MicroMax 384 v. 3.0 (9 Mar 2007)

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3 MicroMax 384 with FluorEssence Operation Manual Rev. 3 i

4 Copyright HORIBA Jobin Yvon Inc. All rights reserved. No part of this work may be reproduced, stored, in a retrieval system, or transmitted in any form by any means, including electronic or mechanical, photocopying and recording, without prior written permission from HORIBA Jobin Yvon Inc. Requests for permission should be requested in writing. Origin is a registered trademark of OriginLab Corporation. Windows is a trademark of Microsoft Corporation. Information in this manual is subject to change without notice, and does not represent a commitment on the part of the vendor. March 2007 Part Number ii

5 Table of Contents 1: Introduction About the MicroMax Disclaimer Safety summary Risks of ultraviolet exposure Additional risks of xenon lamps : Requirements & Installation Requirements Installation Calibration and alignment of F-3000 or F adapter Calibration and alignment of MicroMax : Operation of the MicroMax Manual control of the MicroMax Running experiments with the MicroMax Constant-wavelength analysis with the MicroMax Changing the type of microwell plate Batch jobs : Daily Calibration Verification Introduction Method : Tutorials Introduction Tutorial 1: Full-spectrum analysis Tutorial 2: Constant-wavelength analysis : Maintenance Care of the fiber-optic cable Storage : Troubleshooting : Technical Specifications Computer requirements Software requirements Physical requirements Electrical requirements Fiber-optics requirements : Compliance Information Declaration of Conformity : Index iii

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7 1: Introduction About the MicroMax 384 Introduction The MicroMax 384 is a microwell-plate reader able to accept plates with up to 384 wells. It connects to the FluoroMax and Fluorolog spectrofluorometers. MicroMax 384 s high speed allows it to scan completely a 96-well 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 monochromator is carried via a fiber-optic bundle to the MicroMax 384. The fluorescent response of the samples in the microwells is returned to the emission monochromator via the same fiber-optic bundle. 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 FluorEssence software. Custom selection of specific microwells on a plate is also controlled from the sofware. Note: Keep this and the other reference manuals near the system. 1-1

8 Disclaimer By setting up or starting to use any HORIBA Jobin Yvon product, you are accepting the following terms: Introduction You are responsible for understanding the information contained in this document. You should not rely on this information as absolute or all-encompassing; there may be local issues (in your environment) not addressed in this document that you may need to address, and there may be issues or procedures discussed that may not apply to your situation. If you do not follow the instructions or procedures contained in this document, you are responsible for yourself and your actions and all resulting consequences. If you rely on the information contained in this document, you are responsible for: Adhering to safety procedures Following all precautions Referring to additional safety documentation, such as Material Safety Data Sheets (MSDS), when advised As a condition of purchase, you agree to use safe operating procedures in the use of all products supplied by HORIBA Jobin Yvon, including those specified in the MSDS provided with any chemicals and all warning and cautionary notices, and to use all safety devices and guards when operating equipment. You agree to indemnify and hold HORIBA Jobin Yvon harmless from any liability or obligation arising from your use or misuse of any such products, including, without limitation, to persons injured directly or indirectly in connection with your use or operation of the products. The foregoing indemnification shall in no event be deemed to have expanded HORIBA Jobin Yvon s liability for the products. HORIBA Jobin Yvon products are not intended for any general cosmetic, drug, food, or household application, but may be used for analytical measurements or research in these fields. A condition of HORIBA Jobin Yvon s acceptance of a purchase order is that only qualified individuals, trained and familiar with procedures suitable for the products ordered, will handle them. Training and maintenance procedures may be purchased from HORIBA Jobin Yvon at an additional cost. HORIBA Jobin Yvon cannot be held responsible for actions your employer or contractor may take without proper training. Due to HORIBA Jobin Yvon s efforts to continuously improve our products, all specifications, dimensions, internal workings, and operating procedures are subject to change without notice. All specifications and measurements are approximate, based on a standard configuration; results may vary with the application and environment. Any software manufactured by HORIBA Jobin Yvon is also under constant development and subject to change without notice. Any warranties and remedies with respect to our products are limited to those provided in writing as to a particular product. In no event shall HORIBA Jobin Yvon be held li- 1-2

