ng_biopro-user-manual
Contents
1. 4 Sensor Ox8f4 Sensor Ox8f5 E Reference 0 Reference 0 e NEH E EL gi yyy o yes al x On trigger rel 8 00 On trigger rel 8 00 E Offtrigger rel 2 00 Offtrigger rel 2 00 Sensors and inputs Os 1white Us 1white Bi eeeece X Sensor 0x8ed normal output normal output P raranca O Shutdown flag On trigger rel 8 00 7 Out delay msec o Out delay msec 0 Offtrigger rel 200 El Timeout msec 100000 Z Timeout msec 100000 Connected to state 1 E OS 1lwhite nB m pi 7 normal output Out delay msec 3330 Out delay msec 3330 ts a E Timeout msec 82000 Timeout msec 82000 IA UU Actors and outputs Pin B3_WS_J3 2 0x1daPin B3_WS_J3 3 0x1 db 00 del EM nn del 00 ce HH del deel Actors and outputs gt E level one A Take reference value D gt E level one A Take reference value gt The upper section of the Properties window left image shows the sen sors chosen for this particular level one element a spreader As is shown in more detail in section 4 2 6 two sensors and three actors have to be specified In the picture above only two actors are specified yet at the left image and no actors in the right image For each sensor an intensity value can be spec ified at which the sensor tells the level one element that the existence of the ON2. c_status f status Present a status window c_read_vbase f_read_vbase Read presets from VBase c_write_vbase T write_vbase Store presets in VBase NG_OBJECT_FOOT 5 3 2 2 Vosskuehler camera 5 3 2 3 Andor camera 5 3 2 4 Cascade Il camera 5 3 3 BioModule driver 5 3 4 Bio Fox driver 5 3 5 Pumps driver 5 3 6 MereGen board 53 Chapter 6 Miscelleanous 6 1 GTK 2 installation instructions for MAC OSX and other UNIX derivates After installing XCode Tools and X11 development environment the follow ing packages all are available from public domain online sources e g source forge net need to be installed atk 1 9 1 tar bz2 cairo 1 2 6 tar gz jpegsrc v6b tar gz expat 2 0 0 tar gz libXrender 0 9 0 tar gz libpng 1 2 8 tar bz2 fontconfig 2 4 2 tar gz pango 1 12 4 tar bz2 freetype 2 1 4 tar gz pkg config 0 20 tar gz gettext 0 16 1 tar gz glib 2 12 12 tar bz2 render 0 8 tar gz gtk 2 10 12 tar bz2 renderext 0 9 tar gz tiff 3 7 4 tar gz The revisions shown here reflect the situation in May 2007 Later revisions should be unproblematic though if not requirements are given with the configure scripts of the individual packages Unfortunately the sequence of installation is not at all arbitrary pkg config gettext expat jpegsrc tiff libpng have to be installed first Then follows render libXrender freetype and fontconfig The last chunk are glib cairo atk and pango The last package to be
3. 2 let the environment variable NGEN DESIGN point to the newly cre ated sub directory bioenv Verify that HOME points to the users home directory With bourne shells the command looks like export NGEN _ DESIGN home myuser bioenv 3 create a link startbio in a directory which is in the user s search path for executables e g ln s SNGEN_ DESIGN bin startbio SHOME bin startbio The file startbio at the web page is usually the most recent one It can be copied into bioenv bin and replace the already available startbio Please be aware that site specific changes are made in startbio and have to be updated with a new startbio file installed 4 Run the script startbio and provide the missing libraries which are mentioned as error messages at several levels of the startup procedure this step probably has to be accompanied by someone who has root access super user and knows the concept of libraries 1 2 Running the software The usual mode of operation is executing the script startbio This should be done once per experiments day It is not important at which specific place this script is executed All file creations and changes are done below HOME sessions Calling startbio ensures that the user is able to choose from the designs available change further runtime parameters and gets a full log of all operations during the session If startbio is called the first time in
4. knobs from the camera control Generally the camera features used here are optimized in advance for the task given and the user is only confronted with the most important options The camera output shown in this window is only meant for cursory obser 21 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE vation and not for high definition precise microscopic views High precision observations with the camera are reserved for programs specialized on that task Here the focus is on the attachability of a broad range of cameras without the users need to study all the subtleties of the given hardware File Options ace o HI a UB QEAD EJ EE Gain O Min O Avg 145 Max 1023 Y Extended biofox_A 26 biofox_B 26 1 biofox_F 26 2 biofox_AOTF Z biofox_blue 0 7 M Z biofox_red 0 M Toggle output x Oly O0 z 0 ar Satur 100 O NoScale C Diff mode Cra baia Da The scroll bars at the drawing area are dimensioned such that in principle the whole camera output can be viewed by scrolling through the different areas Please note that the actual images picked up from the camera are only from the visible part on the screen Non visible parts do not enter the circular image buffer and are thus not stored in the movies taken When scrolling certain delays might occur when changing the visible area 2 4 1 Camera knobs and status information The main focus on the camera features presented via the ng biopro
5. 5 PROGRAMMERS MANUAL pa _set_tck f set_tck Set TCK at FPGA to value x pa _set_vccf set vcc Activate power supply via parport pa get_initf get_init Get INIT pin status i get_tdo f get_tdo Get value of TDO at FPGA get_tmsf_get_tms Get current TMS value NG _ OBJECT FOOT 5 3 2 Camera driver typedef int c_ get_sens bounds ng_ngen int int typedef int c_set_sens ng_ngen int typedef int c_set_exposure ng_ngen double typedef int c_get_expo_bounds ng_ngen double double typedef int c_rdpicqq8 ng_ngen int int int int pixel8 t typedef unsigned int c_rd_info ng_ngen typedef int c_init_sum_mems ng_ngen int typedef int c_sum_ clear ng_ngen int typedef int c_set_sumarea ng_ngen unsigned int unsigned int unsigned int un signed int unsigned int typedef int c_age_wr_ videoram ng_ngen unsigned int unsigned int unsigned int typedef unsigned int c_ get_sumpixels ng_ngen int typedef unsigned int c_get_sumram ng_ngen int typedef int c_read_sumram ng_ngen int typedef int c_wr_ofs fac ng ngen unsigned int unsigned int typedef int c _ioctl ng_ ngen int typedef int c_min_avg_ max ng ngen int int int typedef int c _image _ size _x void typedef int c_image size y void typedef int c_on ng_ngen typed
6. Download XCS is provided to let the user decide whether a plugged in BioModule should be configured or not If no BioMod ule is attached this check box avoids lots of error messages and the waiting on hardware to respond correctly This check box is working only if the BioModule is configured from the PC starting the startbio script In all other cases especially when using the Bio Fox box the chip will always be configured The ng biopro software supports an arbitrary number of pumps to be addressed This startbio scripts lets the user to choose between zero and four different pumps The control window of these selected pumps is only visible on request see section 2 5 1 for a further explanation Due to the multitude of different hardware types and many of them only with simple serial interfaces a real plug and play philosophy cannot be provided Instead the user is required to choose the hardware components by clicking at the appropriate check boxs given as Device and Camera area In the case shown above par means use of the parallel port of the computer 10 2 1 PERL TK USER INTERFACE EPM USER INTERFACE This is for users connecting the BioModule directly to the parallel port The parallel port must be configured in IEEE1284 MODE COMPAT mode see section 3 1 7 for further specifications If the software is operated with a MereGen board which is controlling large parts of the setup then the user is require
7. TRAP TRIGGER and SPREADER You can find specific descriptions of these level one elements in chapter 4 Chapter 2 User Interface The user interfaces comprises several levels of interaction with the system As a preparation on what the software should do a Perl TK script startbio asks for the hardware to be used during the session A low level interface console based gives the user access to special fea tures and provides debugging facilities in case of failures This low level interface also provides communication with the program ng biopro in case of a missing graphical environment The third user interface level is the graphical user interface with click able buttons and design data presented Further interfaces exist which allow the program to communicate with other programs or to setup a client server operation scheme All these interfaces are described in the sequel 2 1 Perl TK user interface startbio The Perl TK user interface serves two purposes The first one is to provide an easy to use navigation tool allowing the user to choose between different design variants and to tell the software which hardware configuration is to be used In addition path information is defined including a simple session management and logging The second purpose is to provide a user editable script which allows customization to the users need of course a person able to handle Perl scripts is needed for this feature Th
