3. Display of Cytometric Information in the Internet
Cytometry is a fast developing multidisciplinary science.
The high interest in improved diagnostic, prognostic and therapy control
procedures in medicine as well as cellular aspects in immunology, cancer
research, molecular biology, biotechnology and microbiology is concurrent
with the development of cytometric assays for a variety of
biochemically specific cell functions and markers, of instrumentation
for flow and image cytometry and of software for cytometric
multiparameter data processing.
Due to the multidisciplinary nature of the cytometric field it is
increasingly difficult for scientists to overlook the individually
relevant progress due to cytometric contributions in many different
journals and meetings.
At this point, the Internet is attractive for several reasons. A major
interest concerns the potential for fast international information
exchange e.g. by bulletin boards
(3.1)
of scientific societies and user groups for the announcement of meeting,
workshop and courses
( 3.1.1),
by displaying extracts of the scientific literature (abstracts, table of
contents of journals
(3.1.2)
or by providing collections of laboratory procedures
( 3.1.4).
Another even more important aspect concerns the interactive potential
of the Internet for the conceptual development
( 3.2)
of science e.g. by the installation of concept fora
( 3.2.1)
by the preparation of consensus processes
( 3.2.2),
by participating in the discussion of E-mail groups (e.g. Purdue E-mail
box)
( 3.2.3),
by advancing scientific software development
( 3.2.7)
for the standardized establishment of individual patient disease diagnosis
and especially disease prognosis from multiparameter cytometric and
other data and by promoting international quality assurance
( 3.2.2)
programs and educational offers (STEP program, telelearning
( 3.2.6)
in this area.
The Internet provides a significant lead time for the early
recognition of new interest areas in this fast expanding multidisciplinary
research field especially when compared to the traditional time lapse
between scientific concept, experimental phase and result publication.
Due to the rapidly increasing importance of the Internet for the cytometric
field during the last year, it is the goal of the Rimini 1996 tutorial
to provide the essential basic information on Internet access,
operation, diagnostics and performance enhancement
for the interested scientist.
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The Internet is a world wide electronic network. It consists
physically of wire and glassfiber cables supplemented by terrestrial or
satellite radio communication and terrestrial infrared laser links.
Data in this network are exchanged between computers by the standardized
TCP/IP protocol.
The network is mostly accessed from PC, MacIntosh or
Unix computers via fast ethernet lines (250Kbytes/sec), by
digital ISDN telephone lines (8 or 24Kbytes/sec) or by modem
connections via slower analogue telephone lines (3.6Kbyte/sec =
28800baud) using the SLIP or PPP data exchange protocol.
The hardware connection between computer and network is established
by computer bus access via a plug-in network card, via external
interfaces attached to the parallel printer port or via telephone modem
attached to a serial (RS232) port of the computer.
Appropriate driver software establishes the data communication between
computer and network. Telephone modem access to the Internet is assured
by access provider companies. Such companies operate gateway
computers to the Internet. Universities or other larger organisation
uses typically the faster ethernet access to the network.
Each network computer is identified and accessed by its unique
URL
(Universal Relocater) address consisting of a mnemonic name e.g.
pcv4.biochem.mpg.de which is transformed into a unique computer address
number e.g. 141.61.1.64 by a name server computer of the network.
Network computers are set up either as clients or as server
computers. Most computers are client computers. Client computers can
demand, display and download information from server
computers and send messages to server and client computers.
Name server computers contain address lists and access
information comparable to e.g. telephone books. This information is
required to identify and access each computer in the network.
Gateway computers provide network access from multiclient and server
environments of large institutions like universities, hospitals,
administrations, access providers or companies.
Routing computers distribute the network traffic within the network
in an optimized way.
Network
analysis
programs permit the analysis of network performance to the individual user.
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The Internet offers different services. The most wellknown is the display
of www-pages with text, graphic, audio and video information using the
hypertext transfer protocol (
HTTP) standard. This
includes exchange of E-mail messages, file download by
FTP (file transfer protocol),
off-line
(2.7.4)
display of downloaded www-pages as well as
Gopher
information system with its multitude of Academic and Newsgroups information.
A variety of commercial or other intranets are installed in
the Internet like the Compuserve, America On-Line, Europe On-Line, T-OnLine
etc. The registerd user has access from any of these nets to the
general Internet. Intranets are not accessible, however, to the general public.
