MicroMax 384. Operation Manual Rev Fax: (33) 1/ U.K.: 020/

Transcription

1 MicroMax 384 Operation Manual Rev. 2 In the USA: Jobin Yvon Inc Park Avenue In France: Edison, NJ Jobin Yvon S.A. Tel: , rue du Canal Fax: Longjumeau cedex Germany: 89/ fluorescence@jyhoriba.com Tel: (33) 1/ Italy: 2/ Fax: (33) 1/ U.K.: 020/

2 Copyright 2001 by Jobin Yvon, Inc. All rights reserved. No part of this work may be reproduced or transmitted in any form by any means, including electronic or mechanical, photocopying and recording, without prior written permission from Jobin Yvon, Inc. Requests for permission should be requested in writing. Information in this manual is subject to change without notice and does not represent a commitment on the part of the vendor. Part Number

3 Contents Introduction... 4 About the MicroMax... 4 Symbols used in this manual... 4 Requirements & Installation... 5 Requirements... 5 Overview of installation... 5 Installation... 6 Software operation DataMax applications specifically for the MicroMax Data collection Daily Calibration Verification Tutorials Introduction Tutorial 1: Full-spectrum analysis Tutorial 2: Constant Wavelength Analysis Additional functions not used in the tutorials Care of Fiber-Optic Cables Troubleshooting Care of the bifurcated cable Storage MicroMax F-3000 fiber-optic adapter and cables Appendix Computer requirements Software requirements Physical specifications Electrical specifications Fiber-optics specifications Index

4 Introduction Introduction About the MicroMax 384 The MicroMax 384 is a microwell-plate reader able to accept plates with up to 384 wells, and connect to FluoroMax or Fluorolog spectrofluorometers. MicroMax 384 s high speed allows it to scan completely a 96-microwell plate in less than one minute. By moving the microwell plate through stationary optics, the MicroMax 384 insures high sensitivity, excellent accuracy, and high reproducibility. Light from the excitation and emission monochromators is carried via a fiber-optic bundle to and from the MicroMax 384, thus the user may scan with the main spectrofluorometer and select any excitation and emission wavelength pair for intensity measurements. All control of the MicroMax 384 is automated through DataMax software; custom selection of microwells on the plate is possible through the software. Symbols used in this manual Certain symbols are used throughout the text for special conditions when operating the instruments: Warning: A hazardous condition exists, or danger exists that could damage the equipment. Jobin Yvon Inc. is not responsible for damage arising out of improper use of the equipment. Note: General information is given concerning operation of the equipment. 4

5 Requirements & Installation Requirements & Installation Requirements Physical requirements Within 0.75 m of the spectrofluorometer sample compartment Allow 10" (25 cm) height minimum including the MicroMax 384, to account for fiber optics. Requires F-3000 fiber-optic adapter accessory Computational requirements Dedicated serial COM port DataMax software Windows 95 or higher or equivalent PC computer Overview of installation 1 Unpack and inspect the components. 2 Read this manual. 3 Install the F-3000 adapter on the spectrofluorometer. 4 Assemble the MicroMax 384 and its accessories. 5 Install the software (only if the MicroMax was purchased separately). 6 Calibrate and align the F-3000 adapter. 7 Calibrate and align the MicroMax. 5

6 Installation Requirements & Installation 1 Unpack and inspect all components. a The following MicroMax 384 components should be included: Quantity Description Part Number 1 MicroMax 384 microwell plate reader 1 MicroMax 384 Operation Manual AC power cord RS-232 communication cable RS-232 female-9-pin-to-male-25-pin adapter Bifurcated fiber-optic cable, 1 m long Lens assembly for fiber-optic cable Fiber-optic interface for the spectrofluorometer (sold separately from the MicroMax 384) F " diskette with calibration offset for allplate.ini (included only if the MicroMax 384 was purchased separately from the spectrofluorometer) b Inspect all the components for signs of damage that may have occurred during transit. If damage is evident, do not continue with the installation. Notify Jobin Yvon Inc. and the shipper at once. Note: Many public carriers do not recognize claims for concealed damage reported later than 15 days after delivery. For a shipping damage claim, inspection by the carrier agent normally is required. Therefore, the original packing material should be kept as evidence. While Jobin Yvon Inc. is not liable for damage occurring during transit, the company will extend every effort to aid and advise. 6

7 Requirements & Installation 2 Read this instruction manual thoroughly before proceeding to install the MicroMax Install the F-3000 fiber-optic adapter on the spectrofluorometer. a Be sure that the MicroMax 384, host computer, and spectrofluorometer are all switched OFF. b Open the lid of the MicroMax 384 and remove the white plastic rectangular plate. This plastic plate was used to prevent the base from moving during shipping. Warning: Operating the MicroMax 384 without removing the white plastic rectangular plate can damage the MicroMax 384! c Remove the existing sample mount from the spectrofluorometer. Save the two brass thumb screws for mounting the F-3000 adapter. d Insert the F-3000 fiber-optic adapter into the spectrofluorometer s sample compartment. One mirror should face the excitation monochromator, and the other should face the emission monochromator. e Secure the base plate by using two brass thumb screws. The lens and baffle should be closer to the excitation monochromator. f Remove the 1⅜"-diameter (3.5-cmdiameter) plastic plug from the front of the spectrofluorometer s sample compartment. If there are any threaded plugs or set screws in the two adjacent mounting holes, remove them as well. g Remove the plastic shipping caps from the fiber-optic cable s ferrules. 7