9 Introduction able for any special, incidental, indirect or consequential damages of any kind, or any damages whatsoever resulting from loss of use, loss of data, or loss of profits, arising out of or in connection with our products or the use or possession thereof. HORIBA Jobin Yvon is also in no event liable for damages on any theory of liability arising out of, or in connection with, the use or performance of our hardware or software, regardless of whether you have been advised of the possibility of damage. 1-3

10 Safety summary Introduction The following general safety precautions must be observed during all phases of operation of this instrument. Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design, manufacture and intended use of instrument. HORIBA Jobin Yvon assumes no liability for the customer s failure to comply with these requirements. Certain symbols are used throughout the text for special conditions when operating the instruments: Warning: A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or similar that, if incorrectly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met. HORIBA Jobin Yvon Inc. is not responsible for damage arising out of improper use of the equipment. Caution: A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or similar that, if incorrectly performed or adhered to, could result in damage to the product. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met. HORIBA Jobin Yvon Inc. is not responsible for damage arising out of improper use of the equipment. Caution: Ultraviolet light! Wear protective goggles, fullface shield, skin-protection clothing, and UVblocking gloves. Do not stare into light. Caution: Intense ultraviolet, visible, or infrared light! Wear light-protective goggles, full-face shield, skin-protection clothing, and light-blocking gloves. Do not stare into light. Warning: Danger to fingers! This symbol warns the user that the equipment is heavy, and can crush or injure the hand if precautions are not taken. 1-4

11 Introduction Caution: This symbol cautions the user that excessive humidity, if present, can damage certain equipment. Read this manual before using or servicing the instrument. Wear protective gloves. Wear appropriate safety goggles to protect the eyes. Wear an appropriate face-shield to protect the face. Note: General information is given concerning operation of the equipment. 1-5

12 Risks of ultraviolet exposure Introduction Caution: This instrument is used in conjunction with ultraviolet light. Exposure to these radiations, even reflected or diffused, can result in serious, and sometimes irreversible, eye and skin injuries. Overexposure to ultraviolet rays threatens human health by causing: Immediate painful sunburn Skin cancer Eye damage Immune-system suppression Premature aging Do not aim the UV light at anyone. Do not look directly into the light. Always wear protective goggles, full-face shield and skin protection clothing and gloves when using the light source. Light is subdivided into visible light, ranging from 400 nm (violet) to 700 nm (red); longer infrared, above red or > 700nm, also called heat; and shorter ultraviolet radiation (UVR), below violet or < 400nm. UVR is further subdivided into UV-A or near-uv ( nm), also called black (invisible) light; UV-B or mid-uv ( nm), which is more skin penetrating; and UV-C or far-uv (< 290 nm). Health effects of exposure to UV light are familiar to anyone who has had sunburn. However, the UV light level around some UV equipment greatly exceeds the level found in nature. Acute (short-term) effects include redness or ulceration of the skin. At high levels of exposure, these burns can be serious. For chronic exposures, there is also a cumulative risk of harm. This risk depends upon the amount of exposure during your lifetime. The long-term risks for large cumulative exposure include premature aging of the skin, wrinkles and, most seriously, skin cancer and cataract. Damage to vision is likely following exposure to high-intensity UV radiation. In adults, more than 99% of UV radiation is absorbed by the anterior structures of the eye. UVR can contribute to the development of age-related cataract, pterygium, photodermatitis, and cancer of the skin around the eye. It may also contribute to age-related macular degeneration. Like the skin, the covering of the eye or the cornea, is epithelial tissue. The danger to the eye is enhanced by the fact that light can enter from all angles around the eye and not only in the direction of vision. This is especially true while working in a dark environment, as the pupil is wide open. The lens can also be damaged, but because the cornea acts as a filter, the chances are re- 1-6

