facman

8/8/2019 FACMan

1/10

F AC (F lu or escen ce An a lys is ) Curr ent Version: 8

Authors : Dean Brown, Perkin ElmerDave Fr azer, Flu orescent Microspher e Resour ce Center

Comm ent s/Corr ections : frazer @u.wa sh ington.edu

Manual revised: J anua ry 22, 1996

I n t r o d u c t i o n

FAC (Fluorescence Ana lysis) is designed to read th e fluorescence in multiple sam ples u sing a Perk in Elmer LS50 or Per kin E lmer LS-50B luminescence spectrophotometer. F AC provides bookkeeping for mu ltiple samp lewith a num ber of different fluorescent dyes in each sample. Measur ement s are written to screen and a texfile, with optiona l output to a printer. The resulta nt t ext files are forma tted su ch th at t hey can be easilyimported into spreadsheet s on either DOS/Windows or Macint osh compu ters. The pr ogram works for thecuvett e reader , th e original 96-wellplate reader, a nd t he configur able new wellplat e reader . The pr ogram alsocalculates flow to each piece (ml/min) if a reference blood sample was obtained.

FAC is pu blic doma in softwa re. Only th e execut able file is available for dist ribu tion. FAC will evolve to meetth e needs of resear chers measu ring organ blood flow with fluorescent microspheres. Updat ed versions will beavailable through the FMRC.

F e a t u r e s

FAC is a menu driven program t hat runs under DOS. It ru ns separa tely from the F luorescent Dat a Man ager(FLDM), available from Perk in Elmer. FAC is restr icted to read ing th e fluorescent intensities of samples atup t o 10 different excitat ion/emission wa velength pairs. FAC does not pr oduce spectra or allow for synchronousscanning. However, FAC assists the user to setup a 'method' tha t, when stored, can be recalled an d usedwithout reentering the desired para meters du ring subsequent a nalyses. Storable parameters include excitationand emission slit widths, emission filter settings, the PMT voltage and the excitation/emission wavelengthpairs. Dur ing analysis fluorescent values ar e written to both screen an d text file, with an option to print a ha rd

copy. When using cuvettes th e user has th e option of accepting or rejecting the value after each samp le is readOnly accepted values are writt en to th e text file an d printer . There is a comment line tha t can be u sed for eachsam ple as well. The out put t o th e text file can be format ted for either DOS (tab delimited, car riageret ur n/linefeed) or Macintosh (ta b delimited, carr iage ret ur n). The user ha s t he opt ion of calculat ing blood flow(volum e/time) if a r eference blood sample h as been obta ined a nd t he fluorescence measu red.

I n s t a l li n g F A C

FAC can be installed anywhere on your hard disk, as long as locations for the program and preference file arepresen t as well as dat a files an d meth od files locations. These subdirectories mu st exist prior to run ning F ACfor th e first tim e. An accompan ying INSTALL.BAT file creat es th e needed su bdirectories on th e chosen dr iveTo use the INSTALL.BAT file, type i n s t a l l followed by the drive (e.g., i n s t a l l c ) to which you wish to instalFAC and the needed subdirectories.

The p rogra m comes configured initia lly as follows:

C:I_____FA...........................Progra m an d prefer ence file (C:\ FA)

I_____METHODS .........Meth od files (C:\ FA\ METH ODS)I_____DATA................Dat a files (C:\ FA\ DATA)

FAC (Fluorescence Analysis) Manual Page 1

8/8/2019 FACMan

2/10

F AC O p e r a t i on

The program is initiat ed by typing FAC while in th e same directory as the F AC program. Once initiateduser inpu t is menu dr iven. By convention, the choice enclosed by squa red bra ckets is th e defau lt choice an dcan be chosen by simply pressing th e retu rn or enter k ey.