8 Requirements & Installation h Thread the two slit-ended fiber bundles through the hole in the sample compartment. Insert the fiber bundle labeled excitation fibers into the excitation port, and then insert the other (unlabeled) fiber bundle into the other port, as shown below. Warning: Avoid twisting the fiberoptic cables too much. Breakage of the glass fibers may occur with excessive strain. Secure them loosely, with the narrow thumb screws, on the F-3000 adapter, in their respective mounts. Mount the plate and gasket on the outside of the compartment, as shown in this photograph: gasket (underneath plate) light-shield plate 8

9 4 Assemble the MicroMax 384 and accessories. Requirements & Installation a Place the MicroMax 384 on a level surface near the spectrofluorometer s sample compartment, so that the fiber-optic bundle reaches the both sample and MicroMax 384. b Verify that the MicroMax 384 is set for the proper line voltage for your country. The power entry module has a white arrow that indicates the AC voltage set by the factory. c Connect the AC power cord to the receptacle on the MicroMax 384. d Attach one end of the RS-232 communication cable to the I/O connector on the MicroMax 384. Attach the other end of the RS- 232 cable to the free COM port on the back of the computer. Use the enclosed 9-pin 25-pin adapter as necessary. Note: Which COM port chosen depends on the settings in the file mw.ini, described later. 9

10 Requirements & Installation e Insert the combined end of the fiber-optic cable into the lens cell. The cable should reach to the bottom of the cell. Secure the cable to the lens cell by tightening the set screw on the lens cell. f Insert the lens-cell assembly into the receptacle at the top of the MicroMax so that it is firmly seated. The top of the lens cell should protrude about 1/8" (~2 mm) from the receptacle. Warning: Never pull the cable itself the ferrule may loosen from the cable. Hold the cable by the metal ferrules only. g Tighten the nylon thumbscrew on the lens receptacle. This secures the lens-cell assembly in place. h Plug the free end of the power cord into a properly-rated AC receptacle. This is the initial position for alignment. 5 Install the software. (only if the MicroMax 384 was purchased separately from a spectrofluorometer) a For FluoroMax users: i. Find the layout directory on the computer s hard drive, C:\Datamax\layout. ii. Find the readme.txt file, which describes the layout file configuration. 10

11 Requirements & Installation iii. Open readme.txt using Notepad; find the line containing Fluoromax with MicroMax, and its corresponding layout file. For example, in line 2, it says, Fluoromax with MicroMax, Layout2.lay. iv. Copy this layout file (e.g., layout2.lay) into the ISA_INI folder, C:\Datamax\ISA_INI. v. Find the mw.ini file in the folder C:\Datamax\ISA_INI.ini. Read the file using Notepad, and find the paragraph [COMMUNICATION] TYPE=1 CHAN_NUM=2 CHAN_NUM sets the communications port used by the computer. The default setting is for COM2, i.e., CHAN_NUM=2. Set the appropriate port number. For example, to use COM3, change the CHAN_NUM to CHAN_NUM=3. vi. Insert the 3.5" diskette sent with the MicroMax 384 into the computer. From the diskette, copy the allplate.ini file into C:\Datamax\ISA_INI\ to replace the original allplate.ini file. This step enters the calibration data. b For Fluorolog users: Contact Spex service to get the layout file to operate the MicroMax. With the layout file, continue as for part (a) above, steps (iv) through (vi). 6 Calibrate and align the F-3000 adapter. a Be sure that the DataMax software is properly installed on your computer. Turn on the MicroMax 384 using its power switch. 11

12 Requirements & Installation b From Windows, start the DataMax software by clicking on the icon. Select the layout for your spectrofluorometer that includes the MicroMax 384. This initializes the system. At the end of the boot-up process, the MicroMax 384 makes a characteristic sound as it resets to the home position. The Instrument Control Center appears. c Click on the Visual Instrument Setup icon to open the Visual Instrument Setup dialog box. Note: The instrument configuration shown may vary, depending on the particular accessories. d Click on this symbol of the MicroMax 384. The Sample Compartment Accessories dialog box appears: e Click on this Image, and the Plate Reader Control dialog box appears: 12

13 Requirements & Installation f Enter home to reset the MicroMax 384 to the home position. Click on the Move button. g Insert the microwell plate into the MicroMax 384 sample compartment. Insert the plate by first setting the left side in, then the right side. Warning: If the microwell plate overlaps the left or right sides of the platen, alignment is impaired. h Click on this icon to open the Real Time Display dialog box: i Make sure that the Monos dialog box and the Band Pass dialog box are visible by clicking Options in the toolbar. 13

14 Requirements & Installation j In the Monos (monochromators) box, set the excitation monochromator to 500 nm (blue-green light) by typing in 500 here. k Click the EX1 (excitation) button, and set the Band Pass to 5 nm. Open the shutter. l Remove the top of the spectrofluorometer s sample compartment. m With a dental mirror, check to see that the bright blue-green, slit-shaped beam falls properly on the slit-shaped entrance to the excitation fibers. Reduce the Band Pass slits to 2 nm, and examine the beam, then reduce them to 1 nm and examine the beam. In all cases the beam should fall directly on the fibers: end of ferrule slit-shaped bundle of fibers beam of light n If the beam is not in the center, remove the fiber-optic cables and F-3000 adapter, and insert an Allen wrench in the central hole between the mounting screws. Loosen the front mirror set screw, and adjust the mirror s left-right position slightly. Loosen the interior mirror set screw, and adjust the mirror s front-back position slightly. Replace the F-3000 and fiber optics in the sample compartment, and examine the beam again. Repeat this procedure until the beam falls properly on the entrance of the fiber bundle. o Remove the F-3000, 14

