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Using  large-­‐scale  Deconvolution  service  over  Internet  at  Imaging  and  Bioinformatics,  LIACS  

Dome  Potikanond  Imaging  and  Bioinformatics,  LIACS  Leiden  University,  Leiden,  the  Netherlands.  

Version  1.4,  April  2012  

In  this  paper,  we  provide  instructions  for  the  use  of  Huygens  Remote  Manager;  a  web  interface  that  allows  users   to   do   deconvolution   on   large   volumes   of   image   data   using   the   Huygens   software   developed   by  Scientific  Volume  Imaging  B.V.,  the  Netherlands  The  software  is  available  for  researchers  and  students  via  a  server  maintained   by   Imaging   and   Bioinformatics   section,   Leiden   Institute   of   Advanced   Computer   Science  (LIACS).    

Introduction  Deconvolution  is  an  image  restoration  method  to  recover  an  original  signal  from  an  observation  degraded  by  the  sensor  system.  Image  enhancement,  on  the  other  hand,  is  designed  to  emphasize  features  of  interest  of  the  image  but  not  necessarily  to  produce  realistic  data  from  a  scientific  point  of  view.  It  does  not  require  an  a  priori  model  of  the  process  that  created  the  image.  Deconvolution  is  capable  of  removing  noise,   increasing  contrast  and  increasing  resolution,  especially  in  the  axial  direction.  

Huygens  Remote  Manager  (HRM)  is  an  open-­‐source,  web-­‐based  interface  to  the  Huygens  software  for  doing  parallel   batch   deconvolution   (Ponti   et   al.,   2007).   HRM   allows   to   use   the   Huygens   software   for   doing  deconvolution   on   2D,   3D   and   time   series   image   data   set   acquired   by   various   types   of   microscopy,   i.e.,  widefield,  confocal  or  spinning  disk  fluorescent  microscopes.  User  provides  the  HRM  with  the  image  and  the  restoration   parameters,  which   are   stored   as   settings   in   a   back-­‐end   database.   Subsequently,   user  may   use  these   settings   multiple   times   on   different   deconvolution   jobs.   The   restored   images   can   be  reviewed/inspected  via  web  browser  before  downloading  the  files.    It  is  also  possible  to  download  a  preview  of  3D  stack  as  an  AVI  movie.  

This  manual  guides  through  all  necessary  steps  on  how  to  access  and  to  use  HRM  service  as  provided  by  our  research  group,  Imaging  and  Bioinformatics,  LIACS,  Leiden  University.    

Material  and  Methods  Section   Imaging   and   Bioinformatics,   LIACS,   provides   service   for   doing   deconvolution   via   HRM   on   our  dedicated  server.    In  order  to  gain  access  to  this  service,  user  needs  to  have  an  HRM  account.  Accounts  are  provided  in  correspondence  with  Fons  Verbeek  (http://www.liacs.nl/~fverbeek).    Accounts  are  personal  and  should   not   be   shared   with   others.   After   having   an   account,   the   user   can   access   the   service   through   the  following  URL:  https://deconvolution.liacs.nl/  [cf.  Figure  1].    

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Figure  1:  HRM  at  Imaging  and  Bioinformatics,  LIACS  –  login  page    

STEP  0:  Log  in  After  login  to  HRM  with  a  provided  account,  the  user  will  see  the  home  screen  of  the  system  [cf.  Figure  2].  User   is   able   to   modify   account   information   by   going   to   the   Your   account   section.   In   this   account  management  page,  user  will  be  able  to  change  email  address  and  set  a  password  [cf.  Figure  3].  It  is  strongly  recommended  to  change  password  after  the  first  login.  

 

   

Figure  2:  Home  screen  -­‐  Manage  user  account  by  going  to  the  Your  account  menu    

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Figure  3:  Account  Management    

STEP  1:  Upload  input  images  Before   starting   a   new   deconvolution   job,   input   image(s)   need   to   be   provided   by   uploading   them   to   the  deconvolution  server.  To  do  that,  from  the  home  screen  go  to  the  File  manager  section  [cf.  Figure  4].    

 

   

Figure  4:  File  Manager    

In   this  section,   there  are  two  system  folder  pages:  Raw   images  and  Deconvolved   images.  The  Raw  images  folder   stores   all   the   uploaded   input   images   ready   to   be   deconvolved   [cf.   Figure   5].   The   user   can   select  multiple  files  to  upload  with  the  maximum  single  file  size  of  2048MB.  By  clicking  on  each  image  file,  user  will  see  its  preview  in  the  panel  on  the  right.  The  Deconvolved  images  folder  stores  all  the  deconvolution  results  and  user  can  download  these  restored  images  if  satisfy.  We  will  discuss  about  this  folder  again  in  the  “review  and  get  back  the  results”  section.  