8. X windows based graphical user interface is opened see chapter 2 for further details A multitude of error checks are undertaken and it might very well be that a lot of error messages do occur if something with the hardware or the designs fail When the fluidic and the electronic design is visible in one window and the current camera image in another window as well the software is operational 1 3 Feedback loops and level one elements The major objective of the integrated system environment ng biopro is to provide the ability on using electronics to directly control the biochemistry happening in the micro fluidic channels It is the central feature of the PACE project concerning its embedding in the IST framework This objective requires full access to the images produced by the camera and as much control as possible on all actuators available in the system With the Bio Fox box many of these actuators can be accessed The direct electronic control of the biochemistry immediately allows the creation of a multitude of feedback loops in the system The fluorescence images from the camera are evaluated at certain regions of interest ROI intensity values are calculated and due to several already defined or yet to be defined regulatory elements all written in software commands for the actuators electrodes xy table aotf filters etc are derived Currently six different simple feedback loop elements are defined CATCHER BARRIER NEURON
9. a day it creates a new session date folder and all logs and data files are stored in this directory At start up of startbio a Perl TK window opens and lets the user choose parameters Take your time and play with these parameters Essentially the user is asked about the hardware setup whether and how many pumps are attached to the computer what the interface between computer and the BioModule is whether a new sessions directory is to be created if it is already available and whether the BioModul should be configured or not See section 2 1 for a detailed description of all the features of this interface With the button Start the real ng biopro software is launched With the button Exit the Perl TK script startbio finishes and gives the control back to the user When ng biopro is launched the startbio scripts goes suspend meaning it is no longer reacting to user events With launching ng biopro a big Xterm window is opened executing the ng biopro software with the attached runtime parameters Through this Xterm window the ng_biopro software can be controlled on a console level 7 1 3 FEEDBACK LOOPS AND LEVEL HYPHERMENND RODUCTION a simple prompt using the readline library is provided see section 3 7 for an explanation of all possible commands and features During the boot process of the ng biopro software specified design files are read in contact is established to the defined hardware resources and an
10. condition is fullfilled and a second intensity value can be specified below which the sensor activates the OFF condition The sensors intensity values are dimensionless and relative to a reference value which can be adjusted by pressing the Take reference value button This reference value is simply the average intensity of the current camera view Because the sensors might have a considerable distance to the site where the actual decision has to be made a delay in milli seconds can be specified after which the recognized ON or OFF event is evaluated in the software If the sensor when it is just actively looked at this depends on the type of the level one element does not see an event in a specified time a timeout event is generated and the level one element s state machine is reset see section 4 for further details on how level one elements are implemented Each sensore can be specified in the context of a certain filter If the S checkbox is active then before a new measurement is done the according filter is moved into the microscope beam to assure that the measured values are really corresponding to the parameters set With the S box not checked the filter definition only has a function as commenting the context how the intensity threshold have to be interpreted Instead of using a sensor as input another already existing level one 36 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE elem
11. control and definition There are several possibilities to connect pumps to the computer and to access them via serial interfaces The number of pumps addressable is arbitrary and the user defines in the Perl TK script how many pumps to be shown when pressing the pumps button The pump devices are specified in the ng biopro_c ecmd file which will be described in section 3 7 30 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE al Comment Comment Y End switch Fine End switch Fine 8 inj v EJ SelCode Max active f EEN Y Max active ithdraw 7 SyrDia mm e la Stop 2 40 gt a Close ca The number entered in the field SelCode defines the absolute identifier of the pump This identifier is either specified in a ROM area of the pump or a specified hardware address given in ng_biopro_c cmd Furthermore the inner diameter of the syringe used and the wanted injection rate can be chosen For fine tuning a slider is available which has a 10 times smaller effect than the min tic at the rate entry Currently only the Inject button is active Inject and Stop affect all pumps at the same time Each pump can be deactivated locally when the icon R is visible With the icon 7 the according pump can be restarted again About every second the current state of the pumps is updated the ab solute number of revolutions per second is shown as well as the absolute position and the m
12. each image 24 2 4 3 Storing snap shots 25 2 4 4 Long term measurements 25 CONTENTS CONTENTS 2 4 4 1 General principle y AA 26 2 4 4 2 How to create a new measurement chain 27 2 4 4 3 Changing the measurement chain 27 2 4 4 4 Moving the measurement chain 28 2 4 4 5 Modifying measurement parameters 28 2 4 5 Synchronizing camera s output with the fluidic design 28 2 5 Design window aia da ee ia ee ee ee A 30 2 5 1 Pumps control and definition 30 2 5 2 Shift register and basic electrode control 32 2 5 3 Level one elements control 34 2 5 4 Sequences control o ee eee 37 3 Interfaces 39 3 1 Hardware Interfaces gx a a a 39 Salado Camera ico R A hae Aaa By 40 3 1 1 1 Firewire camera e g Vosskuehler 40 ZLE Andor CCN eip Soe ee a ee A A Tk 40 3 1 1 3 Cascade II Princeton Imaging 40 3 1 2 BioModule and electrodes 40 gdko PUMPS z sni a aa a ae ee eee iA 40 3 1 4 Filter wheel A a 40 3 1 5 Temperature control lt lt a a a 40 3 1 6 AOTEF and light sources ii aa 40 3 1 7 The parallel port e imc as ke dike ia 40 3 2 Graphical interface ua a ee ae 40 3 3 DPD Interface dissipative particle dynamics 40 3 4 Client Server Interface a eee ee 41 3 5 BioQFox Inter ace o 42 3 6 Runtime parameters sio A RA eg 43 3 7 Scripting Interface ei 4
13. installed is gtk itself In al most all packages configure scripts are provided Sometimes special runtime options have to be provided configure can always be called as configure help resulting in an extensive list of options possible As usual after a successful run of configure make and make install have to be issued Al ternatively fink adapt synaptic or other software installation utilities can be used as well Please be aware that the software from the packages nor mally will be install in the usr local tree whereby fink for examples uses a sw tree This can result in not found libraries and include files and has to be resolved individually 54 6 2 REVISION HISTORY CHAPTER 6 MISCELLEANOUS 6 2 Revision history Version 0 1 was released in April 2006 Version 0 2 was release in May 2007 with further descriptions of hardware interfaces 6 3 Release Notes 59 Index BioModule 13 configure 13 customization 9 design 14 design variants 9 Download XCS 10 MereGen 13 navigation tool 9 pumps 10 ReScan button 12 Select button 12 Use existing session 10 user interface level 9 56 Bibliography
14. is hard coded in the starting script startbio and last but not least the design environment HOME bioenv which contains all binaries and design files usable by the software the name bioenv is not mandatory but has to be defined in the environment variable NGEN_ DESIGN Updates of the soft ware will change or add files below this directory Further directories which are provided during the installation procedure are HOME sessions session_ template a directory which contains native design files used to start the software NGEN DESIGN bin the directory tree containing all available binaries of that revision e g ng _biopro_ i686 NGEN_ DESIGN design a directory tree containing all design files avail able you can add further design files at all times if you obey certain naming conventions see section 3 9 and NGEN_DESIGN icons the full collec tions of icons used by the software The current version of the software is available on line in the members section of the PACE website at PACE Working Area Movement_ simulator BioPRO_ Software bioenv Three container files are provided bioenv_shipped tar gz sessions shipped tar gz bioenv_ fluidic tar gz All three of them should be unpacked in the same di rectory A typical installation comprises the following steps 1 unpack the tar archive in the home directory or where ever the user has write access to 1 2 RUNNING THE SOFTWARE CHAPTER 1 INTRODUCTION