13 Introduction duced. This should not lessen the concern over lens damage however, because cataracts are the direct result of lens damage. Burns to the eyes are usually more painful and serious than a burn to the skin. Make sure your eye protection is appropriate for this work. NORMAL EYEGLASSES OR CONTACTS OFFER VERY LIMITED PROTECTION! Training Warning: UV exposures are not immediately felt. The user may not realize the hazard until it is too late and the damage is done. For the use of UV sources, new users must be trained by another member of the laboratory who, in the opinion of the member of staff in charge of the department, is sufficiently competent to give instruction on the correct procedure. Newly trained users should be overseen for some time by a competent person. 1-7

14 Additional risks of xenon lamps Introduction Warning: Xenon lamps are dangerous. Please read the following precautions. Among the dangers associated with xenon lamps are: Burns caused by contact with a hot xenon lamp. Fire ignited by hot xenon lamp. Interaction of other nearby chemicals with intense ultraviolet, visible, or infrared radiation. Damage caused to apparatus placed close to the xenon lamp. Explosion or mechanical failure of the xenon lamp. Visible radiation Any very bright visible light source will cause a human aversion response: we either blink or turn our head away. Although we may see a retinal afterimage (which can last for several minutes), the aversion response time (about 0.25 seconds) normally protects our vision. This aversion response should be trusted and obeyed. NEVER STARE AT ANY BRIGHT LIGHT-SOURCE FOR AN EXTENDED PERIOD. Overriding the aversion response by forcing yourself to look at a bright light-source may result in permanent injury to the retina. This type of injury can occur during a single prolonged exposure. Excessive exposure to visible light can result in skin and eye damage. Visible light sources that are not bright enough to cause retinal burns are not necessarily safe to view for an extended period. In fact, any sufficiently bright visible light source viewed for an extended period will eventually cause degradation of both night and color vision. Appropriate protective filters are needed for any light source that causes viewing discomfort when viewed for an extended period of time. For these reasons, prolonged viewing of bright light sources should be limited by the use of appropriate filters. The blue-light wavelengths ( nm) present a unique hazard to the retina by causing photochemical effects similar to those found in UV-radiation exposure. Infrared radiation Infrared (or heat) radiation is defined as having a wavelength between 780 nm and 1 mm. Specific biological effectiveness bands have been defined by the CIE (Commission International de l Eclairage or International Commission on Illumination) as follows: IR-A (near IR) ( nm) IR-B (mid IR) ( nm) IR-C (far IR) (3000 nm 1 mm) 1-8

15 Introduction The skin and eyes absorb infrared radiation (IR) as heat. Workers normally notice excessive exposure through heat sensation and pain. Infrared radiation in the IR-A that enters the human eye will reach (and can be focused upon) the sensitive cells of the retina. For high irradiance sources in the IR-A, the retina is the part of the eye that is at risk. For sources in the IR-B and IR-C, both the skin and the cornea may be at risk from flash burns. In addition, the heat deposited in the cornea may be conducted to the lens of the eye. This heating of the lens is believed to be the cause of so called glassblowers cataracts because the heat transfer may cause clouding of the lens. Retinal IR Hazards (780 to 1400 nm): possible retinal lesions from acute high irradiance exposures to small dimension sources. Lens IR Hazards (1400 to 1900 nm): possible cataract induction from chronic lower irradiance exposures. Corneal IR Hazards (1900 nm to 1 mm): possible flashburns from acute high irradiance exposures. Who is likely to be injured? The user and anyone exposed to the radiation or xenon lamp shards as a result of faulty procedures. Injuries may be slight to severe. 1-9

