background image

Operating Basics 

 

Rev. 007 Date 2010-01-13

 

   

CyFlow

®

 space Instrument Operating Manual 

15/26

 

PMT High Voltage, Gain, and Log Amplification 

 

 
Controls 

 

Gain  

 

 

 

 Note

 

Gain 

and 

lin/log 

amplification  mode 
cannot  be  adjusted 
after the acquisition. 

 

 Note 

Be  sure  not  to  use 
too high gain values. 
Increasing  the  gain 
values too much can 
cause a decrease of 
amplification  or  a 
loss  of  signals  due 
to saturation effects. 
 

 Note

 

The gain value does 
not  necessarily  be 
equal  to  the  PMT 
high 

voltage 

nor 

does 

it 

be 

proportional  to  the 
amplification. 
 

Log 
 

Typical 

effect 

of 

change 

 
 

 

DNA  distribution  of 
bull  sperms  with  X- 
and  Y-chromosomes.  

Gain = 445

 
 

 

DNA  distribution  of 
bull  sperms  with  X- 
and  Y-chromosomes. 

Gain = 476.5

 
 
 
 
 
 
 
 
 
 
 

 

EAT 

cells, 

linear 

amplification 
 

 

EAT  cells,  log  3  (3 
decade)  amplification

 

Adjustment

 

 
By means of the gain, the signal amplification can be 
individually  adjusted  in  a  wide  range  for  each 
parameter.  For  the  used  photomultiplier  tubes 
(PMTs)  by  changing  the  gain  value,  the  PMT's  high 
voltage is adjusted.  
 
The  gain  value  of  a  parameter  can  be  selected  by 
clicking into the corresponding gain value field in the 
instrument  settings  box  (see  Fig.  4).  By  using  the 
Right  (Left)  buttons  gain  values  are  increased 
(decreased). 
 
In  case  linear  amplification  is  used,  peaks  are 
expanded to the right (compressed to the left) when 
increasing  (decreasing)  the  gain  (see  example  on 
the left).  
In  case  logarithmic  amplification  is  used,  peaks  are 
moved 

to 

the 

right 

(left) 

when 

increasing 

(decreasing) the gain.  
 
Use  the  gain  to  move  the  peaks  of  interest  to  a 
suited position in the histograms.  
 
Start  with  low  gain  values  around  250.  When 
increasing  the  gain,  make  sure  the  most  intense 
signals  still  appear  in  the  histogram  and  are  not 
moved out of scale. 
Increasing 

(decreasing) 

the 

gain 

by 

50 

approximately  causes  an  increase  (decrease)  of 
amplification by a factor of two. Gain values between 
250 and 600 are a good start for adjustment. 
 
 
For each parameter, you can choose between linear 
or 

logarithmic 

amplification. 

In 

logarithmic 

amplification mode, the signal range can be set to 3 
(log3) or 4 decades (log4), corresponding to a range 
of  1...1000  or  0.1...1000.  Generally,  a  logarithmic 
amplification  is  advisable  if  particles  with  a  broad 
range of intensities above 1:10 are to be analysed. 
 
Click the little arrow, select one of the following 
 

lin

 

(linear) 

log3

 

(logarithmic, 3 decade range) 

log4

 

(logarithmic, 4 decade range) 

 
and click it. 

Summary of Contents for CyFlow space

Page 1: ...CyFlow space Instrument Operating Manual...

Page 2: ...ent Settings 12 The Parameter Setup Dialog Box Pulse Height Area and Width 13 Trigger 14 PMT High Voltage Gain and Log Amplification 15 Sample Speed 16 Threshold Lower Level L L 17 Appendix 18 Install...

Page 3: ...lly any flow cytometric application The applications cover e g Routine and research Immunophenotyping Blood Cell Analysis HIV monitoring Leukocyte Counting Rare Event Analysis Microorganism Analysis F...

Page 4: ...haracteristic colour emission wavelength spectrum This fluorescence light is separated into colour ranges by means of optical filters The intensity of each colour range is analysed for each single cel...

Page 5: ...ometer is operated with 100 240 V AC Switch on main power at the left side of the instrument The 488nm laser has its own power button closeby please also switch on the 488 nm laser All other lasers ar...