15. pointer If there are several types of selectable objects a popup menu lets the user choose which of these types are to be selected Se lected objects are highlighted The highlighting color can be changed see paragraph 2 3 1 on how to do this Already selected objects are deselected If this should not hap pen then press the CTRL button at the same time when the clicking is done If already a rectangular area has been drawn the effective selecting area Of the mouse button is increased to this size meaning that all objects inside this rectangle are selected Again a popup menu ap pears if different types of objects are to be selected After selection the rectangular area disappears There is a special feature for electrodes or pins which lets the user define the polarity of these electrodes When selecting an electrode the default polarity is positive usually 3 3V with Spartan XL FPGAs Pressing and halting the SHIFT button before clicking on the elec trode reverses the polarity in our example resulting in OV potential at the electrode Just clicking on the electrodes does not actually mean activation of the electrodes They stay in the state possessed before see section 2 3 5 for further details Every simple object has one selection point This selection point is at the center of the electrodes and at the upper left edge of the sensors see section 2 3 5 for details If sensors or electrodes are overlapping unfavorably then zoom
16. regions of interest as well as some reference areas which can later be used to calibrate the interesting measurements Secondly the individual measurement area should not be too small to be overwhelmed by noise and not to large to incorporate inhomogenities in the measurements 2 4 4 3 Changing the measurement chain Changing the measurement chain can be done as long as this measurement is not active or ongoing The number of elements in that chain can be adjusted easily when turning at the spinner wheel denoted with sensors Typical suitable values range between five and 20 of these rectangles Without changing the size manually the rectangles sizes are automati cally adapted to cover a maximum area when doing the measurement When turning the spinner wheel called size pixels the size of each rectangle is specified in pixels by the value of the spinner wheel This manual adjustment 27 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE might be useful when trying to use unconventional measurement areas 2 4 4 4 Moving the measurement chain The measurement chain can be moved into arbitrary parts of the camera window Size and orientation can be changed as well Moving the chain Select one or two of the sensors rectangles in the middle of the chain press the SHIFT button on your keyboard and move the mouse while pressing the left mouse button into to the direction of your targeted middle position of the chain Cha
17. selectable object results in a bunch of further information given in the session log This information is meant for debugging strange behavior and is usually not needed 2 3 3 3 The right mouse button typically means inversion of op eration e When clicking the right mouse button inside a zoom state then a zoom out operation occurs After zoom out the rectangular area of the for mer zoom in rectangle is visible see section 2 3 2 for further details If a rectangular area is visible then this area is removed e If no zoom out is possible and no rectangular area visible then all al ready selected objects are deselected e Double click of the right mouse button always means deselection of all selected objects 2 3 4 Data base export and import of objects The ng biopro software is equipped with an integrated database The main purpose for this database is to store all the complex configuration options and user defined control structures to allow for a seaming less continuation in case of halting the program This database is either stored in a binary con tainer e g ng biopro 10506 Vbase db_0 or a human readable ASCII file close Vbase The database itself is a distributed object oriented database system Especially for client server operations the communication between client and server is mediated via this database For reasons as backup and debugging it is possible to export the internal database into a human readable ASCI
18. y z n n coordinates with x y z defined passive_pol 0 1 2 polarity of non active puls phase pulsewidth percentage of active electrode per puls rate ul h flowrate of fluid state 0 1 2 state is zero or one or undefined 3 4 Client Server Interface The purpose of the client server interface is to allow external operators scripts to access the hardware and provide a full remote control of the experiments The interface declaration is specified in an additional document with name 41 3 5 BIO FOX INTERFACE CHAPTER 3 INTERFACES ext_interface pdf The communication is realized via a TCP connection at port 8023 3 5 Bio Fox Interface A y The Bio Fox board shown is presented without the protecting cover It consists of an embeeded 100MHz micro controller Fox board from ACME Systems Srl with Linux as operating system and several hardware interfaces See the hardware manual of the Bio Fox board for further information The communication is typically done via Ethernet RJ45 connector and a TCP socket connection between the ng biopro software executed on the Bio Fox board and the ng biopro software executed at the controlling PC Low level communication with the Bio Fox board is either done via a telnet or ssh connection or directly using the console output at the serial interface ttySO at the left side in the figure 42 3 6 RUNTIME PARAMETERS CHAPTER 3 INTERFACES 3 6 Runtime parameters Ru
19. 00 images in the ring buffer the resulting video sequence is 40 seconds long 2 4 2 1 Information appended to each image Each image produced gets an individual annotation with several informa tions e current time in micro seconds resolution t e all available temperatures s filter wheel states f light states currently light is controlled by an AOTF acusto optical transmission filter l e the current position of the xy table and the z stage p e absolute counter of the images taken in this session r It follows an example of the data appended to each image t 1178095092 124578 Wed May 2 10 38 12 2007 f 1 emission 1white l 0 biofox blue 0 l 1 biofox_ red 0 p 0 x 2450955 p 1 y 59685 p 2 PI 39985 s 0 biofox_ A 2645 s 1 biofox_ B 24645 s 2 biofox_ F 355 r 2682 24 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE 2 4 3 Storing snap shots 8 There might be situations where the video images taken in section2 4 2 do not show all interesting features at the same time and the maximum possible camera view is needed For these occasions a full cameras screen dump is stored as a pgm file on disk As already described in section 2 4 2 the pixel values are stored in 16 bit pgm format P5 and the file name is derived from the current date and time Two different operations are possible either s
20. 3 3 7 1 ng biopro_ce emd tel a A A 43 3 8 Wivase Interface sos ese be RTE Oe Sk eS a 43 38 1 Veonf filens ea ice Sue we B MOR wep EB ne A eke et 43 3 8 2 vbase ASCII hago odiaba bea ws 43 3 8 3 Message passing to VBase o 43 3 9 Design environment in detail 43 4 Level One 44 4 1 Introduction of level one elements 45 42 gt Basic elements a eao e e a de 45 dL BARRIER lt a d R a E sage ms od E a 45 CONTENTS CONTENTS A CATCHER piri neag di das A 46 ANDES Neuro Saio Soh eR Be Sd ee ed ee 47 ADA A ate She Sete iw te Thee Sense QE chp nike Rip Tale Bo the Die 47 2 IRIS a Ba hi Sins Bae des 48 EDO SPREADER lt a ita 48 Aids NetWork aia e a de io da A Ge 49 5 Programmers Manual 50 5 1 ng_biopro Library eri a 50 5 2 Client Server message passing 00 50 5 3 Drivers interface 2 a a 50 5 3 1 Parallels pott e st ae MOK ae ela ee wy he oe we 50 5 3 2 Camera driver 0 a 52 5 3 2 1 Camera data structure 52 5 3 2 2 Vosskuehler camera 53 5 3 2 3 Andor camera 2 cd a ti o 53 5 3 2 4 Cascade ll camera 53 5 3 3 BioModule driver 53 5 3 4 Bio Fox driver o 53 Dida gt JPUMIpSAdTIVer Cl a hee ad as 53 5 3 6 MereGen board aooaa a a 000000 ee 53 6 Miscelleanous 54 6 1 GTK 2 installation instructions for MAC OSX and other UNIX derivates KE a A a ee 54 6 2 Revi
21. I form The syntax is LISP like with all objects and their corresponding data is written in clear text as is shown 19 2 3 WINDOWS AS USER INTERFACHAPTER 2 USER INTERFACE in the file close Vbase Of course importing such an ASCII database is also possible Import and export is available below the FILE drop down menu in the upper menu bar Remark the binary form of the database is neutral to little and big endian machines 2 3 5 Sensors and actors Besides the pure hardware control like pumps camera and shutters the elec tronic control of electrodes is at the heart of the ng biopro software Sensors in the sense of this section have to be distinguished from e g temperature sensors which are also controlled by this software but do have a clear physical representation The sensors in this section and if not otherwise noted in the whole document are simple rectangular areas in the top level windows In the camera window these rectangular areas are regions of intensity integration All intensity values of pixels in this region are summed up and divided by the number of pixels Arbitrary many sensors in the current version is an upper limit of 512 active sensors can be defined and used to derive decisions on whether to switch on electrodes or not The sensors in the design window are exactly the same with only two exceptions firstly they are pre generated and placed along interesting areas of the fluidic channels this eases the sel
22. Rainbow Color 14 Color 15 Linear Gamma 2 There are two different types of colors to be specified Discrete colors which are used for selected items switched on or off states of elec trodes sensor elements and fluidic channel representations 16 different colors can be specified in this way What color is used for what func tionality in the user interface is currently hard coded in the ng_biopro software and is not customizable The second type of colors is a palette which might be a definable gradi ent a random collection of colors or a rainbow This palette is effective for the camera window where many different colors are needed A suit ably defined color space allows the experimenter to enhance certain aspects of the camera screen The colors defined here only affect what the user sees on the display and do not influence what is stored on disk as a sequence of images see section 2 4 2 for further information on video sequences RE e Load fonts and map them for textual output You only need to provide a suitable name for the font With xfontsel you will find out which fonts are currently available 15 2 3 WIND OWS AS USER INTERFACHAPTER 2 USER INTERFACE Defi niti Fixed fonts Font 3 Font 4 Font 5 Font 6 Font 7 Default test Font 1 Font 2 e Flip drawing area horizontally Only t he view is flipped The internal calculations are not affected L Flip drawing area vertic