16 Introduction 1-10

17 Requirements & Installation 2: Requirements & Installation Requirements Physical requirements Located within 0.75 m of the spectrofluorometer sample compartments Needs 10 (25 cm) height minimum including the MicroMax 384, to account for the fiber-optic bundle. Requires F-3000 (Fluorolog -3) or F (FluoroMax -4) fiber-optic adapter accessory Computer requirements Dedicated serial COM port FluorEssence software version 2.1 Windows XP Pro software or equivalent PC as host computer 2-1

18 Requirements & Installation Installation 1 Unpack and inspect all components a The following MicroMax 384 components should be included: Warning: Watch your fingers! Quantity Description Part Number 1 MicroMax 384 microwell plate reader 1 MicroMax 384 with FluorEssence Operation Manual AC (mains) power cable RS-232 communications 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 spectrofluorometer (sold separately from MicroMax 384) F-3000 (for Fluorolog ) or F (for FluoroMax -4) b Inspect all components for signs of damage that may have occurred during transit. If damage is evident, do not continue with the installation. Instead, notify HORIBA Jobin Yvon Inc. and the shipper at once. Note: Many public carriers will not recognize a claim for concealed damage if it is reported later than 15 days after delivery. In case of a claim, inspection by an agent of the carrier is required. For this reason, the original packing material should be retained as evidence of alleged mishandling or abuse. While HORIBA Jobin Yvon Inc. assumes no responsibility for damage occurring during transit, the company will make every effort to aid and advise. Caution: The MicroMax 384 is a delicate instrument. Mishandling may seriously damage its components. 2-2

19 Requirements & Installation 2 Read this instruction manual thoroughly before proceeding to install the MicroMax Install the F-3000 or F fiberoptic 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 plate was used to prevent the base from moving during shipping. Caution: 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 s sample chamber. Save the two brass thumbscrews for mounting the fiber-optic adapter. d Insert the F-3000 or F 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 baseplate by using two brass thumbscrews. The lens and baffle should be closer to the excitation monochromator. For Fluorolog -3 users: f Remove the 1⅜ (3.5 cm) diameter plastic plug from the front of the spectrofluorometer s sample compartment. If there are any threaded plugs for set screws in the two adjacent mounting holes, remove them, too. g Remove the plastic shipping caps from the fiber-optic cable s ferrules. 2-3

20 Requirements & Installation h Thread the two slit-ended fiber bundles through the hole in the sample compartment. i 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: Caution: Avoid twisting the fiber-optic bundles too much. Breakage of the glass fibers may occur with excessive strain. j Secure the fiber-optic bundles loosely with the two narrow thumbscrews on the F-3000 or F adapter, in their respective mounts. k Mount the plate and gasket on the outside of the sample compartment, as shown in this photograph: Gasket (underneath light-shield plate) Light-shield plate 2-4

21 Requirements & Installation For FluoroMax -4 users: f With a 9/64 Allen key, remove the 4 inside cap screws from the front wall of the sample compartment. The existing blue plate detaches from the sample mount. g Remove the 1⅜ (3.5 cm) diameter plastic plug from the new blue plate. Insert the rectangular gasket into the rectangular depression. h Attach the new blue plate with gasket to the sample mount, using the 4 cap screws. Thread the two slit-ended fiber bundles through the hole in the sample compartment. i 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: 2-5

22 Requirements & Installation Caution: Avoid twisting the fiber-optic bundles too much. Breakage of the glass fibers may occur with excessive strain. j Secure the fiber-optic bundles loosely with the two narrow thumbscrews on the F-3000 or F adapter, in their respective mounts. 4 Assemble the MicroMax 384 and accessories. a Place the MicroMax 384 on a level surface near the spectrofluorometer s sample compartment, so that the fiber-optic bundles reach both sample and MicroMax 384. b Verify that the MicroMax 384 is set for the proper line voltage. The power (mains) receptacle has a white arrow that indicates the AC voltage set by the factory. c Connect the AC power cord (mains) 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. e Attach the other end of the RS- 232 cable to the free COM port on the back of the host computer. Use the enclosed 9-pin 25-pin adapter as necessary. Note: Which COM port chosen depends on the settings in the instrument configuration, described later. 2-6