15 rotate 90 counterclockwise, Requirements & Installation and replace it in the sample compartment so that the emission beam now is placed in the excitation-fiber position. p With the dental mirror, examine the slit-shaped beam as in step m. Make sure that the light falls in the middle of the emission ferrule s end. Adjust the mirror as in step n as needed. (A later procedure will tune the emission signal-level.) q Remove the F-3000 adapter, rotate 90 clockwise back to its original position, and fasten it down with the two thumb screws. Note: If the F-3000 adapter was purchased with the spectrofluorometer, the mirrors now should be aligned. If the F-3000 was purchased separately, continue aligning the mirrors below. r s Start the spectrofluorometer. t Turn on the MicroMax 384 using its power switch. Be sure that the DataMax software is properly installed on the computer. The plate holder moves automatically to the Home position. u Insert a clean, unused microwell plate containing the sample at the proper volume, i.e., the typical volume for the samples. Jobin Yvon recommends an initial calibration with 200 µl of 100-nM fluorescein (extinction coefficient ε ~ 15

16 Requirements & Installation ) in 0.1-N NaOH in one of the wells of the microplate. In addition, prepare a blank solution of 0.1-N NaOH, and put 200 µl of the blank in another well. The fluorescein gives an absorbance A = at 490 nm above the blank. The following steps assume that fluorescein is the sample used to calibrate and align. Other fluorescent solutions may require different settings. Note: Be sure to use fluorescein in basic solution. Acidified solutions of fluorescein have a low quantum yield, so the spectrofluorometer s sensitivity will be far lower than expected. v Close the sample compartment lid. w From Windows, start the DataMax software by clicking on the icon. Select the layout for your spectrofluorometer that includes the MicroMax 384. This initializes the system. At the end of the boot-up process, the MicroMax 384 makes a characteristic sound as it resets to the home position. The Instrument Control Center appears. x Click on the Visual Instrument Setup icon to open the Visual Instrument Setup dialog box: y Click on this symbol of the MicroMax 384. The Sample Compartment Accessories dialog box appears: 16

17 Requirements & Installation z Click on this Image, and the Plate Reader Control dialog box appears: aa Enter the position of the sample, e.g., A12, in order to move the MicroMax 384 to that position. Click the Move button to activate the plate s motion. bb Click on this icon to open the Real Time Display dialog box: cc Click Options to make sure that the Monos dialog box is visible, the Intensity dialog box is visible, and the S signal detector is enabled (in the Intensity dialog box). 17

18 Requirements & Installation dd In the Monos (monochromators) box, set the excitation monochromator to 485 nm by typing in 485 here. Set the emission monochromator to 513 nm by typing in 513 here. Set the excitation and emission Band Pass to 2 nm. Open the excitation shutter. ee Watch the S (signal) detector output (cps) in the Intensity box. If, at any time during alignment, the signal is greater than cps (counts per second), reduce the slit widths. gg Adjust the front set-screw for the emission mirror. This controls the pitch (frontback fine rotation) of the mirror. Set the position to maximize the signal in the Intensity box. ff Adjust the side set-screw for the emission mirror on the F This controls the left-right fine rotation of the mirror. Set the position to maximize the signal in the Intensity box. Note: Do not adjust the signal for the excitation mirror. If you have trouble adjusting the position of the light beam on the ferrules, or maximizing the signal, contact Spex service. 7 The mirrors should now be aligned. 18

19 8 Calibrate and align the MicroMax 384. Requirements & Installation a Be sure that the DataMax software is properly installed on your computer. b Start the spectrofluorometer. c Turn on the MicroMax 384 using its power switch. The plate holder will move automatically to the Home position. d Insert a clean, unused microwell plate containing the sample at the proper volume, i.e., the typical volume for the samples. Jobin Yvon recommends an initial calibration with 200 µl of 100-nM fluorescein (extinction coefficient ε ~ ) in 0.1-N NaOH in one of the wells of the microplate. In addition, prepare a blank solution of 0.1-N NaOH, and put 200 µl of the blank in another well. The fluorescein gives an absorbance A = at 490 nm above the blank. The following steps assume that fluorescein is the sample used to calibrate and align. Other fluorescent solutions may require different settings. e Close the sample compartment lid. Note: Be sure to use fluorescein in basic solution. Acidified solutions of fluorescein have a low quantum yield, so the spectrofluorometer s sensitivity will be far lower than expected. 19

20 f Requirements & Installation From Windows, start the DataMax software by clicking on the icon. Select the layout for the spectrofluorometer that includes the MicroMax 384. This initializes the system. At the end of the boot-up process, the MicroMax 384 makes a characteristic sound as it resets to the home position. The Instrument Control Center toolbar appears. g Click on the Visual Instrument Setup icon to open the Visual Instrument Setup dialog box: h Click on this symbol of the MicroMax 384. The Sample Compartment Accessories dialog box appears: i Click on this Image, and the Plate Reader Control dialog box appears: 20