23. User s Manual of BioPRO Software Uwe Tangen August 15 2008 Ruhr University Bochum c o BMZ Otto Hahn Str 15 44227 Dortmund Version 0 3 Contents 1 Introduction 5 1 1 Installation instructions 26 48 we OS a 5 1 1 1 Operating system requirements 6 1 1 2 Directory trees and installation 6 1 2 Running the software ee ge ited Pek Aa 7 1 3 Feedback loops and level one elements 8 2 User Interface 9 2 1 Perl TK user interface startbio 9 2 1 1 Session tab of Gob ay arg ak anna Se Rare Ses 10 2 1 2 Path tab iid ae BU peo Ue et US ee a Bh ute Uk 11 A A NO 12 2 1 4 FPGAs and bit files o 13 2 1 5 Post processing AA A A A 13 2 2 Console terminal sis ce E A oot ee 14 2 3 Windows as user interface ooo a 14 2 3 1 Colors and fonts ete mall al ae 14 OR OQ gra a as E EA a ly BO e A 17 2 3 3 Mouse buttons and cursor keys 17 2 3 3 1 Left mouse button 17 2 3 3 2 The center mouse button 19 2 3 3 3 The right mouse button typically means in version of operation 0 19 2 3 4 Data base export and import of objects 19 2 3 5 Sensors and actors o 20 2s Camera wind OW anes che 2 wkd ee coat Ow Ae Oe bd 21 2 4 1 Camera knobs and status information 22 2 4 2 Storing video sequences 4 23 2 4 2 1 Information appended to
24. ally Only t L coe a log book entry Enter a log book entry 2 he view is flipped The internal calculations are not affected The log book entry popup window gives the experimenter the ability to add notes at any time during the experiment It is just a very simple editable text field After pressing the Close button the contents of the window is printed in the log book of this experiment accompanied by two time stamps one for opening the log book entry form and the second for the time of closing the window 16 2 3 WINDOWS AS USER INTERFACHAPTER 2 USER INTERFACE 2 3 2 Zooming Zooming is only possible in the design window There is no restriction on the depth of zooming The procedure to zoom into a region of the design is two draw a rectangular area with the left mouse button see section 2 3 3 for notations and then after releasing the left mouse button clicking the middle button Zooming out is done with the right button just clicking it at the design window Then the zoom jumps back to the former rectangular area The sketch of this area is shown in addition Zooming into this now old region is done as before just clicking the middle mouse button There is no limit apart from memory considerations in how many zoom levels can be created The once created rectangular area can be modified Pressing the left mouse button inside the area means moving it pressing the left mouse button in direct vicinity
25. conds resolution time stamp attached When pressing the Video button this ring buffer is written onto the disk as a series of pgm files The name of the files is derived from the current date and time and is sequentially numbered The pgm files are not compressed and the pixel values do have a depth of 16 bit images are raw unmodified camera data This means that the visible image which is subjected to a further contrast and brightness operation might significantly differ from what is stored in the pgm files This apparent difference is the price for allowing a later precise evaluation of the pgm files With the post processing procedures mentioned in section 2 1 5 the pgm files can be compressed losslessly or transformed into mpeg streams with mapped in electrodes if the camera window had been synchronized with the design window see section 2 4 5 With special tools these electrodes can be inserted in the mpeg stream even later without having synchronized both windows 23 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE Be aware this type of storing video sequences means storing the history of an interesting event If the experimenter realizes some interesting behavior he or she might wait a little further and then press the Video button It depends on the camera and the effective exposure time used of how many seconds of the experiment can be captured via this procedure e g with a frame rate of 10 images per second and 4
26. d to check the meregen button With no BioModule attached nodev should be checked Checking the button cli client and providing the appropriate name of the Bio Fox box in principle IP addresses might be sufficient as well but some peculiar socket issues will result sometimes in error messages if not given a real name opens the connection to the according Bio Fox controller In principle the ng biopro software supports several different types of cameras Check the button no if no camera is currently available If an Andor camera is provided check the button andor and have a look into sec tion 3 1 1 2 for further information on the software development kit used in this implementation Even though firewire IEEE 1394 is a generally de fined camera bus each firewire camera requires special control flags to be set The current ng_ biopro software supports the Vosskuehler camera With a Vosskuehler camera attached check the button vosskuehler With proto life the driver for a Cascade II camera from Princeton Instruments is se lected 2 1 2 Path tab Startbio File Mode Info Session Design FPGA Postproc Fluidic S S aT ReScan Select Default u utangen bioenv designfuidic Bitfiles 7 5 ReScan Select Default u utangen bioenv designfpga Electrodes x S ReScan Select Default ufutangen bioeny design electric If the user doesn t have a special environment configured
27. e and an impedance of about one M ohm is already interpreted as fully connected Furthermore due to requirements of CMOS logic potentials electrodes should not float in the 0 8V to 2 0V range because arbitrary large currents could occur This means that the manufacturer artificially clamps the voltage in the high impedance regime to about 2 0V if the electrode had a high potential before or OV if the elec 32 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE trode had been in the OV state and worse the impedance is set to about 300kOhm These values are not normed and might change from one output driver technology to the next Electrode control in the micro fluidics area has to suffer from a mismatch of technology and physics Nevertheless a clever usage of the electrodes timing behavior can mask these sorts of problems Charges gt Cycle normal active Ref 170s ad med 1 10ms 25 80 100 400 Stop A Clean HU Disable Close The electrodes control described in this section is at the lowest possible level Electrodes which have been selected beforehand see sections 2 3 3 and 2 3 5 can be switched on or off These electrodes can be subjected to a periodic switching between states The switching frequency of the period is defined with the slider Ref in tenths of milliseconds This means e g for a value of 400 every 40 milliseconds the period starts anew A period has two characteristics the normal duty cycle a
28. e ng_biopro software uses several different input interfaces Two of them are utilized by this startbio script For runtime parameters of the program see section 3 6 and for the scripting interface of the ng biopro software see section 3 7 When starting the startbio script the Session tab is displayed 2 1 PERL TK USER INTERFACE EPM USER INTERFACE 2 1 1 Session tab File Mode Help im Info Path Design FPGA Postproc Select Default home rihm utangen sessions todays session Select Default session_20070530 W Use existing session _ Download XCS Pumps 40 412 3 1 4 Device Z par nodev meregen w cli fox2 0000 Camera no andor vosskuehler protolife E a This is the first window seen after the startbio script has been launched The user is allowed to define a directory in which all the logging and further definitions of the software behaviors are saved The two paths in the upper half of the window are preset with default values which might be changed via the buttons Select on the right side The directory chosen in the first path entry has to exist already it is thought as the general container of all experiments done with this software The second will be created if not already existent By switching off the check box Use existing session a new directory is being created each time the ng biopro software is started anew A further check box
29. e to be guessed The principle alignment procedure is as follows 28 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE A Pressing the Adjustment button initiates the alignment proce dure The ng_biopro software highlights two electrodes actors in opposite corners of the design window It might be that these electrodes are not immediately visible the user has to scroll the design window till he or she sees these highlighted actors typically these are quite big black filled circles The next step is to find the according region in the camera window Then the user is requested to click on one of the two black circles which is then immediately vanishing and the user in the sequel clicks at the center of the corresponding electrode in the camera window A blue square at that position will blink once to indicate that this position has been acquainted This procedure is repeated with the second black circle With these two reference points the ng_biopro software rotates and maps the camera window and hither two both windows are aligned Sensors in the design window can now be used with the correct inten sity values recorded In addition the electrodes outlines are shown in the camera window to let the user verify the correct mapping Rotating the camera window at each image is a time consuming procedure and slows down the camera acquisition If only the syn chronization is needed and not the exact camera view the on line rotati