23 f Insert the combined end of the fiber-optic cable into the lens cell. The cable should reach the bottom of the cell. Requirements & Installation g Secure the cable to the lens by tightening the set screw on the lens cell. h Insert the lens-cell assembly into the receptacle at the top of the MicroMax 384, so that it is firmly sealed. The top of the lens should protrude about ⅛ (~2 mm) from the receptacle. ~ 2 mm i j Caution: Never pull the calbe itself the Tighten the ferrule may loosen from the cable. Hold the cable by the metal ferrules only.. nylon thumbscrew on the lens receptacle. This secures the lens-cell assembly in place. Plug the free end of the power (mains) cord into a properly rated AC receptacle. Initial positioning for alignment is complete. 2-7

24 Requirements & Installation 5 Create a new instrument configuration. (only if the MicroMax 384 was purchased separately from the spectrofluorometer, otherwise skip to step 6) Note: If you plan to use plates with different numbers of microwells (e.g., 384 or 96), create a separate instrument configuration for each type of plate. a Double-click the FluorEssence icon on the desktop to start the FluorEssence software. The main FluorEssence window appears. b Choose the Collect menu. A drop-down menu appears. c Choose Experiment Setup. The Select Hardware Configuration window appears. 2-8

25 Requirements & Installation d Choose the New button. The System Configuration Wizard window appears. e Choose the Standard Instrument Configuration radio button, and click Next >>. f The System Selection Page window appears. Choose the main instrument to which the MicroMax 384 is to be connected, and its model (as necessary) from the dropdown menu, then click Next >. 2-9

26 The Configuration Name window appears. Requirements & Installation g Enter a distinguishing name for this instrument configuration, and click Next >. The Fluoromax Configuration window appears. h Activate the MicroMax checkbox, and click Next >. i The MicroMax Communication Settings window appears. Choose a free COM port, and click the OK button. A Dialog window appears. 2-10

27 j From the dropdown menu, choose the type of plate, and click Add >>. The plate appears in the empty box on the right. k On the left side of the accessory, read the offset values and enter them into the X Origin and Y Origin fields, then click the OK button. Requirements & Installation The Summary window appears. This window is not editable, but shows the instrument configuration to be activated. Note: Even if the offset shows negative values, enter positive values. l If this configuration is correct, click the Finish button. m To use another type of microwell plate, repeat step 5. 6 Calibrate and align the F-3000 or F adapter. This procedure is explained in the next section of this chapter. 2-11

28 Requirements & Installation 7 Calibrate and align the MicroMax 384. This procedure is explained in the section after calibrating the F-3000 or F adapter. 2-12

29 Requirements & Installation Calibration and alignment of F-3000 or F adapter 1 Start the MicroMax 384 and FluorEssence software. a Be sure that FluorEssence is installed on the host computer. b Start the spectrofluorometer. c Turn on the MicroMax 384 via its power switch. d Double-click the FluorEssence icon on the desktop to start the FluorEssence software. The main FluorEssence window appears. e Click the Experiment Menu button. The Select Hardware Configuration window appears. 2-13

30 Requirements & Installation f Choose the appropriate instrument configuration with a MicroMax 384 attached. Note: If you have only one instrument configuration, this window does not appear. The FluorEssence Main Experiment Menu opens. g Choose the type of experiment to run. The Experiment Type window asks what subtype of experiment to set up. h Choose the desired subtype, and click Next >>. The Experiment Setup window appears. Note: Grayed-out buttons are not available on your instrument configuration. 2-14

31 2 Activate the MicroMax 384. a Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. Requirements & Installation The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. 2-15