The
World Wide Web Consortium,
a vendor neutral industry consortium develops and promotes new hardware and
software standards for the Internet.
Ethernet access to the network is typical for universities and
other public research institutions. The local computer center
will usually provide the necessary help for new users. Telephone modem
access from home occurs either through large Internet access providers like Compuserve,
America On-line, T-OnLine or smaller local companies, identified through
the Yellow Pages or the Internet search services.
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The client's software access to the network is assured by either freely
downloadable network browser programs (2.4.2) like Netscape, Mosaic,
Microsoft Explorer or by commerically available programs like e.g. Chameleon.
These programs operate from a user surface like Windows3.x, 95 or NT in PC's
or from the MacIntosh MAC-OS operating system. For proper operation they
additionally require software for proper TCP/IP network communication
or telephone modem functionality.
The actual features and download possibilities for a variety of web
browsers can be obtained e.g. by a
Lycos
search for: Mosaic Download Site using the: Match All Words
option to find software download sites or by the direct download of
Netscape,
Mosaic
or
Microsoft Explorer
browser software. It is advisable to download Netscape Gold versions
because the most essential plug-in drivers for multimedia applications
(2.5.3.2)
are enclosed in the download.
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The potential for the display of information in the Internet requires the
presence of a www-server computer. Server computers are installed
in the majority of universities or large public research centers and mostly
set up under a Unix operation system. Departments, laboratories or
individual researchers can localize their www-pages either on the central
www-server computer or they can install separate www-servers e.g. in PCs by
appropriate programs.
A comparatively cheap possibility for the installation of personalized
www-pages outside public institutions is provided on the www-server
computers of access providers or in commercial nets like Compuserve,
America On-line etc. The installation of www-servers on PCs accessible by
telephone modem is also possible but the data link is slow and the telephone
costs are high for continuous server operation. Access is faster
by ISDN telephone lines (64Kbit/sec i.e. 8 Kbyte/sec) but telephone
costs are still significant i.e. the operation of www-servers outside
public institutioons is mostly restricted to commercial access providers.
The display of www-pages requires the establishment of electronic
pages in HTML (hypertext markup language,
NCSA,
WWW-Consortium).
The textual, graphic, video and audio information of HTML files is
displayed by network browser programs like Netscape of Mosaic. The files
are editable by normal editors and contain ASCII text intermixed
with display and link commands to outside computers, contained within: < >
brackets. Examples of cytometric web pages are provided in fig.1-3
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In addition to text formatting that can make information on the computer
monitor appear like the familiar pages in books or journals, the Web is a
unique computer-based communication medium in that it affords the use of
still images, animation, and sound. All can be used on the same page, but
may require the use of special programs that work with the
browser. There are two types: helper applications that work with files accessed by the browser,
and plug-ins that work with the browser and extend the capabilities of the
browser. Many are available at no charge from the sites that supply browsers.
Graphics may consist of two types: in-line graphics (fig.4)
and full-sized graphics (fig.5) In-line graphics are small to large
images that are embedded in the web page. Small scale images
(so-called thumbnail images) may be used to show the user a sample a
larger scale image that is typically accessed by clicking on the thumbnail.
Virtually all browsers will display the image directly, although some
image types require a plug-in.
Image maps (fig.6) are graphics that contain embedded
reference points that can be selected with the mouse. Such reference
points contain hypertext links to other files or images. This is useful
for organizing menus or for the examination of features of complex images.
An excellent introduction to images can be seen on the Internet at:
Cornell University
or at the
www-Consortium
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Images may contain anything graphical from drawings, plots, charts,
photomicrographs, etc. (fig.7)
For the Web, all images are displayed as bitmapped images. This
means, the image is made up as a mosaic of PIcture ELementS (pixels) on
the computer monitor. Several familiar formats include TIFF (tagged
interchange file format), GIF (graphical interchange format),
JPEG (joint photographic experts group). All three formats use file
size compression. This is important because it reduces transfer
time via the Internet. (As an example, an image file that contains
large numbers of the same pixel (areas of black or white) can save
information about the numbers and location of the same pixel
and reduce the size of the file substantially; a ten-fold reduction in
size is not unusual.)