30. easured rate A pictogram indicates the left slider going up and the right slider going down these pictogram are only resembles the meaningful with MMT pumps and the pictogram opposite case whereby denotes the pumps being stopped There is a software based end position detection which can be deactivated by decheck ing the End switch button This end position can only be detected if the software is running A further entry field is a comment area which helps the experimenter to remember which chemicals are driven by which pump The button Close closes the window The pumps themselves are not affected by this closing Remark 31 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE Exiting the program does not change the pumps status if they are active they stay active Of course a change of the pumps rate is then not possible apart from manu ally regulating the pumps After restarting the program a short pressing of the Inject button reestablishes the contact of the software with the pumps This was imple mented as a security feature because strange flow rates only become active after the Inject button be pressed A further security measure has been implemented to avoid incidentally high flow rates With the default setting of the check box Max active the flow rate is restricted to 1001 per hour Please be alert especially when starting the program anew and adjusting the pump parameters to verify that the s
31. ection considerably And secondly the intensity values are only available after camera and design window had been synchronized How this is done is explained in section 2 4 5 20 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE Plot BioPRO Reactor gt File Options Qe Bao aA GS Colors Fonts Control Series Sequence Log Shifts Pumps Actors on the other side are simple electrodes which are electrically ac cessible for the fluids in the channels Not all electrodes are always accessible because certain fluidic designs do not allow arbitrary dense channel networks Though in the image above not only sensors green rectangular areas and ac tors brown rectangular areas are shown but also blue traces which actually are wires connecting the electrodes with the controlling FPGA The galvanic accessibility is only given for the electrodes All other parts of the design are covered with an isolating SiN or 5103 layer Of course the isolation layer is quite thin but for the frequencies and areas used here no visible influence during the experiments can be observed 2 4 Camera window The camera window is the first top level window Essentially it is a combina tion of four parts a menu bar with two drop down menus File and Options and a Help button an icon menu bar with often used functions a drawing area with the cameras output and some status information and refinement
32. ef int c_off ng_ngen typedef int c_status ng_ngen ng_grafik typedef int c_read_ vbase Vbase typedef int c_ write_vbase Vbase 5 3 2 1 Camera data structure struct s_ng cam NG_OBJECT_HDR p_data gt p_ana p_user gt c_get sens bounds T got sens bounds Get sensitivity bounds c_get_expo_bounds T get _expo_ bounds Get bounds of exposures c_set_sens f_set sens Set sensitivity gain c_set_exposure f set exposure Set exposure time c_rdpicqq8 f_rdpicqq8 Read an byte image c_rd_infof rd_info Get info about camera c_init_sum_mems f init_sum_mems Initialize summen memories c_sum_ clear f sum clear Clear summen memories c_set_sumarea f set_sumarea Define a summen area c_age_wr_videoram T age _wr_videoram Write something into cambuf 52 5 3 DRIVERS INTERFACECHAPTER 5 PROGRAMMERS MANUAL c_get_sumpixels f_get_sumpixels Get size of summen area c_get_sumram f get_sumram Get integrated value of sum c_read_sumram f read_sumram Read value of sum and clean c_wr_ofs fac T wr oa fac Define offset factor c_ioctl f_ioctl 1 O control c min avg max T min avg max Readout min avg and max va c_image_size_xf image size_x Return the cameras nr x pi c_image_size_y f image _size_y Return the cammers nr y pi c_onf_on Switch the camera on c_off f_off Switch the camera off
33. ent can be chosen See an example in the right image of the figure above where the spreader eeeeee serves as input for the spreader qqqq The output of this level one element is then taken as input for this level one element and provides the basis for building networks of low level regulators 2 5 4 Sequences control Generating complex time series of electrode potentials can be realized with sequence elements To create a new sequence element at least one actor has to be selected highlighted in the design window Pressing the button New element popups a window which asks for the name of the new sequence element Selected sequence elements can be switched on or off using the On Off button Each sequence element can contain arbitrary many different actors These additional actors have to be selected and then via the button Add actor added Without any further specification in the according property window of the selected sequence element each actor always is in the high impedance state Sequence element def a sequence 1O New element On Off After pressing the button Properties the following window pop s up in 37 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE which the user is able to define the polarity at each discrete time step Take the pull down menu below the actual electrode names to define the polarity at the selected time step The value z means high impedance Time is ru
34. in the hierarchy of possible control structures Several different types can be specified of which currently six are possible the spreader the catcher the neuron the trap the trigger and the barrier The general idea of these level one elements is to provide one layer of control These level one elements are constructed such that they can be combined to networks of regulation The connection between these level one elements is realized purely in software Physical delays of remote elements can be dealt with Additionally several timeout features are available to allow for the buildup of robust regulators 34 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE Control element definit a spreader TOS New element On Off All known level one elements in this design are shown in a simple list A graphical representation of the connections is not yet available A new element can be created to the element sensors and actors can be added as long as not all requirements are fulfilled Note Before you can define a new level one element you first have to select means highlighting a sensor which should be part of the level one element The buttons Put and Pop are currently not implemented When pressing the Properties button a popup window of the following type appears 35 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE Properties of eeeeee a spre Sensors and inputs
35. ing into the region usually helps to distinguish these objects e Pressing the left mouse button Some objects mostly sensors are allowed to be moved around The usual procedure to accomplish this is to select the object you see the highlighted color and then while pressing the SHIFT button in addition dragging the object over the screen Typically you will see a rectangular area emerging which is removed after the object has been moved With a rectangle is existing the behavior depends on the position of the mouse pointer If the mouse pointer is well inside the rectangular area then the user is able to move this rectangular area by dragging the pointer while pressing the left mouse button If the mouse pointer is fairly precise at an edge or a vertex of the rectangular area then the size of that rectangular area can be changed by dragging the pointer while pressing the left mouse button 18 2 3 WINDOWS AS USER INTERFACHAPTER 2 USER INTERFACE e Double clicking the left mouse button Currently no functionality behind this event 2 3 3 2 The center mouse button e Zooming in into an already given rectangular area when clicking It is not necessary to have the pointer inside this rectangular area just clicking is sufficient All other possible events pressing double clicking and in conjunction with other keys are not implemented e Pressing the shift button while clicking the center mouse button and being located over a
36. mera this is an and then by the procedure name giving hints on the real functionality All prefixes and the procedure names are connected via underscores _ Each type of hardware has a special data structure containing all specific details Local data only relevant for the specific driver has to be defined as static in the drivers source file as long as this static declaration does not interfere with multiple driver instantiations In these type specific data structures each function does have an entry point which is used by the ng biopro software to operate the hardware An initialization function has to be provided by the hardware interface which initializes this data structure with the appropriate procedure calls All procedures are to be of type integer with a 1 return indicating an error condition This error condition usually lets the program stop with a series of error messages indicating the cause of this error Return codes less then 1 indicate warnings which are not further evaluated but can be used 39 3 2 GRAPHICAL INTERFACE CHAPTER 3 INTERFACES for self test software In addition when obeying the rules user interactions can be modified Simple buttons scales popup windows and so on are available see section 3 2 for further definitions 3 1 1 Camera The camera interface certainly is the most important and far reaching inter face because of the performance requirements involved 3 1 1 1 Fi
