From owner-drosophila@net.bio.net Tue Nov 23 22:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!warwick!not-for-mail From: lsrei@csv.warwick.ac.uk (Dr S T May) Newsgroups: bionet.drosophila Subject: Re: confocal microscopes Message-ID: <2d00rq$8cj@parmesan.csv.warwick.ac.uk> Date: 24 Nov 93 16:07:22 GMT References: <3098.2cf0f8bf@mbcl.rutgers.edu> Organization: University of Warwick, Coventry, UK Lines: 136 NNTP-Posting-Host: parmesan.csv.warwick.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit GETTING CONFOCAL IMAGES ONTO YOUR PC/mac I wrote this little piece a few months ago planning to put it into the newsgroup, but lost my nerve :) Now that we have had a request for fly pictures and for info on confocals here it is..It is a description of how to get images from a BIORAD MRC600 into your local computer for analysis or presentation (even printing). The examples given (as a FTP address), are pictures of Dros (a head and some 3rds). I will also post in a few days (when I find it and unless someone tells me not to ;) ), a 'step by step' guide to dissecting out dros brains (head as well as thorax/abdomen), which suggests an (I think) efficient way of getting out whole brains in about 3-4 mins without damage. This will including the appropriate diagram montage as a GIF/JPEG (or I'll give an FTP address if the file is large - although I suspect it will compact well) =========================================================== Some of you may have seen my recent plea for help with the manipulation (e.g. labelling) of BIORAD confocal microscope pictures in the Internet Newsgroup alt.binaries.pictures.misc. I received several helpful suggestions and promised some enquirers that I would post one or more solutions to the net - here they are: (*NOTE* at least one place in the USA got a PIC to TIFF conversion utility from BIORAD themselves, it may be worth trying to contact them directly, although I wrote to them in the UK twice with no success - the following solutions work fine anyway.) *NOTE* If you wish to convert pictures from the confocal to your local machine, and most of the below is pure gibberish to you (it would have been to me not all that long ago), then show this mailing to your local computer support person(s), or as a last resort, mail me. 1) A Macintosh solution. T.Trusk (ttrusk@its.mcw.edu) and S. Cavrak (sjc@moose.uvm.edu) both suggested using NIH IMAGE (available by anonymous FTP or Gopher from several major Mac archive sites) Unfortunately the versions that I found would not run on my machine (an LCII) because they require a maths co-processor or suitable INIT. Apparently however, IMAGE has a macro that allows for the direct conversion of BIORAD PICs (single or multiple images), in addition, it has 3D image manipulation. NIH IMAGE is FREEWARE. 2) An alternative solution (full instructions below the 'semi-technical' data (I'm not being condescending - just trying not to frighten people off :) ). **semi-technical data - you probably don't need to know this** **Several people (see below) suggested using the pgm/ppm file **format utilities (PBMPLUS toolkit) which are available in most **machine formats (and as portable C code). **This is the most universal solution. **With these utilities, one can strip the header (76b) from the **PIC file and read the crude data directly. You have to tell the conversion program, the exact x, y picture size (pixel range). This can either be taken from the first four bytes of the header (with a suitable editor), or calculated when saving the image (see below). The syntax of the command is (on our UNIX system, this may be different according to your local system or computer): rawtopgm -headerskip 76 X Y 'infile' | ppmtogif > 'outfile' where: X and Y = the horizontal and vertical pixel size; 'infile' = name of the file to be translated (e.g. test.pic) 'outfile' = name of the destination file (e.g. test.gif) | = symbol meaning - output from 'left' becomes input to 'right' > = symbol meaning - put output to this file (instead of the screen) *NOTE* ppmtogif can be replaced by ppmtojpg, ppmtotif, etc depending on the final format that is most convenient to you. STANDARD SIZES for the BIORAD MRC600 (X;Y): Rectangle 1(768;512), 2(384;256), 3(255;170), 4(192;128) Square (X=Y) 1(512), 2(256), 3(170), 4(128) For custom sizes, note the box co-ordinates on the bottom of the screen during the eXtract (e.g. 22;41 to 189;158 => X=168;Y=118, i.e the co-ordinates are INCLUSIVE ). If you don't understand, try eXtracting and watch the co-ordinates on the bottom of the screen. OR TRY saving the file over the top of a pre-existing file of a different size and noting the original file size on the ERROR message that you get. For a file test.pic, full screen (i.e. rectangle 1) translated to test.gif: rawtopgm -headerskip 76 768 512 test.pic | ppmtogif > test.gif It only takes a few seconds! (+ Thanks to: Dennis Anthony (Warwick); Matt Gibson (Warwick); Paul Chamberlain (IBM)) VIEWING + EDITING That's really up to you, there are several freeware and shareware (small donation - based on honesty) packages widely available on the internet (or on the cover of many computer magazines) that are perfectly good or adequate for image manipulation - there really is no need to spend hundred/s of pounds, these are available for everything from the Amiga to SUNs. I mainly use GraphicConverter (by Thorsten Lemke) on the Mac Paintshop pro/JPEGview (view + save only) on the IBM, and XV / Xpaint on the SUN. Remember, if the image is not as good on your terminal as it was on the confocal screen, this is due to display limitations not data degradation. You can edit the picture on your low end unexpanded PC, but you need something with a better video card, or a mac (preferably a big mac), or ideally, a workstation such as a SUN SPARCstation to see the finished product in all of its glory. SENDING/RECEIVING IMAGES VIA MAIL If you want to know how, ask around, read the Newsgroups (alt.binaries.pictures.misc.d or alt.binaries.pictures.utilities - esp. the FAQs: frequently asked questions), or if you don't have any luck, mail me - if I can help I will. If you want to test your receiving skills, then E-mail me for a picture. Some sample images are also available (temporarily) as anonymous FTP from lemming.uvm.edu /incoming as: fly.uue - uuencoded BIORAD pic file (converted into a GIF) larvae.jpg - imaged, converted and edited from a confocal video source Happy imaging, Sean Dr. S.T.May lsrei@csv.warwick.ac.uk ys@dna.bio.warwick.ac.uk .