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Instead of graphical objects on a Web page, one can also display entire
pages as graphics. Adobe developed
Acrobat
to provide page images in PostScript. This format has the advantage
that it is scaleable (that is, you can enlarge the image and see
more detail unlike bit mapped formats where you see only bigger pixels).
Thus you can display any type of information (text or pictures) at
any resolution. The disadvantage is that you need a special viewer
to see Acrobat (pdf : portable document format) files although the
viewer is available at no cost. (You can get a plug-in (see below), the
Acrobat Amber reader, for Netscape web browsers that allow you to view pdf
documents within Netscape). A further disadvantage is that the contents are
not accessible. That is, you cannot capture part of the image and keep the
same resolution; you can only capture the image that is on the computer
monitor.
This kind of information has time structure; some can have non-linear
sequences. All time-based media costs more to create and to use, but if
used well, has greater value than traditional "mono"-media. Streaming video
or audio provides sound/video as continuous delivery. That is, there is no need
to download and play back the entire file. Streaming video requires very large
transfer rates to be useful. That is, individual images must have sufficient
spatial resolution and must be transferred at a sufficient speed to give an
adequate illusion of motion. Such speed is only possible with a direct
(i.e., T1) Internet connection.
Animation, on the other hand, can work with few images, but can give
the illusion of speed. There are several instance in cytometry where
animation can provide more information. Displays of kinetics data are obvious,
but complex multi-parameter data sets that require multiple views may be more
understandable if time is used as a feature.
Most video/animation cannot be viewed directly in a browser, but must
be viewed with another helper application or plug-in. Depending on the file
source, you will need the appropriate viewer. There are several file formats
in use.
MPEG
(motion pictures joint experts group) is one standard that is widely
implemented.
QuickTime is to store, edit and play synchronized graphics, sound,
video, text and music. QuickTime is available for both Macintosh
and PCs. A good source for viewers is:
Lentz or:
Quicktime or at
Apple.
Typical
video/audio
formats for PCs are the .AVI or .WAV format. Practical applications in
cytometry concern the visualization of
intracellular processes
or the display and commenting of critical steps
in cell preparation or staining procedures as well as the demonstration of
instrument setup features like laser focusing or particular flow conditions
in cytometric
analysis or sorting chambers
as well as in microscopic imaging.
Streaming video is available from
VDO
In the future, we may expect
Java
scripts (applets),
JavaScript
or Java programs to deal with animation directly. This can
include various virtual reality displays as well.
QuickTimeVR provides interactive objects for manipulations in space.
Another standard,
VRML
(the virtual reality modeling language), provides for a variety of ways
to use graphical
display and interaction
in time. 3D animation is of interest for the intuitive display of
multidimensional measurements in flow and image cytometry.
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Helper applications are available from a variety of sources like
Netscape ,
NCSA
(Macintosh) or
Windows
Plug-ins are designed to work within the net browser. These are mostly
proprietary formats and there is no standard at this time. Some are quite
useful. An overview and various plug-ins can be obtained at
Netscape
Internet www-pages offer in many instances interactive services e.g.
for sending E-mails, or for filling in search (fig.8) or abstract forms (ISAC
electronic abstract service for the Rimini meeting).
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The functionality of the network can be checked by the individual
user.
Look-up
programs search either the mnemonic name or the numeric
computer addresses.
Ping
programs send a sequence of byte packages
to a distant network computer. The round trip time until the return of
the echo of the Ping byte sequence and the number of lost data
packages are provided by these programs.
Trace
programs provide the exact routing of calls to distant computers by
displaying the numeric and mnemonic addresses of server, gateway and
routing computers (fig.9).
Watch
programs permit fast checks of entire sets
of www-server, gateway and routing computers e.g. for the availability
of intra- and extramural (fig.10 right and left)
Systematic network analysis shows that large distance traffic e.g. over
the atlantic is organized "freeway" like i.e. it mostly proceeds
via the same sequence of commercial network stations in e.g.
the t3answer.net, mci.net, dante.net, dfn.net etc. Final access e.g.
within universities or larger cities, in contrast, may be by "country road"
traffic via variable roads.
"Hotspots" occur presently in some network areas like e.g. San
Francisco but also in certain parts of Europe where network calls may
circle in waiting loops a substantial number of times (e.g. 5-10 times)
between the same routing stations. The call reaches finally either the
wanted station or is terminated by a time out mechanism e.g. of the
network browser.