37. mes available the fluidic module which shows check boxes of the possible detailed variants of the Fan reactor design in this case In total several dozens different fluidic designs are available The preview button is especially useful because it allows the user to look at the designs quickly and to find out which biomodule really is mounted and what the specific design name is These specifications have to be done because otherwise the user almost certainly will be not able to specify which electrodes in the fluidic channels are effective and have contact to fluids 12 2 1 PERL TK USER INTERFACE EPM USER INTERFACE 2 1 4 FPGAs and bit files Startbio gt File Mode Info Path Session Design Agent biopro_agecam_sum4ovt_trex_bsy bit Camera Area Summation Image on demand external Trigger V 0x7 Distri evonet_distriFc bit f r BioPro Camera 16 Bit auslesen y Depending on the attached hardware this tab allows the user to specify the bitstreams used for the configuration of the FPGAs The two upper sections in this tab are only needed when the MereGen board is used as the major hardware control unit The lower section shows the currently available bit files used to configure the FPGA of the BioModule 2 1 5 Post processing File Mode Help O Info Path Session Design FPGA todays session Select Default session_20080804 session history home rihm utange
38. n 2 4 4 5 Furthermore an ongoing measurement can be dynamically visualized on the screen to allow the ex perimenter a fast feedback on how the reactions are going press button Plot in the window Measure element definition 26 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE 2 4 4 2 How to create a new measurement chain Measure element defini ziuziu jojouztuzt jojouztztizgg Ts Changing properties of time interval sec 10 0 frames per measurement 10 Hy sensors 3 a a diagonal line size pixel 12 Before creating a measurement chain a rectangle has to be drawn in the camera window see section 2 3 3 This rectangle roughly shows the covered area of the future measurement areas Pressing the button Add measure the user is asked for a name for this new measurement chain After creating the measurement chain three rectangles one with color violet and the others two with color green are drawn in the camera window with one of these rectangle covering the upper left and the other covering the lower right edge of the user drawn rectangular area This is the default measurement area which now can easily be customized to fit the user s need When pressing the button Properties the right window shown in the figure above pops up and the user is able to adjust the number of elements in the measurement chain It is good practice to let this chain cover the
39. n sessions history Move session files to session history Zip images create tar files Create MPEGs M withelectrode animation M with timestamp Create MPEGs from all dirs in session call filme_machen Create TIFFs from all picts in session call tiff_machen EE E Some simple standard processing steps can be undertaken after experi ments have been conducted For example the videos stored are written as pgm files and probably needed to be converted into a mpeg movie or they 13 2 2 CONSOLE TERMINAL CHAPTER 2 USER INTERFACE should be compressed to save disk space 2 2 Console terminal A basic user interface is given with a console window This console is created as an Xterm window after pressing the Start button in the Perl TK script or the ng biopro software is started itself from a console without any Perl TK script The sole function of the Perl TK script is to provide the wished for environment This environment is defined via two interfaces firstly the pro gram s runtime parameters and secondly with script files which have been created by the Perl TK script Runtime parameters and these script files are open They provide all the necessary data for operation in case of debugging or a non graphical user interface Many additional commands are available on this low level interface which are not needed in typical cases and thus are almost not documented in this user manual Especially when using
40. nd the active duty cycle The normal duty cycle defines with what percentage per period the elec trodes are subjected to the values they have been specified The rest of the period the electrodes do have the opposite polarity the idea behind this is to stop charged molecules from attaching to surfaces This normal duty cycle is controlled by the active duty cycle which in addition defines the percentage of the electrodes being active and not in the high impedance state The following screen shot of a measurement illustrates the real electrode behavior 33 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE 0 MAN 9 iim or cycle start activ til 60 of Ref tri state from 70 Ref regular Elec trode red cycle duration S to Ref 1 10 ms normal duty 60 gt activ duty 70 gt inverted Elec trode blue The normal duty cycle is set to 60 meaning that the electrodes will have the potential they have been specified with in 60 of the reference cycle period of about 15ms After 60 of the reference period are passed the polarity of the electrodes is inverted The active duty cycle in addition is set to 70 meaning that 70 of the reference period the electrodes are active and in the rest of the 30 the electrodes are switched to tristate See the explanations in the screen shot shown above 2 5 3 Level one elements control ca e Kad Level one elements represent the next higher level
41. nging the size and orientation of the chain As with moving the chain select now one of the outer sensors rectangles of the chain visible through yellow color Pressing the SHIFT button and moving with the pressed left mouse button towards the target position of this sensor lets the chain stretch or shrink The opposite boundary sensor remains stationary In the second step the opposite boundary sensor can be selected and moved accordingly to its target position 2 4 4 5 Modifying measurement parameters There are further parameters which can be changed via using the Properties window of a certain measurement time interval and frames per measure ment The time interval defines the time in seconds between two measure ments Frames per measurement defines the number of measurements done before the average of these measurements is written either on disk and or updated in the plot window Default values are 10 seconds break between two measurements and three measurements for one plot update disk write which is done in any case 2 4 5 Synchronizing camera s output with the fluidic design To let the ng_biopro software know which area in the design window exactly corresponds to which area in the camera window a certain procedure has to be undertaken Especially when using fluorescence microscopy edges of electrodes or wires are hard to see and mostly only partially visible More important fluid channels in almost all cases hav
42. nning from top to bottom and the time period is defined in the scale at the bottom right side e g 5 seconds This means every 5 seconds the next step of the time sequence is activated After the bottom step set has been executed the next time step starts from the top anew Adding a new time step set is done with the Add set button All electrode potentials are preset with the default value of high impedance Each selected time step can be removed with the Delete set button Properties of flute a sequi Actors and outputs dd del dd del dd del B3_WS_03 1 B3_WS_03 2 B3_WS_03 3 time interval sec 50 Rip Add set close Delete set 38 Chapter 3 Interfaces The purpose of this chapter is to declare the many different interfaces needed in the ng _biopro software It is meant as a help for developers of drivers to attach new hardware to the ng biopro software The ng biopro software is written in C using an object oriented paradigm Thus the programming style is a mixture between C and C 3 1 Hardware Interfaces Hardware interfaces are realized with a procedure oriented set of functions These function headers will be declared in detail in the current section The naming convention is a two to three letter prefix pointing to the type of hardware e g for camera drivers the prefix is cam followed by a two letter prefix for the specific hardware e g for the Andor ca
43. nput Select In S4 13 Input Auto Feed C1 14 Output Prog 22 Error S3 15 Input Init 21 Initialize C2 16 Output CClk 23 Select C3 17 Output DIN 24 Ground 18 25 GND 29 40 VCC at adapter GP1 ST53 or GP3 ST55 to ST62 connected S7 CO Ci amp C3 signals are inverted ee state of port is Dat 0xff Sts 0x7f Ctl 0xf Flags 0x0 Phase x typedef struct s_ng_parport ng_parport Data of parallel port class typedef int pa_port_ info void typedef int pa_set_all int typedef int pa_set_din int typedef int pa_set_prog int typedef int pa_set_cclk int typedef int pa_set_gck5 int typedef int pa_set_tms int typedef int pa_set_ tdi int typedef int pa_set_tck int typedef int pa_set_vcc int typedef int pa get_init void typedef int pa get_tdo void typedef int pa get _tms void SUS RP HbR p_data gt p_ana p_user gt pa _port_ info T port_info Printout some information of parport pa _set_allf set_all Set all parport pins to value x pa_set_din T sot din Set DIN of FPGA pa_set_ prog f_set_ prog Pull the PROG pin and initialize FPGA pa_set_cclk f_set_cclk Set the CCLK to value x pa_set_gck5 f_set_gck5 Set the global clock 5 GCK5 to x pa_set_tms f_set_tms Set TMS at FPGA to value x pa_set_tdif_set_ tdi Set TDI at FPGA to value x 51 5 3 DRIVERS INTERFACECHAPTER