S E T UP P R O C E DU R EWhen FAC is ru n for th e first tim e, you will be ask ed to init ialize a pr eference file (FAC.PRF ). Onsubsequent program initializations the program skips to the C o n f i r m S e t u p section below. Thepreference file saves u ser su pplied h ar dware an d softwar e configur at ions for fut ur e use. Th e followingquestions a re asked du ring the setu p procedure:

Com p u t e r Disp la y E xp la n a t ion

P r e fe r e n c e s F i l e N ot F o u n dS e t u p R e q u i r e d

F l u o r o m e t er H a r d w a r e1. P e r k in E lm e r LS -502. P e r k in E lm e r LS -50BH a r d w a r e U s e d ( 1-2 ):

Choose which spectr ophotometer you wil l be using.

I s t h e r e a p r i n t e r a v a i l a b l e (Y/N ) : Is th ere a printer atta ched to t he contr ol computer?If so, FAC can create a hard copy of the intensitydat a as th ey are r ead. FAC will prompt th e user totur n t he pr in t er on i f it i s off .

D e f a u l t o u t p u t f il e fo r m a t . M a c /I B M (M /I ) : Select which file format should be selected on adefau lt selection. The only difference between t hetwo is the end of l ine cha ra cter sequence used. Ea choperating system recognizes different sequences toindicate en d of l ine.

Yo u m u s t n o w d e c l a r e w h e r e p r o g r a m f i le s a r e t o b e s t o r e d . T h e p r o g r a mp r e f e r e n c e s fi le m u s t b e i n t h e s a m e l o ca t i o n a s t h e p r o g r a m . I f y o ue v e r m o v e t h e p r o g r a m b e c e r t a i n a n d a l s o m o v e t h e f i le F AC .P R F .T h e r e a r e t w o p a t h n a m e s t o d e c la r e :

D a t a p a t h - s t o r e s f lu o r e s c e n c e c o u n t e r r e s u l t f i le sM e t h o d s p a t h - s t o r e m e t h o d s d a t a f il e s

T h e s e p a t h s m u s t e x i st . T h i s p r o gr a m d o e s n o t cr e a t e t h e m f or y o u .T h e d e f a u l t p a t h s a r e d i s p l a y ed . T o a c c e p t t h e m j u s t p r e s s t h e E N T E Ro r R E T U R N k e y s .

D a t a f i l e p a t h [ C :\ F A \ D AT A] :

Pr ess return to acceptdisplayed setting,otherwise enter avalid path nam e. Ifthe path name thatyou enter does notexist you areprompted to reenteror optionally enter Qto exit th e program sothat you can createthe needed subdirec-tory.

M e t h o d s f i le p a t h [ C :\ F A \ M E T H O D S ]: Press return to accept displayed setting, otherwiseen te r a valid path n ame. If the path name t hat youen te r does not exist you ar e prompted to reenter oroptionally enter Q to exit the program so that youcan create th e needed subdirectory.

This completes the setu p section of th e progra m. Please skip to the M aj or Op t i on s section below.


8/8/2019 FACMan

3/10

CONFIRM SETUP

After the initial setup run , the program initially displays the curr ent set up. An option is present ed toallow th e preferences to be changed as ha rdwar e configur ation or th e user's n eeds vary. With eachsubsequ ent u se of FAC, the curr ent p reference settings ar e displayed as follows:

C u r r e n t P r o gr a m S et u pF l u o r o m e t e r i s LS-50 (LS-50B)A p r i n t e r i s ( is no t ) ava i l ab l eF i l e fo r m a t t i ng i s fo r Macin tosh ( IBM)D a t a fi le p a t h i s C :\ F A \ D A T A \ M e t h o d s f i le p a t h i s C :\ F A \ M E T H O DSCh an ge Se tu p (Y/N) [N] :

If th e setup is correct, accept the default by pressing the retu rn or enter key. To change t he setup, enterY; you will be prompted with th e questions a s listed in section on S e t u p P r o c ed u r e .

MAJ O R O P TI O N S

FAC supports two prima ry options. The first rea ds the sam ple fluorescence an d the second r etrievespreviously acquired dat a and calculates flow per sa mple. The n ext menu allows th e user to choosewh ich FAC option is to be used. See section on Fl ow Ana l ys is for inst ru ctions for t ha t option.