The response time between call and build-up of a wanted www-page on
the computer screen is frequently very significant especially for graphic
information. While in theory network waiting times should
decrease by the installation of faster data lines, the influx of more and more
network users so far outweighs in practice most speed gains. Network
waitings times can be decreased or avoided in various ways:
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The installation of mirror sites (
Cardiff,
ISAC,
Martinsried,
Oviedo),
speeds up information access by reducing transatlantic long
distance network traffic.
The recording of www-pages, file repositories, E-mail box contents and
software on periodically updated
CD-ROMs
eliminates network waiting time. The information on the CD remains fully
accessible with current network browsers. The off-line available
information is read from the CD while on-line calls are
serviced on-line as long as the computer is actively connected to
the network.
In case no network access is given the off-line files are correctly displayed
while on-line calls cause a bypassable error message.
Because CD-ROM updating is limited for practical reasons to six month
or one year periods, a substantial interest for delay free updates
in between remains in certain areas (e.g. meeting and course announcements,
consensus processes). Such updates can be achieved
automatically
if cytometric www-page providers store their pages in compressed
form on FTP-server computers from where they can be collected by
suitable programs (e.g. all
Martinsried
www-pages)
daily, weekly or monthly according to the interest of the individual user.
Following decompression, the received files can be
inspected
with the network browser off-line from harddisk in the same way as
CD-ROM files.
Network searches for cytometry and cytometry related topics are of
fundamental importance to find specific subjects in the
fastly growing number of Internet server sites.
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Searches can be initially started from the Directory menu e.g. of the
Netscape
browser. Consulting various databases for the term: cytometry
provides quite different results. While some databases like
Alta Vista,
Lycos or
Infoseek
provide a high number of entries, other databases cover cytometry at
present only comparatively scarcely like:
Yahoo or
WWWW.
Lycos has a good tendency to find realistic answers to complex queries and
Alta Vista performs frequent updates on screened www-sites.
Due to the fast development of the cytometric field and the high activities
on the
Purdue E-mail box,
searches have usually to be narrowed down e.g.
cytometry AND calcium AND intracellular like in
Alta Vista Advanced Query
searches.
The search databases are updated by robot search programs which
continuously explore thousands of www-homepages and subdirectories per
day for their actual information content. The results of these searches are
indexed and introduced into the above searchable databases.
The characteristics of some
robot
programs for various purposes are available. Besides identified
robot program, the network is also searched by an unknown number of
unidentified robot programs. The routine access of web pages
by known robot programs can be
blocked
by inclusion of an appropriate ASCII-text file at the beginning of the
first directory branch of a web site.
Computer viruses may be imported from the download of infected programs
but also from text documents containing program bound but operating
system independent Macro Viruses.
It is useful to periodically check computers with Internet access by
anti-virus programs and to follow the
documention
on the appearence of new viruses.
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The variety of media that are supported on the Internet provide good
opportunities for the exchange of information and for the interaction among
individuals and groups involved in cytometry throughout the world.
Announcements via the Internet are instantaneously accessible and
www-pages can be frequently updated if required.
Websites may entertain an E-mail forum to ask questions or
exchange comments with colleagues, and to view or discuss results. The
future will see the formation of "electronic" meetings or workshops
taking place.
Currently there are several cytometric societies international and
national on-line. These are listed at
ISAC,
Martinsried,
Purdue University
[Purdue CD-ROM],
and at the
Wiley Website
Access to several websites has been made more rapid by regional
mirror sites. Such mirrors duplicate the contents of other websites
on remote web sites. Examples include mirrors for the ISAC WWW site in
Germany, Spain and the UK. For Europe the
Cytorelay Node
at the Max Plank Institute, Martinsried web site has mirrors of ISAC,
Purdue University, Scripps Research Institute, and JCSMR in Australia.
For North America, the
ISAC
Web site will soon have a mirror for the
Sociedad Iberica de Citometria
(Iberian Society for Cytometry) in Oviedo.
Notification of upcoming meetings, some meeting abstracts (
ISAC,
Deutsche Gesellschaft für Zytometrie (DGZ),
Europ.Soc.Anal.Cell.Pathology (ESACP),
Abstract Text
1995, workshops, and courses are available at regional web sites
(ISAC,
Purdue
and
Martinsried).
