>Hi Guys, > >Every single Flow Cytometry textbook and manual states that fluorescence >is measured on a logarithmic scale but never explains exactly why. So, >what is the physical the reason of this particular behavior ? There are at least two reasons for this. 1) The dynamic range of the fluorescence signals vs. the capabilities of the analog measurement systems. Antibody staining can have a dynamic range of four decades (or sometimes more). Typical analog circuitry, in particular, peak detection circuits, fail to work over such a wide range. So log amps were used as a way to compress this wide range into one which could be handled by these circuits. Coulter had designed a clever 2 stage analog circuit, each measuring a part of the range, which allowed the full four decades to be measured using linear amplification. BD uses a 14-bit fast ADC to accomplish the same in the DiVa. 2) Even if measured in a linear amplification system (such as the Coulter or BD one), the presentation of such widely varying data benefits from log scaling, especially since many antibodies stain in a pattern approaching a log normal distribution. Note that not all fluorescence measurements need to be done using log amplification and scaling. Cell cycle data, since it has a dynamic range of less than one decade, is more effectively measured using linear amplification and a linear presentation. > Also, why >do we measure FSC and SSC on linear scales ? Forward scatter for PBL's or cell lines is generally measured using linear amplification and presentation because its dynamic range is low. Side scatter on PBLs is effectively measured using either type of amplification. Marty > >Thank you for your help. > >Thierry Sornasse. > >Content-Type: text/x-vcard; charset=us-ascii; > name="areitsma.vcf" >Content-Transfer-Encoding: 7bit >Content-Description: Card for Andrea Reitsma >Content-Disposition: attachment; > filename="areitsma.vcf" > >Attachment converted: Asimov:areitsma.vcf (TEXT/ttxt) (00069369) -- Marty Bigos Director, Flow Core Gladstone Institute of Virology and Immunology Building 3 SFGH Rm 509 415-695-3832
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