Av a i l a b l e O p t i o n s1 - Us e F l uo r om e t e r2 - F low Ana lys i s3 - Ex i t P r og r amEn ter Op t ion (1-3) [1] :

Us e F lu o r o m e t e r

The first step in m easur ing fluorescence is selection of param eters r equired for m easur ement of eachsam ple. FAC gives th e user th e option of recalling a pr eviously defined m ethod (param eter set ) orcreating a n ew one.

P a r a m e t e r S e le c t io n1. R e ca ll a Me th o d2. Ma k e a Ne w Me th o d

Selec t ion (1-2) [1]:

Opt i on 1 ( Reca l l a M e t hod)

Met h od l is t in g (Y/N) [Y]:

If th e user r esponds with 'Y', th en all of th e meth ods cur rent ly stored in th e meth ods pat h su b

directory will be listed. Oth erwise the user is immediat ely prompted for a user n am e.

M e t h o d N a m e ( Ma x i m u m 8 c h a r a c t e r s - N o E x t e n s i o n s )?

Once the u ser ha s ent ered a method na me, please skip to the Tr ansport device section below.


8/8/2019 FACMan

4/10

Opt i on 2 (M ak e a New M e t hod)

Com p u t e r Disp la y E xp la n a t ion

En t e r Exc i t a t i on S l i t ( 2.5 -15) ?En t e r Em i s s i on S l i t ( 2.5-20) ?

First, the excitation and emission slit widthsare entered.

Em iss ion F i l t e r (Y/N) : Second, the user is asked if an emission filteris to be used . If a filter is to be used , 5 choiceare presented.

S e l e ct o n e o f t h e F o l lo w i n g1. 2 90 n m c u t o ff fi lt e r2. 3 50 n m c u t o ff fi lt e r3. 3 90 n m c u t o ff fi lt e r4. 4 30 n m c u t o ff fi lt e r5. 5 30 n m c u t o ff fi lt e r

Se lec t ion (1-5) :

If you wan t a n em ission filter th en select whichone you want .

Def au l t o r Se l ec t ed P M T vo lt a ge (d / s ): The user can also specify if the default or aselected voltage is to be used for thephotomultiplier tube (PMT).

P MT vol ta ge (0-900): If default PMT voltage (d) is chosen, the PMTvoltage is determined by the spectrophotometer based on th e excitat ion an d emissionslit widths. If th e selected PMT volta ge (s) ischosen, the user enters the voltage at theprompt sh own to the left.

E n t e r N u m b e r o f An a l y t ic a lWave l en g t h s ( M ax 10 ): The number of excitation/emission wavelengthpairs is then ent ered with a ma ximum of 10.

The user is then prompted to enter each pair in a s c e n d i n g wavelength order. Unless th ewavelength s are ent ered in ascending order the pr ogram will not fun ction properly.

E x c i t a tion Wl 1 (200-800 n m ): E m is s ion Wl 1 (200-900 n m ):E x c i t a tion Wl 2 (200-800 n m ): E m is s ion Wl 2 (200-900 n m ):

. .

. .

. .E xc i t a t ion Wl n (200-800 n m ): E m is s io n Wl n (200-900 n m ):

Any Ch an ges (Y/N) : After all wavelength information is enteredthe operator is given the opportunity to makeany changes to the wavelength entries beforesaving the new met hod file.


8/8/2019 FACMan

5/10

E n t e r t h e N u m b e r t o b e ch a n g e d : If yes, the operator is prompted to select theanalytical wavelength pair of values to becha nged. For insta nce, if an invalid num ber isentered in the second emission wavelengthpair entered, you would enter 2. You are thenreprompted to enter both the excitation and

emission wa velength s for t he second pair.

E n t e r M e t h o d N a m e (N o E x t e n s i o n ): After the revised pair of numbers has beenentered the user is asked to select any othercha nges. If no furth er chan ges are to be madethe user is prompted t o select a meth od nam ein which to store th e par amet ers on disk. Anystandard DOS name can be used up to 8cha ra cters an d without file extension.

Now the declaration of the method, or stored parameter set, is complete and available forsubsequent uses by recalling an existing method, as shown above.