32 Requirements & Installation b Choose the MicroMax tab. The grayed-out parameters of the MicroMax tab appear: 2-16

33 Requirements & Installation c Pick a microwell by clicking on a gray circle on the plate schematic. Any microwell may be chosen; this is necessary for the MicroMax 384 to appear in the Real Time Control. d Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. 2-17

34 Requirements & Installation e Click the RTC button to enter the Real Time Control window. 2-18

35 Requirements & Installation f Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. 3 Examine the alignment. a Set the excitation monochromator to 500 nm (blue-green light) by entering 500. b Set the Band Pass to 5 nm. c Open the shutter. d Remove the top of the spectrofluorometer s sample compartment. Caution: Intense ultraviolet, visible, or infrared light may be present when the sample compartment is open. Do not aim fiberoptic bundles onto the skin or eyes. Use extreme caution with the fiber-optic probes. 2-19

36 Requirements & Installation e 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. The beam should fall directly on the fibers: End of ferrule Slit-shaped bundle of fibers Beam of light f If the beam is not in the center, remove the fiber-optic cables and the F or F 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 back position slightly. Replace the F-3000 or F and fiber-optics in the sample compartment, and examine the beam again. Caution: Intense ultraviolet, visible, or infrared light may be present when the sample compartment is open. Do not aim fiberoptic bundles onto the skin or eyes. Use extreme caution with the fiber-optic probes. g Repeat step f until the beam falls properly on the entrance of the fiber bundle. h Reduce the band pass to 2 nm, and examine the beam again. The beam should fall directly on the fibers. If not, repeat step f. i Reduce the band pass to 1 nm, and re-examine the beam. The beam should fall directly on the fibers. If not, repeat step f. 2-20

37 j Remove the F-3000, rotate it 90 counterclockwise, Requirements & Installation and replace it in the sample compartment so that the emission beam now is at the excitation-fiber position. k With the dental mirror, examine the slit-shaped beam as in steps e through i. l Verify that the light falls in the middle Caution: Intense ultraviolet, visible, or infrared light may be present when the sample compartment is open. Do not aim fiberoptic bundles onto the skin or eyes. Use extreme caution with the fiber-optic probes. of the emission s ferrule end. Adjust the mirror as needed. (A later procedure will tune the emission s signal level.) Remove the F-3000 or F adapter, rotate it 90 clockwise back to its original position, and fasten it down with the two thumbscrews. Note: If the F-3000 or F adapter was purchased with the instrument, the mirrors are now aligned. If the F or F was purchased separately, continue aligning the mirrors below. When the MicroMax is turned on and connected to the host computer, the microwell-plate holder automatically moves to the Home position. Note: The following steps assume that fluorescein is the sample used to calibrate and align. Other fluorescent solutions may require different settings. 4 Calibrate the MicroMax 384 with standard and blank. 2-21

38 Requirements & Installation a Insert a clean, unused microwell plate containing: A microwell with 200 µl of 100 nm fluorescein (extinction coefficient ~ ) in 0.1 N NaOH the standard. A microwell with 200 µl of 0.1 N NaOH the blank solution. The fluorescein gives an absorbance A = at 490 nm above the blank. 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. b Close the sample-compartment lid. c Start the FluorEssence software, and load an instrument configuration with the MicroMax 384. At the end of the initialization process, the MicroMax 384 makes a characteristic sound as it resets to the home position. The FluorEssence Main Experiment Menu opens. d Choose a type of experiment to run. If necessary, the Experiment Type window asks what subtype of experiment to set up. e Choose the desired subtype, and click Next >>. The Experiment Setup window appears. Note: Grayed-out buttons are not available on your instrument configuration. 2-22

39 f Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. Requirements & Installation The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. 2-23

40 Requirements & Installation g Pick a microwell by clicking on any gray circle in the plate schematic. This is necessary to activate the MicroMax 384 in the Real Time Control. h Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. 2-24

