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Optical  chromatography  glass  micro-flowcell.  A)  Construction of  the  microfluidic device  showing  the  pathway  for  fluid  (shown  in  gray)  into  and  out  of  the  separation  channel, and  the  laser  beam  focused through  the  channel.   B)  Illustration  of  a  separation  in  which sample  particles  are  constrained  to  the  focal  point  of  the  beam  by  the  size  of  the  separation channel
that is filled by the laser beam. 
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 The polystyrene  (PS)  beads used in  these  experiments were  2 microns  in  diameter (Polysciences, Inc., Warrington,  PA) dispersed in  water at  a concentration of  4×107 particles/mL.   Bacillus  anthracis  Sterne  strain  34F2 (nonpathogenic, vaccine  strain)  was obtained from  Colorado Serum  Co., Denver,  CO.  To  obtain  spores,  overnight  cultures  of  B. anthracis  were  grown on  trypticase  soy  agar  (TSA)  plates (Difco, BD, Franklin  Lakes, NJ)  at 37°C.   A  few  colonies of  each  strain  were  resuspended in  PBS  buffer  pH  7.0 and plated  on 2×SG  sporulation  agar  plates  followed by  incubation at  37°C.   The  spores  were collected  as soon as  the culture reached over  95%  of  phase  bright  spores, usually  after  four  days, and resuspended  and  rinsed in  2 ml  of  cold  sterile  milliQ  water.   The  spores were  further  diluted in water before injection into  the system  to  a concentration of  6×107  spores/mL. Data  collection and analysis  were  performed  using  ImagePro  Plus version 6.0 (Media Cybernetics, Inc., Silver Spring, MD).  Relative  PS  particle  concentrations  were assessed using  automated software routines  for measuring  greyscale levels  in  the flowcell  (particles appear  dark  on a light  background, thus  yielding  a higher  greyscale “concentration” value).   Another  method based upon automated contrast  thresholding  algorithms  available in  the software package was used to  count  the  B.a.  spores.   This  provided a reproducible  method  for achieving  accurate  particle  counts  in  the injected  and laser  concentrated  bands.  3. Results Initial results  obtained  using  the  optical  chromatography  laser  filter  system  indicate  that concentration of  a continuous  stream  or  an entire  injected band are possible. Fig.  2 shows  the process of  laser  retention and sample  concentration.  In  Fig.  2(A), a liquid containing  2  μm polystyrene particles was introduced  as  a constant  flow  (i.e. not  a discreet  injection)  at  a flow rate  of  3.0  μL/hr.  The  objective of  the experiment  was to  “clean up” the particle  laden  stream and in  the process  produce  a  concentrated  band of  2  μm  particles.   With  the  0.9  W  1064  nm laser beam focused into the 50 μm channel in Fig. 2(B), no  particles were visible in  the  channel  as  they  were all  trapped  outside the 50  μm  channel.   After  three minutes  of particle  retention,  the beam  was turned  off  in  Fig.  2(C), and  the concentrated band of  particles was released  (note the concentrated band  of  particles along  the center line of the channel). Additional data  for  a  similar  experiment  (laser  power  =  1.0 W,  fluid flow  =  3.0  μL/hr) showing a six minute laser  retention period  are given in  Fig.  3.  Initially,  in  Fig.  3(A), particles  are  present  at  a  dilute  concentration  in  the  device  (few  particles  visible  in  field  of view)  and then  the laser  is  switched on  at  around  1 minute.  With  the laser  on,  the particle concentration decreases  to  near  zero  (relative concentration  was estimated from  automated analysis  of  greyscale  levels  in  the  video  images).  At  six  minutes  the  laser  was  switched  off and a  concentrated band was  released,  as  indicated  by  the peak  and  the image  in  Fig.  3(C).   After  the  concentrated band  passed  through the  flowcell,  the  stock  concentration was  again seen in the flowcell, Fig. 3(D). 
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C Fig.  2. A)  Stock  concentration  stream  of  2  μm  polymer  particles in  the  channel,  B)  laser  ON and  retaining  all  polymer  particles  (no  particles  in  the  channel),  C)  concentrated  stream  of  2 μm  polymer  particles released  when  the  laser  was switched  off.   Laser  power  was 0.9 W  at 1064 nm, fluid flow rate was 3.0 μL/hr.
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Fig.  3.  Six  minute  laser  concentration  of  2  μm  polystyrene  particles. A)  Sample  of  polymer particles  in  system  with  laser  off,  B)  laser  ON  – flowcell  is  free  of  polymer  particles  with  the laser  retaining  them,  C)  laser  OFF  –  flowcell  is  filled  with  concentrated  stream  of  particles previously  retained  by  the  laser,  D)  return  to  original  dilute  mixture  of  polymer  particles.  Data were background subtracted. 