Journal table of contents for Cytometry (since 1995) are available
from the
Wiley
Cytometry Website . It is expected that tables of contents for Clinical
Communications in Cytometry will follow. Tables of contents for
Analytical Cellular Pathology (ACP) are available at
Martinsried,
and recent volumes at the publisher,
Elsevier Scientific.
In addition, tables of contents for other journals are available
at the Elsevier Science
Tables of Contents
service.
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Some are available at
Purdue
[Purdue CD-ROM],
and more are to be expected at the Wiley Cytometry Website.
A source of information on cytometric methods and procedure is available
from several laboratories on the Internet. Coverage is still uneven, but more
are expected. An informative example is the listing of
Functional Cell Biochemistry by Flow Cytometry.
The Internet provides the opportunity for on-line organized meetings
on specific topics in cytometry. As in traditional meetings, participants may
present results using any variety of graphics, and respond to questions from
the audience. Several are planned for the near future.
Concepts in Clinical Cytometry
at Martinsried has the goal to advance the scientific discussion on the
clinical use of cytometry. This should favor consensus processes and
standardized data classification on an international and interdisciplinary scale.
The ISAC Web Site is planning an on-line forum on cytometric assays
of apoptosis.
The format of these platforms will be to present one or two page
communications on current issues. They will be grouped by themes. The
communications can be backed up by links in greater depth.
E-mail discussion remarks are welcome and will be appended to the
communications.
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The Internet can speed access to
working groups
and proposals for consensus protocols in cytometry. The inclusion of
graphics makes these on-line discussion fora valuable sources of current
thinking and practice in cytometry as well as proposals for new methodologies.
Consensus documents that contain information on several protocols are
available at Martinsried
[ Purdue CD-ROM].
These include immunephenotyping in
leukemia
as well as
DNA flow cytometry
and
DNA morphometry
(fig.12).
Ring trial results on standards and quality control are useful
references and several are on-line at the Martinsried site. They include:
leukocyte and
thrombocyte
immunophenotyping or
DNA flow cytometry.
[ Purdue CD-ROM].
Mailboxes have been very useful as a methodological and instrumental
discussion forum. The Purdue mailbox was started in 1989
as an E-mail service at Purdue University. The display of the messages
on the
Internet
since early 1995 has greatly enhanced the popularity of this
mailbox
(fig.11).
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The goal of the
Purdue CD-ROM project
is to periodically compile the Internet
information in cytometry. This facilitates the access to larger documents like E-
mail archives, video, image and audio sequences, software, data file
repositories which is for their size are slow to transport through the network.
CD sequences will be of interest to easily visualize the scientific progress in
this research area.
The potential for publication on the Internet is large, and just beginning.
The advantages of electronic publication include: rapid publication,
the inclusion of multimedia illustrations, and the potential for swift
commentary and response from readers. Valuable Internet publications will be
those publications that produce quality papers that are peer-reviewed.
Currently the ISAC Web Site has an issue in preparation. Examples of other
biomedical electronic publications include:
Journal of Image Guided Surgery
and the
Global Emergency Medicine Archives.
The
STEP
and
TATIANA Programs are examples for learning and
telelearning programs in cytometry.
There are several sources for cytometry software on the Internet. A
catalog of free flow cytometry software is at
Amherst
[Purdue CD-ROM].
A popular download site is at
Scripps
The image analysis program for the Macintosh, NIH Image, is available
by
FTP
along with a number of image files and associated programs.
In addition, useful Internet software is at
Martinsried
[Purdue CD-ROM].
Furthermore databases and cytometric list mode data can be
on-line
classified (fig.13) and practical application of the
Standardized Multiparameter Data Classification (SMDC) in
research and medicine
are shown.
Data file
repositories
provide histogram or list mode files from various cytometers.
In the future they may additionally provide entire data sets e.g. on typical
measurement, evaluation and classification schemes in clinical
cytometry.
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Off-line Internet, a timesaver !
Download
all Martinsried cytometry pages, check the concept,
follow the installation instructions (PC) and
display text and graphics from your harddisk without network delay
For problems or comments, please contact:
G.Valet E-mail:
valet@vms.biochem.mpg.de ,
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a,
D-82152 Martinsried, Germany,
Tel: +49/89/8578-2518, -2525, Fax: +49/89/8578-2563,
INTERNET address: http://www.biochem.mpg.de/valet/cytorel.html
Last Update: Nov.11, 1996