41 Requirements & Installation i Click the RTC button to enter the Real Time Control window. 2-25

42 Requirements & Installation j Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. k Click the MicroMax tab. 2-26

43 Requirements & Installation l On the sketch of the microwell plate, click the position of the standard in the microwell plate. The value, e.g., A12, should appear in the Current Cell field, and the microwell plate should move to this position. m Click the General icon. 2-27

44 Requirements & Installation n In the Excitation 1 area, set the excitation monochromator s Position to 485 nm by typing in 485 in Position, and the Slits to 2 nm. o In the Emission 1 area, set the emission monochromator s Position to 513 nm by typing in 513 in Position and the Slits to 2 nm. p Move the Shutter Mode switch to Open. This opens the excitation shutter. q Click the Run button. r Watch the S signal intensity in the black graph or the Signal Intensity tab. If, at any time during alignment, the signal > cps, reduce the Slits settings. 2-28

45 Requirements & Installation s Open the sample-compartment cover. t Adjust the front set screw for the emission mirror to maximize the signal intensity. This controls the pitch (frontback fine rotation) of the mirror. u Adjust the side set screw for the emission mirror to maximize the signal intensity. This controls the left-right fine rotation of the mirror. Caution: Intense ultraviolet, visible, or infrared light may be present when the sample compartment is open. Do not aim fiber-optic bundles onto the skin or eyes. Use extreme caution with the fiber-optic probes. 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 Fluorescence Service. The mirrors should now be aligned. 2-29

46 Requirements & Installation Calibration and alignment of MicroMax Be sure that FluorEssence is properly installed on the host computer. 2 Start the spectrofluorometer. 3 Set up the MicroMax 384. a Insert a clean, unused microwell plate containing: A microwell with 200 µl of 100 nm fluorescein (extinction coefficient ~ ) in 0.1 N NaOH the standard. A microwell with 200 µl of 0.1 N NaOH the blank solution. The fluorescein gives an absorbance A = at 490 nm above the blank. 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. b Close the sample-compartment lid. c Start the FluorEssence software, and load an instrument configuration with the MicroMax 384. At the end of the initialization process, the MicroMax 384 makes a characteristic sound as it resets to the home position. The FluorEssence Main Experiment Menu opens. d Choose a type of experiment to run. Note: Grayed-out buttons are not available on your instrument configuration. 2-30

47 If necessary, the Experiment Type window asks what subtype of experiment to set up. e Choose the desired subtype, and click Next >>. The Experiment Setup window appears. Requirements & Installation f Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. 2-31

48 Requirements & Installation g Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. 2-32

49 Requirements & Installation h Pick a microwell by clicking on any gray circle in the plate schematic. This is necessary to activate the MicroMax 384 in the Real Time Control. i Click the RTC button to enter the Real Time Control window. 2-33

50 Requirements & Installation j Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. k Click the MicroMax tab. 2-34

51 Requirements & Installation l On the sketch of the microwell plate, click the position of the standard in the microwell plate. The value, e.g., A12, should appear in the Current Cell field, and the microwell plate should move to this position. m Click the General icon. 2-35

52 Requirements & Installation n In the Excitation 1 area, set the excitation monochromator s Position to 485 nm by typing in 485 here, and the Slits to 2 nm. o In the Emission 1 area, set the emission monochromator s Position to 513 nm by typing in 513 here and the Slits to 2 nm. p Move the Shutter Mode switch to Open. This opens the excitation shutter. q Watch the S signal intensity in the black graph or the Signal Intensity tab. If, at any time during alignment, the signal > cps, reduce the Slits settings. 2-36

53 Requirements & Installation 4 Align the MicroMax 384. a Loosen the nylon thumbscrew on the fiber-optic adapter assembly. b Gently adjust the height of the fiberoptic assembly above the microwell plate, in order to maximize the S signal. c With maximum signal, 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 s volume changes. Note: The background count of a microwell plate is large, therefore we recommend that you subtract a blank scan from the sample scan to give ultimate sensitivity. See the on-line help and the User s Guide to FluorEssence for more information on blank-subtraction. Note: This alignment of the fiber-optic assembly should be performed each time a different sample volume is used. 2-37