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For  a continuous  sample  input  of  fixed  particle  concentration, the amount  of  concentration attainable  depends  on  the  length  of  time  the  laser  irradiates  the  flowcell.   This  can  be  seen  in Fig.  4  where the laser  was left  on  for  0 (no laser  - baseline),  2, 4, 6, 8,  and  10 minutes (Supplemental  Movie 1). With  each subsequent  two minute increase in  laser  collection time the  concentration of  particles increased.   With  respect  to  the data  one can see  the  peak increase in  height  and  width as  the  laser  collection  period increased. What  is  not  obvious  in the plotted data  is  the spatial  concentration of  particles along  the center  of  the flow  channel.   Spatial concentration  of  particles  is  defined  as  the  extent  to  which  particles  are  co-located  in one  region (center  of  the  channel)  by  the  action of  laser  radiation  pressure.   The  image  data clearly  show  a tightly  clustered group of  particles with in  the  channel,  whereas  the  plotted data are  the  result  of  the  entire channel  analysis.   The effect  of  laser concentration time  on  the resulting particle bands is summarized in Table 1. The spatial width (thickness of the particle band in  the center  of  the channel)  increases  from  17  μm  to  28  μm  within  the 100  μm  channel.   Without  the laser,  the particles are distributed uniformly  throughout  the channel  (100  μm width).  At  the longer  laser  concentration times, more  particles necessarily  occupy  a larger width of  the channel  upon exit.  The temporal  band width (length of  the particle  band in  the channel)  increases  almost  three-fold  as  a  function  of  the  laser  concentration  time.   This is  due to  the  particles  exiting  the  laser  separation  channel in  a  somewhat  sequential fashion  (versus  a tight  knit  group)  thus  creating  a longer  temporal  band  width with  increasing  numbers. Concentration of  an important  biological  sample  has been accomplished using  the optical chromatography  laser  filter  system.  Spores  of  Bacillus anthracis  have  been bioenriched using the laser  filter  system.  The graph of  particle  counts  versus  time  in  Fig.  5, shows the effect  of the laser  on an  injected band of  B.a.  spores.  Without  the use of  the laser,  the injected  band  of particles quickly  passes  through  the  imaging  flow  channel  with  a  broad  peak  between  2 and 4 minutes.  With  the laser  operating  at  2.5 W,  and a flow  rate  of  1.2  μL/hr,  the  majority  of  the injected  spores  were  retained  and  a  spatially  and  temporally  concentrated  band  was detected after  the laser  beam  was blocked at  10.5 minutes  (Supplemental  Movie 2).  Using  automated image  analysis  software,  the  number  of  particles  in  both  experiments  was  counted.  With  the laser  off,  178 particles passed  through the system  and were  detected in  the imaging flowcell.   In  the next  experiment  with  the laser  on, 152 spores  were  retained by  the laser  and only  8 spores were  unretained by  the laser  which equals  a  retention efficiency  of  95  %.   In  addition,   excellent  sample  concentration  was achieved.  If  one compares  a hypothetical  fraction  of spores  contained in  the  laser  concentrated  peak  (0.22 min  baseline  to  baseline)  to  the  same period  for  the  injected  sample  peak  (no  laser),  there  is  a  substantial increase  in  spore concentration.    There  were  21  spores  /  nL  in  the  laser  concentrated  peak  versus  4  spores  /  nL in  the injected  peak  (no laser):  a  five-fold laser concentration  factor.   Such a  concentration factor  could have  substantial  benefit  for  spectroscopic applications (fluorescence[21], Raman[22],  etc.)  where signals from  small  numbers  or  even single  particles /  organisms are routinely  measured.   Furthermore  these applications  may  be implemented  on-chip  thus simplifying  the analytical  process. 
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Table 1. Time based laser concentration of particles Laser Concentration Time  (min) (μm) 2 16.5   Temporal Spatial  Width Band  Width  (min)a 0.33 4 19.8 6 23.1 8 26.2 10 28.3 0.54 0.57 0.70 0.91 a  measured at  the baseline 
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Fig.  4.  Laser  concentration  of  2  micron  PS  particles.  A)  Baseline  /  no laser,  B)  2  minute  laser concentration  of  sample, C)  4  minutes  of  laser  concentration, D)  6  minutes  of  laser concentration,  E)  8  minutes  of  laser  concentration,  and  F)  10  minutes  of  laser  concentration. Images  were  taken  from  the  peak  region  for  each  laser  concentration  time. (Supplemental Movie 1)
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Fig.  5. Injection of  ()  B. anthracis  spores  in  water  with  the  laser  off,  and  ()  B.anthracis spores  with  the  laser  on. The  laser  beam  was  blocked  at  10 min,  releasing  a  concentrated  band of B.a. spores. (Supplemental Movie 2) 
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4. Conclusions 
The use of  an optical  chromatography  based system  adapted for  complete  sample interrogation  has been  shown.   Previous  work  using  optical  chromatography  was  limited  by the  inability  to  interrogate  all injected  particles.   Concentration  of  particles  from  continuous input  streams and discreet  injections  have  been  demonstrated using  a new  experimental design.  The carrier  fluid and suspended particles are  forced  through a  flow  channel  which is filled  by  the  mildly  focused  optical  chromatography  laser  beam.   The  ability  to  interrogate  all injected particles represents  a significant  achievement  and advance in  optical  chromatography based  systems.   This  capability  will drastically  facilitate  applications  in  both  detection  and sample  preparation using  this  technology.   Currently  work  is  in  progress to  increase  the number  of  particles  /  microorganisms that  can be optically  retained and separated.   This  will facilitate  coupling  with  many  more  complementary  techniques. 
Acknowledgments 
The authors would like to  acknowledge the Naval  Research Laboratory  (NRL) and  the Defense  Threat  Reduction Agency  (DTRA)  for  support  of  this  research.





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