21 j Enter the position of the sample, e.g., A12, in order to move the MicroMax 384 to that position. Click the Move button to activate the plate s motion. Requirements & Installation k Click on this icon to open the Real Time Display dialog box: l Click on Options to make sure that the Monos dialog box is visible, the Intensity dialog box is visible, and the S signal detector is enabled (in the Intensity dialog box). 21

22 Requirements & Installation m In the Monos (monochromators) box, set the excitation monochromator to 485 nm by typing in 485 here. Set the emission monochromator to 513 nm by typing in 513 here. Set the excitation and emission Band Pass to 2 nm. Open the excitation shutter. n Watch the S (signal) detector output (cps) in the Intensity box. If, at any time during alignment, the signal is greater than cps (counts per second), reduce the slit widths. Fiber-optic adapter assembly Warning: Minimize the amount of time the emission photomultiplier tube (PMT) is exposed to ambient light, when the sample chamber cover is off, to reduce wear and tear on the PMT. 22

23 Requirements & Installation o Loosen the nylon thumbscrew. Gently adjust the height of the fiberoptic assembly above the microwell plate. As with the other ends of the fiber-optic cable, adjust the level of the fiber-optic adapter assembly until it is positioned at the level of greatest S signal. p With maximum intensity at the S detector, tighten the nylon thumbscrew. The MicroMax 384 is now properly aligned for this sample at its current volume. The procedure need not be repeated unless the sample volume changes. Note: The background count of a microwell plate is large, so subtracting a blank scan from the sample scan should be done to give the ultimate sensitivity. Refer to the DataMax manual for more information about blank subtraction. Note: This alignment of the fiber-optic assembly should be performed each time a different sample volume is used. 23

24 Software Operation Software operation DataMax applications specifically for the MicroMax 384 The MicroMax 384 uses four applications within DataMax: Visual Instrument Setup Real Time Display Run Experiment Constant Wavelength Analysis 24

25 Visual Instrument Setup Software Operation Use Visual Instrument Setup to check the control of the MicroMax 384 via the software. Click on the MicroMax 384 icon to move the microwell plate to various positions and determine plate type. Note: All control of the MicroMax 384 is performed through Visual Instrument Setup. 25

26 Software Operation To move the plate in order to examine a particular well 1 Click on the MicroMax 384 icon in the Visual Instrument Setup dialog box. The Sample Compartment Accessories dialog box opens: 2 Click on the rectangular plate icon. The Plate Reader Control dialog box opens. 3 Enter the coordinate of the well to examine, e.g., C4. Click the Move button. The MicroMax 384 moves the microwell plate to the desired position. 4 To load or unload a microwell plate, enter home and click the Move button. The MicroMax 384 moves the plate away from the lens cell and underneath the lid for easy removal. 26

27 To enter the type of microwell plate Software Operation 1 Follow steps 1 and 2 on the previous page to show the Plate Reader Control dialog box: 2 Click on the Properties... button. This brings up the Plate Properties dialog box: 3 Select the appropriate plate type from the list. Scroll up and down the list as needed. 4 Click OK when done. Note: If the brand name of the 384- well plate is not listed, contact Spex Fluorescence Applications. 27

28 Real Time Display Software Operation Use the Real Time Display dialog box to check and adjust settings of the system before running a scan or experiment. When used with Visual Instrument Setup, the signal response of the sample may be verified, to ensure capture of a useful set of scans. Open the slits here Control the monochromators here Monitor detectors intensities here. Adjust slit widths here. Vary integration time here. Note: The operation of Real Time Display is identical with or without the MicroMax 384 installed. For detailed information on the Real Time Display, consult the DataMax manual. 28

29 Run Experiment Software Operation The Run Experiment dialog box provides all scanning capability for the spectrofluorometer, with or without the MicroMax 384. Using the MicroMax 384 on any microwell-plate position or set of positions, all of the following steady-state scans are possible: Emission Excitation Time Base Synchronous Multigroup Note: The following scans are not possible with the MicroMax 384: Polarization Batch Temperature Scan Dynamic (lifetime) acquisitions Multiple sample holders 29

30 Constant Wavelength Analysis Software Operation Manipulate files here. Set excitation and emission wavelength pairs here. Do signal ratioing and timebased kinetics here. Set up the detector here. Constant Wavelength Analysis allows acquisition of excitation and emission pair data at various sample positions and acquisition modes. Results may be plotted or retained as a table. Data may be saved as an Excel worksheet. Up to 32 wavelength pairs and multiple acquisition modes may be selected. Sample positions may be tagged as blanks, unknowns, and standards. Data manipulation and collection includes: Blank subtraction Concentration curves Correction for lamp intensity Variable-time kinetics: acquire scans at different time intervals, and use different integration times. Photobleach mode: Protects light-sensitive samples by closing the shutter during dead time. 30

31 Data collection Software Operation There are three recommended methods to collect data with the MicroMax 384 and spectrofluorometer: Experiments on a single well of the microwell plate, with the Run Experiment dialog box. Microplate matrix scans, for repetitions of the same experiment across multiple wells of one microplate. Constant Wavelength Analysis, to acquire wavelength-pair data. 31

32 To do one experiment on a single well of the microplate: 1 Use Visual Instrument Setup to load and position the sample. 2 Choose Run Experiment. Software Operation 3 Choose Collect in the Toolbar. This allows definition and acquisition of a single set of data for a particular well on a microplate. 32