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Figure  5:  Raw  images  folder  displays  a  list  of  input  images    

STEP  2:  Start  a  job  –  Image  parameters  To  create  a  new  deconvolution  job,  from  the  home  screen  go  to  the  Start  a  job  section.  In  this  section,  HRM  provides  job  creation  wizard  containing  four  steps.  The  first  step  is  to  create  (or  select)  an  Image  parameter  setting.  User  starts  by  choosing  the  setting  to  be  used  for  the  new  deconvolution  job  from  user’s  predefined  settings  or  from  the  templates  created  by  system  administrator  or  create  a  new  image  setting  [cf.  Figure  6].  One  can  also  make  a  copy  of  a  predefined  setting  for  backup  or  quickly  create  a  minor  change.  

   

Figure  6:  Step  1  –  Select  a  predefined  image  parameter  setting  or  create  a  new  setting  

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An   image   parameter   setting   is   composed   of   information   about   format   of   input   image   intended   to   be  deconvolved,   image  geometry,  number  of  channels  and  Point  Spread  Function  (PSF)  related  information  [cf.  Figure   7].   User   can   either   use   a  measured,   aka   experimental,   PSF   acquired   from   a   particular   microscope  setup  or  use  theoretical  PSF  computed  based  optical  parameters  that  can  be  found  in  the  metadata  of  the  input  images.  This  PSF  is  the  priori  model  of  the  process  that  created  the  image  on  each  specific  microscope  setting.  The  more  accurate   information  on  PSF  we  provide,   the  more   likely   it   is   to  get  close  to  the  original  signal.  Ideally,  the  experimental  PSF  is  preferred  since  it  is  more  accurate.  

                           

Figure  7:  Image  parameters    –  Image  format  and  optical  parameters/1    

The  optical  parameters  include  microscope  type  (typically  single  point  confocal),  numerical  aperture  (NA)  of  the   objective   lens,   excitation   and   emission   wavelengths   of   the   lasers   used   in   each   channel   of   the   input  images   (in  nanometer),   type  of  objective   lens   and   the   reflection   index  of   the  medium   that   the   specimen   is  prepared  (if  do  not  know,  use  water/buffer).  Voxel  size  information  (in  nanometer)  is  required  as  well  and  it  can  be  found  in  the  metadata  of  the  input  image  (typically  in  the  unit  of  micrometer)  [Figure  9].  HRM  suggest  as  well  the  optimal  voxel  size  of  the  image  data  since  the  good  result  can  only  be  achieved  when  input  image  has  enough  sampling  density,  not  too  much  undersampling.      

The  time  interval  is  necessary  is  required  only  for  the  time  series  data  set  that  is  when  the  image  geometry  is  either   XY-­‐time   or   XYZ-­‐time.   For   the   practical   session   for   the   Image   Analysis   in   Microscopy   course   this   is  usually  not  the  case.  

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The  back  projected  pinhole  radius  value  (in  nanometer)  is  also  needed,  unfortunately  most  of  the  microscope  manufacturers  do  not  provide  this  information  in  the  standard  way.  Therefore,  user  is  recommended  to  use  the   Backprojected   pinhole   calculator   [cf.   Figure   8]   to   calculate   this   value   based   on   the   model   of   the  microscope  used  to  get  the  input  image.  In  the  practical  session  of  the  Image  Analysis  course  we  use  either  Zeiss  LSM510  or  LSM710  microscope.  The  spherical  aberration  correction  is  the  part  where  the  HRM  provides  PSF  correction  for  the  deconvolution  process  due  to  the  fact  that  the  shape  of  the  PSF  is  not  always  the  same  but  rather  more  elongated  as  the  section  image  is  deeper  in  the  image  stack.  

     

Figure  8:  Image  parameters  –  optical  parameters/2  and  the  backprojected  pinhole  calculator    