T r a n s p o r t d e v i ce

Read i ng fr om cuve t t e , w e l lp l a t e o rl i qu i d h an d l e r ( C /W/L) [C] :

How will samp les be tra nsported to th efluorometer for an alysis? The two primaryoptions are via cuvettes or wellplate readerThe liquid handler is in the experimental andnot ready for u se at this t ime

Wel lp la te If you choose the wellplate option, thefollowing quest ions ar e a sked

New o r Ol d We l lp l a t e r ea de r(N/O)[N]:

Select either the original or new configurablePerkin Elmer wellplate reader.

Ent e r We l l p l a t e Geom e t r y Nam e ( NoE x t e n s i o n ) :

If using the new wellplate, you are promptedto enter the name of the file that describes thegeomet ry of th e wellplate. This is notnecessary for the original reader as thegeomet ry of the pla te is fixed.

A r e a b l a n k a n d s t a n d a r d i n w e l l s 1& 2 (Yes/No )[Y]:

The program is set to read a blank (solventonly) an d a stand ard in wells 1 & 2. This

allows compa rison between wellplates. If youare using this meth od enter Y. If stu dysamples ar e in wells 1 & 2, enter N.


8/8/2019 FACMan

6/10

S t o p a ft e r e a c h w e l l o r e a c h p l a t e(W/P )[P ]:

This gives the operator the option of acceptingor rejecting fluorescent intensities after eachsample is read or waiting until all of thesamp les ar e read. If the operator chooses tostop after each plate, the minimum andmaximum values for each excitation/emission

wavelength pair is displayed at t he en d of th eplate reading.

E n t e r m a x i m u m a c c ep t a b l e v a lu e : If you choose to stop after each plate you areprompted to enter a number above which youwish to receive notificat ions. Ea ch time afluorescent int ensity exceeds this value, a beepis sounded to alert you to the large intensityAfter beeping, the program continues tomeasure the fluorescence in the remainingwells

.

Ou t p u t Op t io n s

O u t p u t r e s u lt s t o p r i n t e r a s c o ll e ct e d(Y/N) [Y]:

The next step is to determine the outputpreferences. If th e initial setup indicat ed th epresence of a printer, the user is asked ifmeasurements should be printed and storedon a disk.

For m a t r e s u l t f i l e f o r M ac i n t os h o r I BM(M/I) [M]:

The user is asked h ow the da ta file should beformat ted; either a Macint osh format or a DOSform at can be selected.

Misce llaneous In form at ion Gath ered

This inform at ion is gath ered to fur th er identify th e samples/study on the disk file and print outs

Am o u n t o f s o lv e n t u s e d p e r s a m p l e (m l ) : Used to keep track of differences which mightbe caused by variation in the amount osolvent u sed.

Scan speed (-1500 to -10, 10-1500) [480n m / m i n ] :

Sets the speed for scans performed during theoperat ion of the pr ogra m.

Re ad va lue t im e (0 .1-100) [1 .0] : This sets the amount time (in seconds) that aread operat ion will avera ge dat a. The longerthe period, the more stable the r esults.

Analys t : Who read t he fluorescence measur ement s?


8/8/2019 FACMan

7/10

S a m p l e I d e n t i f i c a t i o n : Identification (text) for the samples currentlybeing measured.

E n t e r s t a r t i n g s a m p le n u m b e r : This is used to identify each sample. Anynu mber can be enter ed. If the samples beingrun a re a continua tion of a previous dat a setthe true sample number of the first sample tobe read should be used. If you are u sing ablank and s t anda rd in wel ls 1 & 2, the sam plein well 3 corresponds with th is num ber.

St a r t i ng We l l Nu m b er ( 1-96) : If a wellplate reader is being used, theoperator is asked to enter which well to startthe fluorescence reading. All readings areperformed on sequential wells, starting at thisnu mber well. This is th e num ber of th e firstwell to be read whether it contains a blank ora r eal s tudy sample.

E n t e r T o t a l Nu m b e r o f Sa m p l es t o b eR e a d :

This declar es how man y wells will be read. Ithe starting well number plus the totanu mber of samples t o be read exceeds 96, th euser is prompted to put in the next wellplateand r eadings s tar t a t wel l #1 on th at plate .