33 To repeat the same experiment on multiple wells of one microplate: 1 In the DataMax toolbar, choose Collect: Software Operation 2 Under Collect, choose MicroPlate Scan... 3 The MicroPlate Matrix Scan dialog box appears: Choose a predetermined experimental setup with these buttons. Control a series of experiments here. 33

34 To choose a predetermined experimental setup Software Operation Enter the full pathname and filename for an experimental setup here, OR Click on Exp. Browse to browse through a folder of experimental-setup files: From the list, click on a file to use. It will be highlighted in the space. Choose the drive and directory in these lists. List all files of types *.exp or *.* To preview a file, click on Info. 34

35 Software Operation To choose an existing scan or to search for specific output files Click on File Browse to browse through a folder of experimental-setup files. This opens the Select File Name dialog box: From the list, click on a file to use. It will be highlighted in the space. Choose the drive and directory in these lists. List all files of types *.spc or *.* To preview a file, click on Info. 35

36 Software Operation To select the type and location of a group of samples in the microwell plate: Click on Plate Control... to open the MicroPlate Configuration dialog box: Describe the sample as a blank or an unknown. Use this section to create a list of the samples to be scanned. 36

37 To load a previously-defined selection of samples to scan: Software Operation 1 Enter an optional description of the microwell plate in Plate Name/ID. 2 Open the MicroPlate Configuration dialog box as described on the previous page. 3 Click on the Load Set... button. This opens the Read MicroPlate Configuration dialog box. 37

38 Software Operation 4 From the Folders and Drives list, find the appropriate folder in which your set exists. 38

39 To save a previously-defined selection of samples to scan: Software Operation 1 Open the MicroPlate Configuration dialog box as described two pages back. 2 Click on the Save Set... button. This opens the Save MicroPlate Configuration dialog box: 3 From the Folders and Drives list, find the appropriate folder in which to save your configuration. The file is saved with the extension.tpl. 39

40 Software Operation To acquire multiple pairs of excitation-emission data: 1 In the Instrument Control Center dialog box, click on the Constant Wavelength Analysis icon: 2 This brings up the Constant Wavelength Analysis dialog box: Note: In Constant Wavelength Analysis, there is no way to set the bandpass. Constant Wavelength Analysis uses the last setting already defined for the bandpass. If the machine has just been switched on, the bandpass will be defined as 0 nm. To adjust the bandpass, open up the Real Time Display first, set the bandpass, and then open up the Constant Wavelength Analysis dialog box. 40

41 a Set excitation-emission wavelength pairs Software Operation 1 Type in the excitationmonochromator wavelength (in nm) here. 2 Type in the emissionmonochromator wavelength (in nm) here. 3 Continue entering up to 32 wavelength pairs. 4 Edit the wavelength-pair list with these buttons. 5 Choose SB with MicroMax. ( SB stands for Sample Box.) 41

42 Software Operation b Set the acquisition parameters, including kinetics and photobleach mode: 1 Select S for signal detector, R for reference detector, or enter an expression using S and R. 2 Click Add to enter the acquisition expression into the list. 3 Create a list of acquisition expressions here. 4 Use «Remove to delete unwanted acquisition expressions from the list. Note: An example of a list of acquisition parameters would be S (the signal detector) R (the reference detector) S/R (to ratio the signal to the reference) 5 (optional) Choose the Kinetics button to create a kinetics experiment or to choose photobleach mode. Click Define... to bring up the Time-based Acquisition Parameters dialog box (see next page). 42

43 Software Operation To set kinetics and photobleach parameters 1 Choose Kinetics and click Define... as shown on the previous page. The Timebased Acquisition Parameters dialog box appears: 2 Enter the time cycle for a sequence of scans here. 3 Enter the sequence here. 4 Check this box to close the shutter between scans. This minimizes photobleaching effects on light sensitive materials. Note: Up to 23 sequences can be programmed into the table. 43

44 c Set the detector parameters Software Operation (See the DataMax manual for more information.) 1 Set the integration time for the detector. 2 Choose the standard error, in percent. 3 Enter the maximum number of trials. d Click on Proceed to Acquisitions. This starts the CWA Data Display, as shown on the next page. 44

45 d (continued) Software Operation Each cell in the spreadsheet below corresponds to a well in the microwell plate. As the MicroMax scans each well, a data point will be filled in automatically for that well. Go to step (e). e f If the various options in this dialog box are acceptable (see DataMax manual), click on the Start Acq button. This opens the Select Acq Expression To View dialog box, if there are multiple acquisition expressions entered: Select the desired acquisition expression to view. Click OK when finished. This opens up the MicroPlate Configuration dialog box (see next page). 45

46 f (continued) Software Operation In Plate Name/ID, provide an optional description of the microwell plate. 1 Enter the coordinate of each sample to scan here. 2 Tag that sample with the appropriate label (Blank, Unknown, or Standard) here. 3 Add the tagged sample to the list. Notice how the selected samples are highlighted in the schematic of the microwell plate. (Above is shown a 96-well plate.) See the DataMax manual for more information. g Click on Run Plate to start the run. The MicroMax 384 begins the multiple-well scan, with its characteristic sound as the plate moves back and forth. As the scan continues, data gradually appear in the CWA Data Display dialog box, until all the selected wells are scanned. 46