Page 6: ...s of the flow cuvette The CyFlow space Flow Cytometer supports either 2 laser spots when employing FloMax version 2 6 or 2 7 or 3 laser spots when employing FloMax version 3 0 The blue 488 nm laser wh...

Page 7: ...nsert sample tube onto the sample port until you recognize a click The sample should be fully mounted within a second Now the measurement acquisition starts automatically the operating software indica...

Page 8: ...ath fluid tube for 5 seconds Stop the instrument while keeping the tube pinched Incubate for 15 minutes Re start the system by pressing START and let it run to the end Run the system with 1 6 ml Parte...

Page 9: ...ential This requires fast recognition and analysis of the events by electronics and computer All Partec instruments are specifically designed to minimize counting losses by providing direct connection...

Page 10: ...No additional analysis steps e g setting gates for beads are required v Less Expenses No reference beads required Performing True Volumetric Absolute Counting Sedimentation and Count Time Cells or oth...

Page 11: ...rward scatter SSC side scatter FL1 green fluorescence FITC FL2 orange fluorescence PE FL3 red fluorescence I PE Cy5 FL4 far red fluorescence I PE Cy7 FL5 red fluorescence II APC FL6 far red fluorescen...

Page 12: ...ion for the particles of interest The adjustments cover the gains of the optical detectors e g the photomultiplier high voltages the amplification mode lin 3 or 4 decade logarithmic lower and upper le...

Page 13: ...meter label To change parameter names click into the fields and enter the names by using the keyboard For each optical channel you may analyse three different pulse properties 1 The pulse height 2 the...

Page 14: ...l other particles will not be recognized by the instrument The trigger parameter can be used for an efficient exclusion of unwanted particles from the analysis Example Assume vertebrate leukocytes in...

Page 15: ...the instrument settings box see Fig 4 By using the Right Left buttons gain values are increased decreased In case linear amplification is used peaks are expanded to the right compressed to the left w...

Page 16: ...decreased by clicking into the corresponding field in the instrument settings box and using the Right Left buttons see Fig 4 The count rate increases with elevated speed values If the speed is increas...

Page 17: ...of small and unwanted background or noise signals below a threshold The L L value of a parameter can be increased decreased by clicking into the corresponding L L value field in the instrument setting...

Page 18: ...educe smoke dust vibrations direct sunlight and direct neighbourhood of heatings as possible The installation room must be well ventilated and dry Temperature 15 30 C Humidity 20 85 relative non conde...

Page 19: ...space shown from the rearside CyFlow rearside Screen Computer rearside Printer CCD Camera Interface Monitor Printer CCD Computer AC AC AC Air Sheath Mouse Keyboard Sheath Waste AC Waste CyFlow rearsid...

Page 20: ...e supply sheath pressure and sample flow rate The Computer connections of the CyFlow space are located at the rear side of the instrument Connect the CyFlow space to the computer with the interface ca...

Page 21: ...during sample run remove your sample and activate CLEAN several times In this way the flow cuvette is back flushed with sheath fluid Restart the sample and observe peak resolution again If the results...

Page 22: ...lled water and a clean brush and flush with clean destilled water several times Remember cleanliness of sheath fluid reservoir is critical for proper operation If the CyFlow space will not be used for...

Page 23: ...1 2001 Warning Laser light can be emitted if the protection cover for the laser beam is removed and the beam shutter is opened Therefore the system is marked with the following laser safety labels War...

Page 24: ...entation control Particle Concentration Analysis True Volumetric Absolute Counting Particle Size and Fluorescence Distribution Analysis True Volumetric Based on precise counting and mechanical fluid v...

Page 25: ...d precision syringe pump for contamination free sample transport Built in air pressure for sheath fluid Sheath fluid pressure is adjustable from 0 300 mbar Computer controlled Default setting 200 mBar...

Page 26: ...d to 32 gates in free logical combinations Regions quadrants 1P ranges Gating Crosstalk On or offline gating and crosstalk compensation provide adjustments without need Compensation to rerun samples D...

Reviews: