to the material settings the red button on the bottom left closes the display as it is of no use during normal use of Histolab System calibration This stage is fundamental as it conditions the validity of the measurements that will be taken It is used more or less frequently depending on the optical system used e If you often modify the optical system magnification of an objective modification of the position of the camera on the bracket you have to recalibrate the system after each modification using the Define option in the Calibration sub menu of the Measure menu e If you have an optical system with a few pre defined positions objectives of the microscope for example you just have to activate the correct distance scale from those provided by the Calibration sub menu in the Measurement menu e Finally if you use a fixed optical sys
35. u click on the Phase button superimposition of a mask on the thresholded parts of the image or on the Contours button the thresholded parts are bordered Defining a macro A macro is a series of cleverly arranged image transformations it is defined in the following window Filtrage d image o Fermeture 3 Fermeture Ouverture Remplissage Ooo o Guy g od sique 4 Ferm g od sique JOuyv g od sique Remplssage Objets du bord Suppression bords Suppression 4 bords Taille maximum VIDEO 5 PHASE CONTOURS 7 Annuler Definition of a sequence The dialog box that appears shows on the left a list of the transformations that can be applied to the image Here is a brief description of the possible transformations e closing gap filling with contour smoothing e opening removal of the minor artefacts with contour smoothing e geodesic closing gap filling while preserving the contours e geodesic opening removal of the minor artefacts while preserving the contours e filling filling all of the holes whatever their size while preserving the contours e objects on the edge removal of the objects entirely contained in the field e removal of 2 edges removal of the objects intersecting the upper edge or the left edge of the field e removal of 4 edges removal of all of the objects intersecting one of the field edges e maximum size removal of
36. u to observe the sample and to take the measurements The video image and the possibly superimposed profiles can be saved or printed using the corresponding options in the File menu A part of the image can be boxed and then transferred to another software using the Copy option in the Edit menu e The Overview page presents a background history of the fields measured all of the objects plotted are shown superimposed on the image that is under the camera while the measurement is being taken When the system is equipped with an indexed or motorized displacement stage the background history is replaced by a mapping of the sample in which the images and measurements are plotted in their real positions The overview background history or mapping can also be displayed in a separate window and included in the printed report A part of the overview can be boxed copied and then transferred to another software application e The Table shows all of the objects plotted during the study one object per line and a certain number of measurements chosen by the operator for each one The measurements table can be inserted into the report A part of the table can be selected copied and transferred to a spreadsheet program e The Results page allows you to display four separate documents the statistical values calculated for each structure the counting table a distribution histogram as a graph and as a table These four documents c
37. y Operation click on both extremities of an area of interest This area is then automatically analysed according to the defined settings Free outlines e Measurements taken coordinates length width equivalent diameter perimeter surface e Profile click then move the mouse as many times as necessary in order to describe the form to be measured Double click to close the contour again and thus indicate the end of the measurement Histolab smooths the outline Counter e Measurements taken coordinates e Profile click on the location of the point to be marked Thickness e Measurements taken coordinates length Histolab manuel d utilisation page 26 43 e Profile click on both extremities of the segment to be plotted Measuring the intensity An additional measurement is taken for object structures the light intensity of each of the detected or plotted objects It is expressed in different ways depending on the selection made in the box that is displayed via the Displayed intensity expressed item in the Results menu Intensit exprim e Intensit absolue Taux d absorption lumi re blanche Taus d mission fluorescence e the absolute intensity is a value between O and 255 corresponding to the mean value of the grey levels in an object e For an image from a sample highlighted in white light transmission lighting it can be more interesting t
38. y checking the Auto boxes it is possible to allow Histolab to adjust each of the parameters as best it can Histolab manuel d utilisation page 38 43 Exportation e to presentation software via the clipboard click on the statistics title bar on the work sheet When the frame is highlighted select the Copy option from the Edit menu Activate the destination software application and paste in the desired position e to a spreadsheet program via a text format file in the File menu use the Export results sub menu then the counting or statistics item Histolab manuel d utilisation page 39 43 8 Page layout and Report print out The Report page displays the measurements report exactly as it will be printed fl Histolah ae a zy z Fichier Edition Image Mesure R sultats Pr sentation 8 8 20 a Oo za Surface totale v 5 10 aa 20 25 30 35 40 Histofab k Histolab Y Histo Dsrseririsdguss as Jubruss 5 10 15 20 Toul 26 ae Fe acy ted ated b 00D D LOr TED be A eed ere 110 Redo 5 00 0 Or TED by MOTO TION bel urr 210 tend iets k o 4 A x a Cazi wa jf soe jf Machet 2 5x VE 2A RIT 7 Histolab manuel d utilisation page 40 43 The page layout of this document is entirely customisable the size and contents of the header and footer the order and title of the printed documents You access it by pressing this button or via the Report settings
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