47 Daily Calibration Verification Daily Calibration Verification Jobin Yvon Inc. recommends that the operator verify the calibration of the MicroMax 384 and attached spectrofluorometer at the start of each day. 1 Calibrate the spectrofluorometer Perform the normal calibration recommended for the spectrofluorometer in the spectrofluorometer user s manual. For example, check the excitation output with a lamp scan, and then the emission from a water-raman signal in a cuvette. 2 Install the F-3000 adapter Remove the existing sample holder for cuvettes, and replace it with the F-3000 adapter for the fiber-optic bundles. 3 Calibrate the MicroMax 384 Verify the microwell-plate alignment of the MicroMax 384 as listed in the Calibrate and Align the MicroMax 384 section of this manual. 47

48 Tutorials Tutorials Introduction To help acquaint you with the power offered by MicroMax 384, two tutorials are provided. The first tutorial shows how to scan blanks and unknown samples to obtain a blanksubtracted spectrum. The second tutorial demonstrates the use of Constant Wavelength Analysis to scan a large number of wells in a microwell-plate at a particular wavelength. Both tutorials assume that the instrument is set up, calibrated, and running, using a 96-well plate. 48

49 Tutorial 1: Full-spectrum analysis Tutorials The particular sample used in this tutorial is ADIFAB, which is the fluorescent dye acrylodan conjugated to intestinal-fatty-acid binding protein (15 kda). A 96-well microwell plate is already selected. The ADIFAB is in wells A1 and A2, while blanks are in wells B1 and B2. The remainder of the plate is empty. 1 Choose the Run Experiment icon. This opens the main window in DataMax: 2 Under Collect, choose Experiment... This opens the Emission Acquisition dialog box, to set up the experiment: 49

50 Tutorials 3 Click on Exp Type... This opens the Select Experiment Type dialog box: Choose Emission Acquisition; click OK. Return to the Emission Acquisition dialog box. 50

51 Enter the experimental-scan parameters: Tutorials Start the scan at 400 nm Increment the wavelength by 1 nm Use Excitation of 350 nm End the scan at 550 nm Integration Time per data point of 0.5 s 4 Click on the Signals... button. This opens the Signals dialog box: 5 Choose the S (signal) acquisition parameter. Click OK to close the box, and return to the Emission Acquisition dialog box. 51

52 Tutorials 6 Click the Slits... button. This opens the Slits dialog box: 7 Set the Excitation slit to 10 nm, and the Emission slit to 6 nm. Click OK to return to the Emission Acquisition dialog box. 8 Click on Save to save the experimental setup as s1.exp. 52

53 9 In the Run Experiment main menu, choose MicroPlate Scan. Tutorials This opens the MicroPlate Matrix Scan dialog box: 10 Under Exp. Browse, recall the experimental setup s1.exp. 11 Name the experiment itself test.spc. 12 Click on Plate Control... This opens the MicroPlate Configuration dialog box: 13 Select wells A1 to B2. Enter A1 in the From: field Enter B2 in the To: field. 14 Click Add». This adds a new group of four unknowns (samples) the computer is not told that B1 and B2 are actually blanks to the list to be scanned. Therefore, there are now four well positions that the MicroMax 384 will scan: A1, A2, B1, and B2. 53

54 15 Click OK to close the MicroPlate Configuration dialog box. 16 Click OK in the MicroPlate Matrix Scan dialog box to scan the wells Automatic display shows all four spectra overlaid together, and automatically named testa1.spc, testa2.spc, testb1.spc, and testb2.spc, based on the wells coordinates and the overall experiment s name. Tutorials samples A1 and A2 blanks B1 and B2 Note: To automatically subtract blanks from unknowns, place one blank in each group with all unknowns associated with that blank. The software then automatically subtracts the blank s signal from each unknown in that group, and does not display the blank. 54

55 Tutorial 2: Constant Wavelength Analysis Tutorials In this tutorial, there is a 96-well microwell plate in the MicroMax 384, which contains 15 different solutions in wells F1 to H5. There are 3 different blanks in the wells A1 to C1. The rest of the wells are empty. 1 Open the Constant Wavelength Analysis icon. Analysis dialog box: This brings up the Constant Wavelength 2 Enter the wavelengths to monitor: Note: If the system has a T-box, an Emission 2 Monochromator wavelength is needed for every Emission 1 Monochromator wavelength, even if unused. Enter a dummy value say, 600 nm. 55

56 Tutorials Enter 386 for the first excitation-monochromator wavelength, and 432 for the first emission-monochromator wavelength. Then, underneath 386, enter 386 for the second excitation wavelength, and underneath 432, enter 505 for the second emission wavelength. (See note if the system contains a T-box.) 3 Choose SB with MicroMax. 4 Select the signal detector 5 Select Detector Parameters a Choose Acquisition Parameter S b Click Add» a Set the Integration Time to 2 s b Allow 3 Maximum Trials c Make the Standard Error 0.5% 56

57 Tutorials 6 Click on Proceed to Acquisitions. This starts the CWA Data Display, below: 7 Click on the Start Acq button. This opens up the MicroPlate Configuration dialog box (see next page): 57

58 8 Select the samples. Tutorials a Select a New Group». b Choose Blank. c Enter well A1. d Click on Add». e Choose Unknown. f In the Sample Selection area, enter From: F1 and To: F5. g Click on Add». h Select a New Group». i Choose Blank. Note: Only one blank is permitted per group of unknowns. j Enter well B1. k Click on Add». l Choose Unknown. m Enter From: G1 and To: G5. n Click on Add». 58