44. ntime parameters are specified using the standard Unix convention A minus sign plus the type of the parameter then a blank and the parameter value following Strings containing blanks have to be enclosed in double quote signs The available parameters are e cam an integer value 0 This parameter defines the camera interface to be used for image ac quisition Depending on the version of the ng biopro software used different types of cameras can be operated with Use value 0x10 when an Andor camera is attached and 0x20 if a Vosskuehler firewire camera is used e design name of the BioModule design to be used 3 7 Scripting Interface 3 7 1 ng biopro c cmd 3 8 VBase Interface 3 8 1 Vconf file 3 8 2 vbase ASCII base 3 8 3 Message passing to VBase 3 9 Design environment in detail 43 44 4 1 INTRODUCTION OF LEVEL ONE ELKMHANYTER 4 LEVEL ONE Chapter 4 Level One 4 1 Introduction of level one elements 4 2 Basic elements 4 2 1 BARRIER Barrier state diagram gt some material detected Wait for event to start C concentration at 4 still to high G A pickup and transport back 0 still material in the line 45 4 2 BASIC ELEMENTS CHAPTER 4 LEVEL ONE 4 2 2 CATCHER eU tri el tri Wait for event to start cU tri el tri Catcher state diagram some material negatively charged detected concentration at sensor sO in the supply channel is not sufficien
45. of the edges see 2 3 3 for further details means changing size Creating rectangles with the left mouse button in other top level windows is also possible but zooming into these areas is not implemented see 2 3 3 2 3 3 Mouse buttons and cursor keys In general the ng biopro software expects a three button mouse at least This might be a problem for Windows oriented two button mouse users as well as for MAC OS oriented single button mouse users In the sequel the discussion about left and right is based on the assumption that the mouse is used by a right handed user For left handed users and thus exchanged button meanings left and right are reversed 2 3 3 1 Left mouse button Usually the left mouse button serves for selecting objects drawing sensor areas and zoom areas see section 2 3 2 for details on zooming There are essentially four different types of action when using the left mouse button e Press and hold and move With this operation the user creates a rectangular area Currently the minimum size of such a rectangular area is 10 pixels After releasing the left mouse button the rectangular area remains visible The further meaning of the area is then determined by the next mouse click e Clicking the left mouse button Several different possible reactions occur depending on the context 17 2 3 WINDOWS AS USER INTERFACHAPTER 2 USER INTERFACE Usually clicking the left mouse button means selecting what is below the
46. on can be relaxed and the camera view is as before the synchro nization procedure with the exception that the electrodes from the design window are still superposed onto the camera s view Clicking the Adjustment Reverse button a second time switches on the cam era s view rotation again Remark All higher level regulatory feedback elements require the synchronization of the camera window with the design window They cannot be activated if synchronization is missing 29 2 5 DESIGN WINDOW CHAPTER 2 USER INTERFACE 2 5 Design window The design window shows the micro fluidics structure from the computer aided design point of view It is derived from the original data which has been used in the construction of the micro fluidics device As with the camera window is it composed of several sections a menu bar with default pull down menus a menu bar with icons often used and the micro fluidics device s design In contrast to the camera window the user can zoom into the design window arbitrarily often see section 2 3 2 Plot BioPRO Reactor gt Eile Options Qe Bao aA GS Colors Fonts Control Series Sequence Log Shifts Pumps K BSAS AIX INS BNB BIONIb LE a In the following the buttons icons apart from the standard Colors and Fonts buttons see section 2 3 1 are described Navigating in this window is described in section 2 3 3 2 5 1 Pumps
47. r concentration at 1 not sufficient 48 4 3 NETWORKS CHAPTER 4 LEVEL ONE 4 3 Networks o A NU OF say naserial detected Co concentration at 4 not sufficient Co R concentration at 4 st to high base state bacsate Barrier concentrate repell Catcher ed still material in the line transfer 2 pickup and transport back S base state base state concentration at 4 not sufficient rage 0P split as some material detected Trap Spreade concentration at 3 not su ifficient transfer back force apart inia O transfer 5 tion ar 1 not sufficient 49 Chapter 5 Programmers Manual 5 1 ng biopro Library 5 2 Client Server message passing 5 3 Drivers interface 5 3 1 Parallel port Pin assignment of PC parallel port to adapter 50 5 3 DRIVERS INTERFACECHAPTER 5 PROGRAMMERS MANUAL Signal Name Register Bit DB 25 Pin I O Direction Board BioPRO G88 40 Strobe C0 1 Output GCK5 30 Data Bit 0 DO 2 Output TMS 25 Data Bit 1 D1 3 Output TDI 26 Data Bit 2 D2 4 Output TCK 27 Data Bit 3 D3 5 Output gt Vee Data Bit 4 D4 6 Output gt Vee Data Bit 5 D5 7 Output gt Vee Data Bit 6 D6 8 Output gt Vee Data Bit 7 D7 9 Output gt Vec 37 39 Acknowledge S6 10 Input Busy S7 11 Input TDO 28 Paper End S5 12 I
48. rewire camera e g Vosskuehler 3 1 1 2 Andor CCD 3 1 1 3 Cascade II Princeton Imaging 3 1 2 BioModule and electrodes 3 1 3 Pumps 3 1 4 Filter wheel 3 1 5 Temperature control 3 1 6 AOTF and light sources 3 1 7 The parallel port 3 2 Graphical interface 3 3 DPD Interface dissipative particle dynam ics The purpose of the DPD interface is to allow an intimate connection be tween the experimental Omega machine and a simulation tool in this a case Dissipative Particle Dynamics which is a meso scale simulation system Of course any other molecular dynamics code or partial differential equation system would fit as well The role of the ng_biopro software is twofold firstly to give the simulation code the ability to operate the Omega machine especially creating a loop between observation camera output and actua tion electrode control This would allow an informed feedback loop with the simulation providing the none observable details and knowledge of the 40 3 4 CLIENT SERVER INTERFACE CHAPTER 3 INTERFACES chemistry going on in the micro fluidic system The second purpose of this interface is to provide the simulation with the exact geometric and experi mental details of the experiments This is especially useful if the simulation tries to incorporate phase boundaries and inhomogenous geometries Definition of biopro simulator interface by U Tangen and T Maeke 2004 09 15 rev 1 07 Messages are ASCII st
49. rings with items separated by blanks String constants are enclosed in quotation marks Parameters not being numbers are intepreted as names they must not contain white space characters All geometric values are measured in TN ten nanometer All coordinates are written in tripletts 3 dimensions embraced by round brackets The communication is TCP socket based with port number 8085 server gt client client gt server Type A area J Intensity of sensor given back B body Q Abort communication server goes into wait mode C channel element N new design extraction E electrode I fluid input J O fluid output P polygon element Q Q Quit Abort N R reset simulator S sensor T tag U surface V vertex X Finished design extraction sensor name value General message structure type name nr _of_tags tags A name 2 vertices area of interest lower left upper right announces a change of area B name m surfaces constituting this body C name i gt 0 Rol gt 0 input tags output tags E name k tags I name n tags O name n tags P name 1 geom k list of vertices for a line k 2 follow up lines do have the same name S name k tags T name k values of this tag U name n polygons constituting this surface V name m 1 size tags X Special tags nr of args arguments active_pol 0 1 polarity in active puls geom x
50. sion history Sa E E REA E 55 6 3 Release Notes e 55 Bibliography 57 Chapter 1 Introduction This manual is meant for normal users and software developers at the same time using the electronically controlled microfluidic chips BioModules The overall goal for this software is to facilitate and operate these mi crofluidic chips with hundred or more electrodes and many different variants of fluidic designs It has to be admitted that this task is difficult and that the average reader should not expect to utilize the full power of the software and these special chips in a few minutes of training To illustrate the complex ity of the software about two hundred thousands lines of code and several man years of development are behind what is described in this manual The software so far is running on Linux MacOS X and potentially every other Unix like operating system Binaries can be provided on short notice given that the according computer plus compiler are available Essentially three parts are provided with this software a Perl script which operates the ng_biopro software several design files for fluidics electronics and FPGA field programmable gate array 1 1 Installation instructions Currently May 2007 the installation of the software is not as easy as a typical Windows user would expect An experienced Unix manager should install this software Two environment variables are required to be set in advance HOME