STEP  3:  Start  a  job  –  Restoration  parameters  The  2nd  step  is  to  choose  a  restoration  setting.    Again,  user  can  select  the  setting  from  the  predefined  list,  from  the  templates  or  create  a  new  setting  [cf.  Figure  9].  A  restoration  setting  is  a  collection  of  parameters  for   deconvolution   process:   i.e.,   the   deconvolution   algorithm,   the   image’s   signal-­‐to-­‐noise   ratio,   the  background   mode,   and   algorithm   stopping   criteria.   Typically,   it   is   recommended   to   use   the   Classical  Maximum  Likelihood  Estimation  (CMLE)  algorithm  that  gives  a  better  result  compared  to  the  Quick  Maximum  Likelihood  Estimation   (QMLE)  despite  of  the  fact  that   it  usually  takes  more  time.  For  the  signal/noise  ratio,  HRM  provides  also  the  SNR  estimation  that  helps  calculating  SNR  per  channel  from  the  selected  input  image.  There  are  three  background  estimation  methods.  If  user  knows  the  background  value  to  remove,  the  remove  constant   absolute   value   method   generally   gives   better   result.   Deconvolution   is   an   iterative   process.   It  compares   the   quality   of   the   restored   image   from   the   consecutive   iterations   and   stops   when   the   quality  difference   is   less   than   the   quality   change   criteria   otherwise   it   continues   until   it   reaches   the   number   of  iterations.   As   shown   in   Figure   10,   it   possible   to   input   more   than   one   value   of   SNR   and/or   number   of  iterations  separated  by  space.  This  will   result   in  creating  multiple   jobs  with  different  combination  of   those  parameters.      

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Figure  9:  Restoration  parameters  -­‐  Task  setting    

STEP  4:  Start  a  job  –  Select  images  Next  step  is  to  select  image(s)  from  a  list  of  input  files  that  are  already  uploaded  to  the  server  [cf.  Figure  10].  One   can   select  multiple   files   to   be   deconvolved   using   the   same   parameters   setting.   By   clicking   an   image  filename,  the  user  will  be  able  to  preview  an  image  in  the  right  panel  of  the  web  interface.    

 

   

Figure  10:  Select  Images    

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STEP  5:  Start  a  job  –  Create  job  The   last   step   is   to   create   deconvolution   job(s).   All   the   chosen   parameters   that   will   be   used   with  deconvolution  process  are  listed  here  for  user  to  review  before  submitting  jobs  [cf.  Figure  11].    User  can  also  choose  output   file   format  of   the   restored   images.    Be  aware  of   the  output   file   format   since   it  may  not  be  possible  for  further  processing  later  with  certain  software.  For  example,  SCIL  IMAGE  does  not  support  ICS2.  

 

 Figure  11:  Create  deconvolution  job(s)  with  chosen  parameters  on  selected  image(s)  

 

The  newly   submitted   job(s)  will   be  put   in   a  queue  waiting   for   server   to  process.  User   can   view   their   jobs’  status  by  going  to   the  Queue  status   section   from  the  home  screen.  The   job  queue  status  page   lists  all   the  jobs  submitted  to  the  queue  and  shows  whether  a  job  is  running  or  waiting  [cf.  Figure  12].    After  a  job  has  been  processed,  server  will  put  results  in  the  Deconvolved  images  folder  and  remove  the  job  from  the  queue.      

 Figure  12:  Job  queue  

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STEP  6:  Review  and  get  back  the  results  In  the  Deconvolved  images  folder  in  File  manager  section  [cf.  Figure  13],  user  can  have  a  detailed  view  of  the  restored   image   by   following   the   link   above   the   preview   image.   In   this   detailed   view,   HRM   provides   user  different  views  of  the  result.  If  satisfied,  user  can  transfer  the  result  files  back  for  further  processing.  

 Figure  13:  Result  folder  and  result  preview  

 

 

   

Figure  14:  Detail  view  –  Compare  Maximum  Intensity  Projections    

It  is  quite  obvious  from  the  Maximum  Intensity  Projections  (MIP)  that  the  restored  image  has  much  less  out  of  focus  pixels,  especially  in  the  z-­‐direction,  and  significantly  less  background  noise  [cf.  Figure  14].  The  user  can   confirm   the   result   again   from   the   Simulated   Fluorescence   rendering   (SFP)   that   it   is   now   easier   to  

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differentiate  structures  compared  to  in  the  original  image  [cf.  Figure  15].    It  is  even  clearer  if  comparing  slice  by  slice  as  in  the  slicer  view  [cf.  Figure  16].  Note  that  some  signal  in  the  original  image  disappears  because  it  does  not  belong  to  the  actual  signal  in  the  first  place.  

 

   

Figure  15:  Detail  view  –  Compare  Simulated  Fluorescence  rendering    

 

   

Figure  16:  Detail  view  –  Browse  the  z-­‐planes  

   

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Using  Huygens  Essential  over  network  Huygens  Essential   is  an  image  processing  and  visualization  software  package,  part  of  the  Huygens  Software  Suite.  With   specific   licenses   it   is   able   to   do   deconvolution   on  wide   variety   of   images   as  well   as   the  HRM.  However,  at  section  Imaging  and  Bioinformatics,  we  mainly  use  the  Essential  for  creating  visualizations  from  the  deconvolved  data   sets.   It   can  be  used   for   image  cropping   and   removing  undesirable  channels   from  an  original  image  before  doing  deconvolution,  which  could  help  significantly  saving  computation  time.  Another  benefit   from  using  this  software   is   to  disclose   image  parameters   from  an   image   in  the  case  that  user  does  not  have  this  information  at  hand  when  doing  a  deconvolution  with  HRM.  