59 Tutorials o Select a New Group» p Choose Blank. q Enter well C1. r Click on Add». s Choose Unknown. t Enter From: H1 and To: H5. u Click on Add». Three groups of 5 samples each, plus one blank for each group, are now programmed in. 9 Click on Run Plate. Two images of index cards appear: WaveSet 1 (which corresponds to 432 nm) and WaveSet 2 (which corresponds to 505 nm). Click on WaveSet 2 to see 505-nm data. 59

60 10 Click on Save Data to save the data. Tutorials This brings up the Save Data As... dialog box: The data can be saved as an Excel spreadsheet, a CWA file (.cwa) to perform post-experiment analysis, or a text file (.txt). Here are sample data from the run: Excel Spreadsheet F F F F F B G G G G G H H H H H Note: In Excel Spreadsheet and Multi-column Text formats, only the active tab is saved. In CWA Data File format, all tabs are saved. 60

61 Additional functions not used in the tutorials Append Append merges an active, on-screen database with an existing database. For example, a standard curve exists in a file, and new unknowns have just been scanned. These two datasets can be merged in the Append function. Tutorials This opens the Open dialog box. Find the desired file to append to the existing, active one. Click OK when done. 61

62 Options Options creates and fits a standard curve from an active set of standards, and adjusts the display of the spreadsheet on the computer screen. Tutorials This opens the Data Display Settings dialog box: Create a standard curve in this area Change the view of the spreadsheet in this area 62

63 Tutorials To run a fit of standard points, check the Enable Fit box. Choose the type of fit in the Fit Model dropdown list: Select Acquisition Mode View to display the spreadsheet in terms of signal channels. Check Autoscroll Tables to automatically scroll across and down the display as data are recorded. In View Options, Click OK when done. To Recalculate the standard curve, check this box. Under Reference Acq:, choose the signal used as the ordinate to fit a standard curve. Click View Options to choose which items are shown in the spreadsheet. Choose the signal directly from the detectors (Display Raw Detector Data), or use signal algebra (Display Acquisition Mode Data). Check the Display Concentration box to show the abscissa in the standard curve. Display Statistics of the fit in the spreadsheet columns. 63

64 Care of Fiber-Optic Cables Care of Fiber-Optic Cables Troubleshooting If there is suspicion that the fibers in the bifurcated fiber-optic cable are broken, 1 Hold the single end of the bifurcated cable up to a bright light. 2 View each end of the split side carefully to see that the light is coming though the cable. Check that nearly all of the fibers are lit up, and that there are few if any broken fibers. Warning: Never pull the cable itself the ferrule may loosen from the cable. Hold the cable by the metal ferrules only. Care of the bifurcated cable To clean the ends of the bifurcated cable 1 Wet a cotton swab with organic solvent (e.g., acetone). 2 Gently wipe the exposed face of the fibers with the solvent-saturated swab. Warning: Never polish the exposed ends of the fibers. Warning: Do not expose the ends of the cables or the ferrules to aqueous solutions, especially acids or bases. 64

65 Storage Storage MicroMax Simply disconnect the AC power cord and the RS-232 communication cable, remove the sample plates and fiber-optic assembly, and close the lid. F-3000 fiber-optic adapter and cables Store these in a drawer and cabinet. Cover the open ferrules on the ends of the fiber-optic cables with the plastic shipping caps. 65

66 Appendix Appendix Computer requirements or equivalent PC computer with RS-232 port Software requirements Windows 9x or NT4 (SP3 or higher) DataMax Physical specifications 9" (20.3 cm) deep 12" (30.5 cm) wide 7" (17.8 cm) tall. Allow an extra 3" (7.6 cm) of height for fiber optics. Compatible with Fluorolog -3, FluoroMax -3, Fluorolog -2, and FluoroMax -2 Accepts 384-well (24 16) and 96-well (12 8) plates. Scans 96 wells in < 1 minute Typical sensitivity = 25 pm fluorescein with the Fluorolog -322; 10 nm fluorescein with the FluoroMax -3. Electrical specifications Note: For 24-well (6 4), and 6-well (3 2), considered non-standard sizes, contact Spex Fluorescence Service. Line voltage 110 VAC or 220 VAC 0.5 A at 110 VAC or 0.25 A at 220 VAC 50 or 60 Hz, single-phase Important The MicroMax 384 microwell plate reader is designed to comply with the requirements of the Low Voltage Directive 73/23/EEC and the EMC Directive 89/336/EEC, and, as of 1 January 1997, carries the CE marking accordingly. The system was tested using standard (ISA-authorized) components, cables, etc. 66

67 Fiber-optics specifications Appendix Bifurcated-slit-end bundles Concentric ring combination in cable: outer ring is for excitation; inner ring is for emission collection. Length = 1 m Numerical Aperture = 0.12 NA Wavelength range = covers full range of spectrofluorometers from nm. Other wavelength ranges are available. 67