51. software is to have a generalized interface to a multitude of different cameras and not to get out the most advanced features from a specific camera Each 22 Help 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE camera driver has been optimized to provide the relevant information and the really needed fine adjustments On the screen this range is mapped onto an adjustable number of colors e g 128 with an emphasize on the lower intensity regions This might result in an overshooting of the high intensity areas In addition not every image is mapped but a floating average of six images provides the general intensity scale The available features are the following e Gain Each camera has a different dynamical range Many CCD cameras provide a considerable dynamical range e g 12 bits or even more An adjustable preamplifier even can increase the sensitivity traded with noise in the image e Exposure time Some cameras in addition allow the specification of an exposure time The scale shown here is in seconds of exposure What exactly seconds means depends on the camera The camera driver tries to map the special camera features to allow the definition of an effective exposure time which is comparable to other camera types 2 4 2 Storing video sequences The ng_biopro software maintains a ring buffer of images taken from the camera output In the current version this image buffer comprises 400 images with an exact in micro se
52. t e0 3 3V base state el 0V concentrate x concentration of labeled molecules e0 electrode next to molecule supply el electrode next to e0 sO sensor in supply channel f0 x sl sensor in concentration channel f1 x el 3 3V concentration at sensor sl is not sufficient e0 0V fi el 3 3V SUL lower threshold 10 of sensor in supply channel sOu upper threshold of sensor 90 in supply channel s11 lower threshold of sensor 10 in concentration channel slu upper threshold of sensor 90 in concentration channel 46 4 2 BASIC ELEMENTS CHAPTER 4 LEVEL ONE 4 2 3 Neuron 4 2 4 TRAP The trap Wait for event to start E base state some material detected concentration at 4 not sufficient signal to following stage that material is free release concentrate material not yet released concentration at 3 not sufficient transfer nn transfer de concentration at 2 not sufficient 47 4 2 BASIC ELEMENTS CHAPTER 4 LEVEL ONE 4 2 5 TRIGGER Trigger state diagram some material detected Wait for event to start C concentration at 4 not sufficient base state concentrate signal to following state that event happened 4 2 6 SPREADER Wait for event to start some material detected base state concentration at 4 not sufficient spli concentrate concentration at 3 not sufficient concentration at 3 not sufficient force apart transfe
53. the ng biopro software in server mode the main user interface is provided with the ng biopro software working in client mode somewhere else in the world This client server communication is re alized via a socket based TCP IP communication channel see section 3 4 2 3 Windows as user interface The major communication interface with the ng biopro software is realized via typically two different graphical windows One of these provide an on line camera screen and the other the electronic and fluidic design to allow the user to navigate to areas of interest and to provide her with full low level control of the electrodes Each of these windows has different functionalities comprised in a menu area at the top of each window Certain icons with corresponding short texts usually raise new windows with specialized functions Each of these top level windows in addition contain two buttons each for specifying colors and text fonts 2 3 1 Colors and fonts etc Defining colors and fonts is done via two popup windows shown below The icon shown represents the according functionality in the menu bar of these top level windows 14 2 3 WINDOWS AS USER INTERFACHAPTER 2 USER INTERFACE MAA Define and change colors Definition of colors 2 Fixed colors Palette colors Background Highlight 128 On FirstCol Off Undefined LastCol Special Color 6 Color 7 Color 8 Color 9 Color 10 Color 11 Color 12 Color 13
54. the paths given in this section probably would not need any modifications In case you want to test different designs which are not given in the standard installation these paths let you define what electrode designs to be used where the bit 11 2 1 PERL TK USER INTERFACE EPM USER INTERFACE streams for the FPGAs to be found and what the required other fluidic variants are The user is allowed to specify different design directory paths using the Select button on the right side If something changed in these directories during runtime of the startbio script the user might press the ReScan button The startbio script then regenerates the scripts used when launch ing the ng_biopro software see section 3 7 2 1 3 Design tab File Mode Help E Info Path Session FPGA Postproc Z Biopro2 Biopro2_PCB y Biopro2 tiny uy Biopro3 y Biopro3 tiny wv Biopro4 y Biopro5 single okd y BioproS double y Biopro5 single new Bioproceram y other Fluidic Design Z Standard Fluidic Modul ay M3 y M4 y M5 y M6 v M7 m8 Preview EN EN Depending on the BioModule attached and the concrete fluidic variant realized the user has to specify the design e g Biopro3 After defining the correct type the Perl TK interface provides the available fluidic designs in this example Bubble Fan SegmFlow or Standard With the fluidic design chosen e g Fan a further section beco
55. tore a single snapshot on the disk or create a series of snapshots each many seconds apart to allow a long term observation of what is going on in the system As was described in section 2 4 2 1 additional information for later image processing is appended at the end of the snapshots 2 4 4 Long term measurements Arbitrary many measurements with recorded intensity values can be under taken These measurements directly work on the images retrieved from the camera Each measurement consists of a line with at least three rectangular areas All pixel intensities in each of the areas are summed up and divided by the number of pixels 25 2 4 CAMERA WINDOW CHAPTER 2 USER INTERFACE 2 4 4 1 General principle File Options Help Sw s lt Na COXA El gt Gain O S Min O Avg 140 Max 1023 Extended biofox_A 43 biofox_B 43 1 biofox_F 43 2 biofox_AOTF biofox_blue o SU Z biofox_red o E m Toggle output An example of such a measurement chain is shown above The violet square is area zero and the bottom square area four When starting a mea surement an output file is created with the current time in the file name additional information concerning the position of the measurement areas is given as well After the specified time a new measurement is scheduled and all averaged intensity values are written in a single line with the exact time of the measurement stated see sectio
56. which points to the home directory of the user running this software and NGEN DESIGN pointing to the design tree with all binaries and design files see section 1 1 2 Furthermore it is expected that the user has a directory in his executable search path which is owned by himself which typically is SHOME bin 1 1 INSTALLATION INSTRUCTIONSCHAPTER 1 INTRODUCTION 1 1 1 Operating system requirements The operating system requirements for Linux Version 9 0 and higher are available in most of the distributions The software uses X11 GTK 2 pango atk freetype fontconfig readline and Perl TK With MacOS things are more complicated because the programming environment by de fault usually only supports X11 Perl TK and GTK 2 have to be compiled and installed separately This can become quite tedious and cumbersome and is usually only be manageable by an experienced system manager the Xcode utilities and the X11 development environment from Apple have to be installed prior see 6 1 1 1 2 Directory trees and installation A default installation would look like this HOME an environment variable containing the path of the user s data area SHOME bin a directory which is in the search path for executables the name bin is not mandatory every other name would suffice also HOME sessions a directory which will be created during installation and which serves as a root for sessions using this software the name sessions
57. yringe diameter is the correct one If you are changing the syringe from 1 03mm diameter to 4 16mm without adjusting the value in the parameters window huge flow rates can be generated which jeopardize the attached micro fluidic structure 2 5 2 Shift register and basic electrode control The main purpose of the ng biopro software is to provide control of in princi ple arbitrary many electrodes It is implicitly supposed that these electrodes are of digital nature and that individual voltage levels are fixed Generally all electrodes are able to express three different states OV Vss with XCS20 3 3V and tristate high impedance regime These three states are in a cer tain ideal state The potential of OV is quite accurate typical voltage levels are below 50mV Vss with 3 3V usually exerts voltages between 3 0V and 3 2V depending on the concrete supply voltage of the FPGA With Spartan 4 circuits up to five different voltage levels can be supplied varying between 1 2V and 3 3V These voltage levels are generated from outside the FPGAs The most peculiar state is the high impedance state tristate Ideally there should be no potential and the impedance between one electrode and any other electrode of the chip should be infinite In classical digital electronics this is not a real problem because impedances from more than 1M Ohm can be interpreted as infinite In microfluidic scenarios typical fluidic resistances are in the several M Ohm regim
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