E n t e r F i le n a m e f o r D a t a (N o E x t e n s i o n ): The user is then asked for a filename for thedat a. The extension '.FA' will a ut omatically beappended to the chosen na me.

F i l e N a m e A lr e a d y E x i s t sS e l e c t o n e o f t h e F o l lo w i n g

1. R en a m e F ile2. Ove r wr it e File3. Ap p en d F ile

Se lec t ion (1-3) :

If the filename already exists, the user ispresented with appropriate options. The last

option is used when all of the samples from agiven experiment cannot be run in a singlesession. This allows lar ge nu mber s osam ples to be compiled in a single text file. Ithe appropriate st arting sam ple number waschosen, the sample numbers in the large textfile will be cont inu ous.

R e a d i n g F l u o r e s c e n c e

So far t he compu ter h as neith er sent n or received any signals from the fluorometer. At this point t hecomput er commun icat es with th e spectrophotometer to set it up as the user has specified duringth e setup a nd initialization procedur es. Durin g this period the compu ter displays th e following:

S e t t i n g -u p I n s t r u m e n t ...P l e a s e Wa i t

The screen is then initialized with column s for th e sample nu mber a nd ea ch excitat ion/emissionwavelength pa ir. An exam ple is shown below:

490 530 565S a m p le 506 552 598


8/8/2019 FACMan

8/10

Inst ru ctions ar e displayed at th e bott om of th e screen to prompt the user . The first prompt directsth e user to ready a new device for sa mpling (either wellplate or cuvette).

P l a c e N e w C u v e t t e i n t o S a m p l e H o l d e r ! C o n t i n u e o r Q u i t (C /Q ):

or

P l a c e N e w P l a t e i n t o t h e W e ll R e a d e r ! C o n t i n u e o r Q u i t ( C /Q ):

The user sh ould th en enter either a 'C' to start reading the sa mple(s) or a 'Q' to quit FAC.

The program h an dles sample fluorescence measu remen ts different ly, depending on whet her t hesamples are pr esented in cuvettes or th e new or old wellplate. In both th e old wellplate a nd t hecuvette modes each wavelength p air is "read" for a well before moving on. Out put is immediatelydisplayed on screen and print ed as the wells are r ead. In th is insta nce the screen output is asshown below. The fluorescence at ea ch excitat ion/emission wa velength pair will be rea d an ddisplayed on t he screen:

490 530 565S a m p le 506 552 598

1 32 456 2312 23 498 174

At the completion of reading all wavelength s for a well th e values ar e displayed on t he pr inter ifrequest ed. At th e completion of each cuvette mea sur ement cycle th e following prompt is displayed.

En t e r A [accep t ] , R [ r e j ec t ] , S [s t a n da r d ] (A/R/ S) [ A] :

If the user selects 'A' (accept) the values ar e written to the t ext file an d th e printer if the pr interoption was selected. By selecting R (reject) the user can r erun th e same sam ple after a ltering thesam ple (e.g., diluting th e sam ple if th e fluorescence was out of an accepta ble ran ge). It isrecommen ded to read st an dar ds [S] periodically throughout a session t o verify tha t th espectrophotometer r emain s stable throughout th e session. When th e user selects [S] th e readingsar e written t o a .CTL file which is stored in th e same locat ion as t he .FA data file. The nu mber ofthe curr ent sa mple is used for the sta ndar ds reading in th e .CTL to enable the user to locate wh enthe s tan dard was r ead, but the sample num ber is not increased.

As each color completes, th e screen is erased a nd t he n ext color's values a ppear on th e screen.

Once a cuvette sam ple is accepted, th e user is asked if a commen t is t o be added to this sa mple.

C o m m e n t [ No n e ] :

This is helpful for noting where a sam ple is from, how much it wa s diluted, or an y informat ionabout the sam ple. The comment is written t o the text file only. Asterisks are printed on the ha rdcopy if a comment has been entered.The user is th en reprompted t o place a new sam ple in t hecuvett e h older.

