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1995-03-09
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H_PROG Manual Page i:
H_PROG V1.1
Contents:
Introduction......................................................Page 1
Menu 1, starting and ending remote mode ..........................Page 3
Initialisation Menu, changing ports and default directories.......Page 4
Menu 2, Staring Calcium measurements and saving data files........Page 5
Menu 3, Manually setting parameters...............................Page 7
Menu 4 Starting single wavelength measurements....................Page 8
Problems, Technical stuff.........................................Page 9
This program is freeware, and you are encouraged to copy it and give
it to people who can use it (see the last page for more information).
Ian Musgrave Ph.D.
Prince Henry's Institute of Medical Research
PO Box 5152, Clayton 3168
Victoria, Australia.
email Ian.Musgrave@med.monash.edu.au
phone +61 3 550 4286 fax +61 3 550 6125
H_PROG Manual Page 1:
INTRODUCTION:
This is a simple program for controlling the Hitachi F-2000
spectrophotometer, imaginatively called H_PROG.EXE. It is intended
mainly for measurement of intracellular ions using either ratio
(eg FURA2, BCECF) or single wavelength (eg. FLUO3, Sodium green) dyes.
BASIC INFORMATION:
This is a very simple program. Basically you can only start a
measurement, stop it, save the data in spreadsheet compatible CSV format
and draw a crude graph of the ratio values. It cannot SET any
parameters, due to the nature of the Hitachi command set. To do this you
have to return control of the Hitachi F-2000 to itself and set them (eg
excitation and emission wavelengths) manually. Sorry about that, but that
is how Hitachi wrote the command language.
Currently you can measure either a single ratio dye (using the CALCIUM
protocol) or a single wavelength dye. The program cannot do wavelength
scans, although it is possible to program it in and I may do it at a
later stage.
WHAT THE PROGRAM DOESN'T DO.
There is no printing facility as I assume you will transfer the CSV files
to EXCEL, QATTROPRO or something similar for post processing. These have
better graphing algorithms than I could ever write.
I have also not written a ratio to calcium section for the same reason
(also peak detection problems). I have written a suite of EXCEL files
(which should be LOTUS and QUATTROPRO compatible) to do just that. They
should be included with this program
There is no autofluorescence correction, as this is quite tricky to do at
the moment (again, you can fix it up in a spreadsheet).
KNOWN PROBLEMS:
The subroutine that checks that a file already exists
may not work properly on all BIOSes. If it says a file known to exist
is not there, I can provide a version for the variant BIOS.
The algorithm that does the online graphing (as opposed to the
graphing of saved data) doesn't purge old data, so that you get bits of
graph carrying over. I have tried unsuccessfully to fix this. The Graphs
are VERY crude and ugly, but they were only designed to give a rough idea
of what is happening.
MACHINE COMMUNICATION PARAMETERS:
The Hitachi should be set to 4800 baud, no parity, 8 data bits one stop
bit for optimum efficiency. You can choose other parameters though. You do
need either a null modem cable or a null modem adapter to connect the
Hitachi to your computer (see page 4-13 of the manual for a confusing
description). These are available in virtually every computer
and electronics store. Otherwise see the cable wiring diagram in section
4 of the Hitachi manual and get your workshop to make it.
H_PROG Manual Page 2:
STARTING THE PROGRAM:
It is usually best for the Hitachi F-2000 to be already on, as the power
spike generated when the lamp is turned on may scramble you computer.
Alternatively make sure a power conditioner is in the circuit.
To start the program, merely type H_PROG at the DOS prompt in the
subdirectory where H_PROG.EXE has been installed. H_PROG can be run under
Windows (tm) by using the H_PROG.PIF file that has been provided. The program
has two switches, /ver, which gives you the version of the program, and /H
which gives VERY brief help.
The program will load up and display Menu 1, the opening menu. The menu
items can be chosen by pressing the menu number or the associated hot key
(the highlighted letter). You can also use the space bar or arrow key to
move to the appropriate item and then press enter. Help is available by
pressing F1 in all menus except the initialisation menu,
where, for obscure technical reasons, you have to use F1 then spacebar.
USING THE HELP READER:
Pressing the Page up key pages up, pressing the
Page down key pages down. The arrow , home and end keys do just what
you would expect them to. The escape key [ESC] returns you to the
program.
MENUS:
There are 5 menus associated with the program, their general descriptions
are below.
---------------------------------------------------------------------
Menu 1, Main Menu:
This starts (or stops) remote control of the Hitachi F-2000, then sends
you to either the measurement menu, the parameter menu or the single
wavelength menu. There is also the ability to display saved files
as a graph of the ratios and the option to change communication
parameters and default directories via the initialisation menu.
---------------------------------------------------------------------
Menu 2, Measurement menu:
Most of the menu items are self explanatory, the program assumes you
are only measuring one ratio reagent, be it FURA, SBFI or BCECF.
---------------------------------------------------------------------
Menu 3, Parameter menu:
Allows you to change the parameters for Ratio measurement
---------------------------------------------------------------------
Menu 4, Single Wavelength Dye:
Measures intensity changes over time for a single wavelength dye
(eg. FLUO3, Sodium Green, bisoxynol). The setup is basically
similar to the ratio measurements
H_PROG Manual Page 3:
MENU 1, MAIN MENU:
1) Start Remote Control: This starts remote control of the Hitachi F-2000,
the Hitachi MUST be in remote control mode before any other command is sent.
2) End Remote Control: This stops remote control of the Hitachi F-2000,
allowing you to change parameters or otherwise operate the Hitachi F-2000
manually.
3) Intracellular Calcium: Sends you to menu 2, the intracellular calcium
measurement menu. You can of course measure virtually any ratio dye from
this menu. (eg. SBFI, BCECF).
4) Change Calcium Parameters: Sends you to menu 3. Allows you to manually
change the ratio measurement parameters.
5) Single Wavelength Dye: Sends you to Menu 4. Allows you to measure any
single wavelength dye (eg. FLUO 3).
6) Graph Saved Data. Graphs RATIO data saved in CSV format. NOTE! this
CANNOT graph single wavelength data (different file formats).
7) Change Default Setup. Sends you to the initialisation menu, where you
can change communications parameters and default directories. You can use
these changes in the one session or save them to an INI file.
8) Quit: Exits H_PROG. The program creates a file called lastrun.sav which
records wether you had saved file 0 before exiting, and some other
housekeeping info (i.e., If you were using Single wavelength or ratio mode
and if the Hitachi is turned on).
H_PROG Manual Page 4:
INITIALISATION MENU:
This allows you to change the default communications parameters and the
default directories of the program. These parameters are stored in a
file is called HITACHI.INI which resides in the program directory. This file
should have been created during setup. You can alter this file from the menu,
or with a text editor. Please note that the entries MUST