In  order  to  use  Huygens  Essential  on  our  server,  other  than  having  an  HRM  user  account,  the  user  is  required  to  have  a  system  account  on  the  deconvolution  server  as  well.  Basically,  the  HRM  account  is  used  to  access  HRM   service   via   web   browser   for   doing   deconvolution   on   images   while   the   system   account   is   used   for  remote   login  on   the   server  via  a   secure   shell   (SSH)   connection   in  order   to   run   the  Essential  package.     The  application  transfers  windows  back  to  the  user  on   local  computer.  For  Mac  OS  X  and  Linux  systems  having  the  X  server  running,  using  Essential  software  on  the  server  is  very  straightforward.  On  Windows  machines,  it  is  necessary  to  have  some  additional  software  installed.  

Running  software  from  Mac  OS  X  and  Linux  machines  1. Start   terminal  emulator   software   that   supports  X   forwarding   option.   For  example,  Terminal   is   the  

preferred  software  on  Mac  since  it  is  part  of  Mac  operating  systems  therefore  user  does  not  need  to  install  additional  software.  On  Ubuntu  Desktop  Linux,  gnome-­‐terminal  is  the  preferred  software  for  the  same  reason.    

2. In   the   terminal   emulator,   use   ssh   command   to   create   secure   shell   connection   to   the   server,  deconvolution.liacs.nl,  with  X  forwarding  option  (-­‐X),  provided  username  and  password.    

3. After  successful  login,  start  Huygens  Essential  by  using  the  essential  command  [cf.  Figure  17].  

     

Figure  17:  Running  Huygens  Essential  on  server  using  terminal  emulator  on  Mac  OS  X  and  Ubuntu  Desktop  linux    

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Running  software  from  Windows  machines  1. Install   X-­‐server   software   for   Windows.   One   of   the   freely   available   software   is   Xming  

(http://www.straightrunning.com/XmingNotes/).    2. Install   terminal  emulator  software   for  Windows.  PuTTy   is  preferable  since   it  supports  X   forwarding  

option  and  freely  available.  (http://www.chiark.greenend.org.uk/~sgtatham/putty/download.html)  3. Start  Xming  and  PuTTy   respectively.  Note  that   if  the  user  starts  them  in  different  order,  the  server  

will  not  be  able  to  send  the  Essential’s  windows  back  to  local  computer.  If  Xming  starts  successfully,  

user  would  be  able  to  see  a  small  Xming  icon  ( )  in  the  Windows’  notification  area.  

 

             

Figure  18:  PuTTy  Configuration  dialog    

 

   

Figure  19:  Running  Huygens  Essential  on  server  using  PuTTy  on  Windows  

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4. When  PuTTy  started,  the  configuration  dialog  box  appears  [cf.  Figure  18].  Sets  the  Host  Name  to  the  address  of  the  server:  deconvolution.liacs.nl.  Set  Port  to  22  then  save  the  session.  

5. Next  go  to  X11  options  in  the  category  tree  on  the  left  panel.  On  the  right  panel,  check  the  Enable  X11  forwarding  checkbox  then  go  back  to  the  session  and  save  the  configuration  again.  

6. Making  a  secure  shell  connection  to  the  server  by  clicking  the  Open  button.  7. Login  to  the  server  with  provided  username  and  password.  8. After  successful   login,  run  Huygens  Essential  software  by   issuing  the  command  essential   [cf.  Figure  

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If   there   is  no  problem   in  any  steps,   there  should  be   the  main  window  of  Huygens  Essential  appear  on   the  screen  of  user’s  local  computer  [cf.  Figure  20].  For  more  information  on  how  to  use  the  Essential  for  creating  3D  visualization,  please  refer  to  the  Huygens  Essential  -­‐  Visualization  and  Analysis  user  guide.  This  is  available  for  perusal  at  the  offices  of  the  Imaging  &  Bioinformatics  group.  

 

 

   

Figure  20:  Huygens  Essential  –  main  window  

References  • Ponti,  A.  Gulati,  V.  Bäcker  and  P.  Schwarb.  

Huygens  Remote  Manager,  a  Web  Interface  for  High-­‐Volume  Batch  Deconvolution.  Imaging  &  Microscopy  (2007)  V  9,  No.  2,  pp  57-­‐58  

• Huygens  Software  by  SVI,  http://www.svi.nl/    • SVI-­‐wiki,  http://support.svi.nl/wiki/  • Huygens  Remote  Manager,  http://huygens-­‐rm.org/