When u sing th e new wellplate, a single wavelength is selected in t he LS-50 or LS-50B and a ll wellsar e read for th at color before monocromat ors are reset t o th e next color. This saves wear a nd tea ron the monocroma tors and dr am at ically lowers the rea d time for the entire plate. As th emeasur ements ar e made they are displayed on th e screen in the order shown. The program is setupfor usin g a 96-wellplat e. We can provide oth er configura tions if requ ested .

1 9 17 25 33 41 49 57 65 73 81 892 10 18 26 34 42 50 58 66 74 82 903 11 19 27 35 43 51 59 67 75 83 914 12 20 28 36 44 52 60 68 76 84 925 13 21 29 37 45 53 61 69 77 85 936 14 22 30 38 46 54 62 70 78 86 947 15 23 31 39 47 55 63 71 79 87 958 16 24 32 40 48 56 64 72 80 88 96


8/8/2019 FACMan

9/10

When all the sam ples have been read, th e user is asked if s/he wan ts to run another set of samplesfrom th e same experimen t, begin r eading th e samp les of a differen t experiment , or quit t o DOS.

1. R u n a n o t h e r se t of s a m p le s fr o m t h e sa m e e xp e r i m e n t2. S ta r t a Ne w Ex pe r im e n t3. F lo w An a ly sis4. E xi t P r ogr a m

Se l ec t On e o f t h e Above ( 1 -4 ):

FLOW ANALYSIS

If, at t he beginnin g of FAC, the user chooses the option to an alyze flow, th e following quest ions ar edisplayed:

P a t h n a m e o f d a t a fi le s :N a m e o f d a t a fi le ( n o e x t e n s i on s ) :

FAC reads th e requested dat a fi le into memory an d displays the h eader a nd n umber of samples in th eretrieved file.

N u m ber of Wa velen gt h s 4A n a l ys t Da veE xci t a tion S li t 4E m ission S l i t 4Cu t of f Fi l t er 2PMT Volta ge 820S a m p le Id en t i f i ca t ion 1Da t e/T im e 12-21-1992 15:33:33ex 430 490 530 600ex 467 506 552 635 Com m en t N um ber o f s am p l e s i n f i l e : 9I s t h i s th e f i l e (Y/N) :

If this is th e correct file, the user is then asked to enter the fluorescent intensity of the r eference blood

sam ple at ea ch of the excitat ion/emission wa velength pairs.

P ai r 1 430/467:P a i r 2 490/506:P a i r 3 530/552:P a i r 4 600/635:

The user is then pr ompted to enter t he flow ra te of th e referen ce blood withdr awa l sample.

F l o w r a t e o f w i t h d r a w a l s a m p l e ( m l /m i n ):

The flow to each sam ple dur ing each injected color is calculat ed a nd writt en t o a t ext file specified by th euser.

Nam e o f f low f i l e (No ex t en s i ons ) :

The ext ension, '.FR', is appen ded t o each flow file.

FAC His to r y

FAC7E - Add support for st anda rds.FAC7B - Add read time a nd scan speed support


8/8/2019 FACMan

10/10

S u p p o r t f or F AC

In a n a ttem pt t o limit th e nu mber of different versions of th is program a nd t o coordina te its evolut ion for a ll users,th e Fluorescent Microsphere Resource Center will support FAC. Dean Br own of Perk in Elmer will also support t heprogram, but requests should be made t hr ough th e Fluorescent Microsphere Resource Center by one of the followingroutes:

I nt er n et : glen n y@p ele.p ulm cc.wa sh in gt on .ed uPhone: (206) 543-7063FAX: (206) 685-8673Mail: Robb Glenny

University of WashingtonDiv. of Pulm onar y & Critical Car e MedicineBox 356522Sea tt le, WA 98195-6522U.S.A.

The FMRC is keenly interest ed in mak ing FAC useful to resear chers u sing fluorescent m icrospheres. Anysuggestions t o enhan ce the program or th is docum enta tion will be gladly considered.

FAC (Fluorescence Analysis) Manual Pa ge 10

facman

Documents