68 Index Index A absorbance 16, 19 acetone 64 Acquisition Mode View 63 acquisition modes 30 Acquisition Parameter 56 adapter 5, 6, 7, 8, 9, 11, 14, 15, 23, 47, 65 Add 53, 56, 58, 59 Add 42 Allen wrench 14, 23 allplate.ini 6, 11 Append 61 Autoscroll Tables 63 B Band Pass 13, 14, 18, 22 blank 16, 19, 30, 36 Blank 46, 58, 59 blank subtraction 30 browse 34, 35 C cable 6, 7, 9, 10, 14, 23, 65, 66, 67 calibration 11, 15, 19, 47 care 64 CE 66 CHAN_NUM 11 clean 64 cnts 18, 22 Collect 32, 33, 49 COM 5, 9, 11 computer 5, 7, 9, 10, 11, 15, 19, 66 concentration curve 30 Constant Wavelength Analysis 24, 30, 31, 40, 48, 55 cotton swab 64 cps 18, 22 cwa 60 CWA Data Display 44, 46, 57 D damage 4, 6 danger 4 Data Display Settings 62 DataMax 4, 5, 11, 12, 15, 16, 19, 20, 24, 33, 44, 45, 46, 49, 66 Define 42, 43 detector 18, 22, 28, 44 Detector Parameters 56 directory 10, 34, 35 diskette 6, 11 Display Acquisition Mode Data 63 Display Concentration 63 Display Raw Detector Data 63 Display Statistics 63 drive 10, 34, 35 Drives 38, 39 E EEC 66 emission 4, 7, 15, 18, 22, 29, 30, 40, 41, 47, 52, 67 Emission Acquisition 49, 50, 51, 52 Enable Fit 63 Excel 30, 60 excitation 4, 7, 8, 14, 15, 18, 22, 29, 30, 40, 41, 47, 52, 67 Excitation 51 exp 34 Exp Type Exp. Browse 34, 53 Experiment 49 extinction coefficient 15, 19 F F , 6, 7, 8, 11, 14, 15, 47, 65 fiber optics 4, 5, 6, 7, 9, 10, 14, 23, 47, 64, 65, 66 File Browse 35 Fit Model 63 fluorescein 15, 16, 19, 66 Fluorolog 4, 11, 66 FluoroMax 4, 10, 66 Folders 38, 39 gasket 8 G H home 12, 13, 15, 16, 19, 20, 26 I image (of plate reader) 12, 17, 20 Increment 51 Info 34, 35 Installation 5, 6 68

69 Instrument Control Center 12, 16, 20, 40 Instrument Setup 24, 25, 28, 32 integration time 28, 30, 44 Integration Time 51, 56 Intensity 17, 18, 21, 22 ISA_INI 11 K kinetics 30, 42, 43 Kinetics 42, 43 L lamp intensity 30 layout 10, 11, 12, 16, 20 lens 6, 7, 10, 26 load 26, 32, 37 Load Set 37 Low Voltage Directive 66 M Maximum Trials 56 MicroPlate Configuration 36, 37, 39, 45, 53, 54, 57 MicroPlate Matrix Scan 33, 53, 54 Microplate matrix scans 31 MicroPlate Scan 33, 53 mirror 7, 14, 15 monochromator 4, 7, 14, 18, 22, 28, 41 Monos 13, 14, 17, 18, 21, 22 Move 13, 17, 21, 26 multigroup 29 mw.ini 11 N NaOH 16, 19 New Group 58, 59 Notepad 11 Numerical Aperture 67 O OK 45, 61, 63 Open 61 optics 4 Options 62 organic solvent 64 output 18, 22, 35 P pathname 34 photobleach 30, 42, 43 Index plate 4, 6, 7, 8, 13, 15, 19, 23, 26, 27, 29, 31, 36, 37, 45, 46, 47, 48, 49, 55, 66 Plate Control 36, 53 Plate Name/ID 37, 46 Plate Properties 27 Plate Reader Control 12, 17, 20, 26, 27 plates 65, 66 power cord 6, 9, 10, 65 preview 34, 35 Proceed to Acquisitions 44, 57 Properties 27 R R 42 Raman 47 readme.txt 10, 11 Real Time Display 13, 17, 21, 24, 28 Recalculate 63 Reference Acq 63 Remove 42 Requirements 5 RS-232 6, 9, 65, 66 Run Experiment 24, 29, 31, 32, 49, 53 Run Plate 46, 59 S S 17, 18, 21, 22, 23, 42, 51, 56 Sample Box 41 sample compartment 5, 7, 8, 9, 13, 14, 15, 16, 19 Sample Compartment Accessories 12, 16, 20, 26 Sample Selection 58 save 39, 60 Save 52 Save Data 60 Save Data As Save MicroPlate Configuration 39 Save Set 39 SB with MicroMax 41, 56 Select Acq Expression To View 45 Select Experiment Type 50 Select File Name 35 sensitivity 4, 66 service 11 set screws 23 setup 34 shutter 14, 18, 22, 30, 43 Signals 51 slits 14, 18, 22, 28, 52 software 4, 5, 10, 11, 12, 15, 16, 19, 20, 24, 25, 66 spc 35 standard 30 Standard 46 standard error 44 Standard Error 56 Start Acq 45, 57 Storage 65 symbols 4 synchronous 29 69

70 T T-box 56 thumb screws 7, 8, 10, 15 time base 29 Time-based Acquisition Parameters 42, 43 tpl 39 troubleshooting 64 Tutorials 48 txt 60 Unknown 46, 58, 59 unload 26 V View Options 63 View Options 63 Visual Instrument Setup 12, 16, 20, 26 voltage 9, 66 Index U unknown 30, 36 W Windows 5, 12, 16, 20 70