Date: Wed Sep 13 2006 - 16:00:42 EDT
Hi Guy, Since you asked... You're correct that the ImageStream system is configured like a traditional widefield microscope, not a confocal scope. Our 0.75 NA objective results in an optical depth of field of about 1 micron. This, combined with hydrodynamic focussing of the cells with a positional accuracy of ~1.5 microns, results in a very consistent equatorial "coffee ring" image of membrane-bound fluorescence signals. I would say that the change from a sharp "coffee ring" staining pattern to one that is uniformly distributed over a relatively wide band of cytoplasm is pretty good evidence of internalization, and in my opinion it can be done well with our system. However, I would also agree that any methodology relying on an uncontrolled assessment of fluorescence distribution alone would benefit from a decreased depth of field like that provided by confocal imaging. In my opinion, it's preferable to use an internal control for surface localization. The way we generally assess internalization is to label each cell with a known surface-bound control marker in one color along with the experimental marker which is being assessed for internalization in a second color. We then perform a cross-correlation of the two images of each cell and look for a decrease in image correlation as evidence that the experimental marker is redistributing. This methodology detects capping and/or internalization because any relative signal redistribution between the two images that exceeds 0.5 microns (our pixel size) is reliably detected. It won't replace FRET if you need Angstrom or nanometer-scale co-localization (like detecting the inner versus outer leaflet membrane positioning), but by the same token, it is very easy to implement and has a much wider "spatial dynamic range" than FRET. The cross-correlation methodology also be used to determine compartmentalization if you label the endosomes, lysosomes, or golgi. Nuclear translocation of transcription factors is assessed by labeling the nucleus with a DNA binding dye and cross-correlating the nuclear image to the image of the transcription factor. There are a number of studies published and in press that utilize this methodology. If you're interested, I would recommend the following for a more detailed description of the cross-correlation approach itself: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dop t=Abstract&list_uids=16563425&query_hl=1&itool=pubmed_docsum Best Regards, David David Basiji, Ph.D. CEO, Amnis Corporation 2505 Third Ave., Suite 210 Seattle, WA 98121 (206) 374-7165 direct (206) 919-3342 mobile (206) 576-6895 fax www.amnis.com ________________________________ From: Guy Hermans [mailto:Guy.Hermans@ablynx.com] Sent: Wednesday, September 13, 2006 2:43 AM To: Cytometry Mailing List Subject: RE: receptor internalisation analysis Hi V., could you please elaborate on that? This is the type of discussion I continuously see when critically evaluating data re: cell membrane binding versus diffuse internalization. True sceptics will dismiss classical fluorescence microscopy images showing cytoplasmic staining (diffuse or punctate), arguing you really need confocal to decide whether you're seeing surface stain versus intracellular - and I do see their point. The way I understand the Amnis device works, confocal is really not an option. Seeing the individual cell's staining pattern may therefore be nice to have above and beyond the dotplots/histo's and whatnot outputs as from a conventional cytometer, but shouldn't be used as a definitive argument for internalization. I would therefore argue you can use the Amnis to gather more data from your cells, but for this particular application having the macrofocal image shouldn't make the decisive difference. Doing the "stripped cells" experiment, using fluor-specific secundaries, quenching and/or pH sensitive dyes will therefore be as necessary as when using a non-imaging device. Just my two cents, and purely based on my limited knowledge of the Amnis of course. If it does do confocal as well - hey guys, if you read this, write it in block capitals on all your flyers and put me down for one machine to start with! ;-) Guy Next generation therapeutic antibodies <http://www.ablynx.com/> Guy Hermans, PhD Senior Scientist Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 Add me to your address book... <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Want a signature like this? <http://www.plaxo.com/signature> -----Original Message----- From: rozenkov@netscape.net [mailto:rozenkov@netscape.net] Sent: Tuesday, September 12, 2006 7:12 AM To: Cytometry Mailing List Subject: Re: receptor internalisation analysis An ImageStream flow cytometer from Amnis could do this with no additional procedures... Regards. Vladislav Rozenkov -----Original Message----- From: julie.bertout@ibl.fr To: cyto-inbox Sent: Fri, 8 Sep 2006 9:56 PM Subject: receptor internalisation analysis Hi, I have a researcher interested in quantitating receptor internalisation after different treatments. Here is the way she used to do it : - label receptors of interest with primary antibody and secondary antibody - do the treatment she wants to test - remove label from non-internalised receptor with a slightly acid treatment (so only internalised receptors will still be fluorescent) - analyse her samples fluorescence to see a difference. the problem is that, as antibody/receptor afinity is different from one receptor to another, she can't do her positive and negative controls... for example, the receptor which she used as control (which internalisation shouldn't change with/without treatment) has such a high affinity with its antibody that she can't remove it with the acid treatment. So I would like to know if there is any way to quench fluorescence for the receptor that are not internalised or if anybody has a protocol to quantitate internalised receptor? I thought of comparing non-permeabilised vs permeabilised samples but PFA fixation permeabilise cells a little, doesn't it? Thank you for your answers, Julie Bertout cytometry lab Institut Pasteur de Lille 1 rue du professeur Calmette 59800 Lille France ________________________________ Check Out the new free AIM(R) Mail <http://pr.atwola.com/promoclk/100122638x1081283466x1074645346/aol?redir =http%3A%2F%2Fwww%2Eaim%2Ecom%2Ffun%2Fmail%2F> -- 2 GB of storage and industry-leading spam and email virus protection. ________________________________ This e-mail message is intended only for the use of the individual or entity to which it is addressed and may contain information that is privileged, confidential and exempt from disclosure. 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