54 Requirements & Installation 2-38

55 Operation of the MicroMax 384 3: Operation of the MicroMax 384 Manual control of the MicroMax 384 Manual control of the MicroMax 384 is found in the Real Time Control, where you can move the microwell plate to particular wells and see the resulting fluorescence in real time. 1 Start the FluorEssence software, and load an instrument configuration with the MicroMax 384. At the end of the initialization process, the MicroMax 384 makes a characteristic sound as it resets to the home position. The FluorEssence Main Experiment Menu opens. 2 Choose a type of experiment to run. Note: Grayed-out buttons are not available on your instrument configuration. The MicroMax 384 cannot be used with the AutoTitrator or temperature-bath accessories. If necessary, the Experiment Type window asks what subtype of experiment to set up. 3 Choose the desired subtype, and click Next >>. The Experiment Setup window appears. 3-1

56 Operation of the MicroMax Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. 3-2

57 Operation of the MicroMax 384 d Pick a microwell by clicking on any gray circle in the plate schematic. This is necessary to activate the MicroMax 384 in the Real Time Control. 5 Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. 3-3

58 Operation of the MicroMax Click the RTC button to enter the Real Time Control window. 3-4

59 Operation of the MicroMax Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. 8 Click the MicroMax tab. 3-5

60 Operation of the MicroMax On the sketch of the microwell plate, click the position of the standard in the microwell plate. The value, e.g., A12, should appear in the Current Cell field, and the microwell plate should move to this position. 10 To return to the home position, click the Home button. 3-6

61 Operation of the MicroMax 384 Running experiments with the MicroMax 384 Introduction The Experiment Setup window provides all scanning capability for the spectrofluorometer, with or without the MicroMax 384. The 3D scan-type is not possible with the MicroMax 384. Method Note: The MicroMax 384 cannot be used with the AutoTitrator or temperature-bath accessories. 1 In the main FluorEssence window, click the Experiment Menu button. 3-7

62 Operation of the MicroMax 384 The Select Hardware Configuration window appears. 2 Choose the appropriate instrument configuration with a MicroMax 384 attached. Note: If you have only one instrument configuration, this window does not appear. The FluorEssence Main Experiment Menu opens. 3 Choose the type of experiment to run. Note: Grayed-out buttons are not available on your instrument configuration. The Experiment Type window asks what subtype of experiment to set up. 4 As necessary, choose the desired subtype, and click Next >>. The Experiment Setup window appears. 3-8

63 Operation of the MicroMax Activate the MicroMax 384. a Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. 3-9

64 Operation of the MicroMax 384 b Choose the MicroMax tab. The grayed-out parameters of the MicroMax tab appear: 3-10

65 Operation of the MicroMax 384 c Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. 3-11

66 Operation of the MicroMax 384 d Choose the radio button for the type of sample in each well: Radio button Color code on Type of sample diagram Empty radio button gray No sample is in the well, or unscanned Blank radio blue Blank sample button Standard radio green Standard sample button Unknown radio button red Unknown sample to be determined by curve fitting or other means To choose a microwell, click on a corner of the diagram s area to be denoted as a particular sample type and drag through the area, or click on each individual microwell. To select all microwells, click the Select All button. To clear all microwells, click the Clear All button. As microwells are designated, their position and type appear in the table on the right side of the window. 3-12

67 Operation of the MicroMax 384 To delete a row from the table, click in the undesired row(s), and then click the Delete Row(s) button. Below is a sample window with many microwells designated for scanning: e The radio buttons in the Curve Fitting area are inactive in the current version of FluorEssence. f Double-click the Concentration field in each Standard s row, type its concentration, then hit the Enter key. 6 Click the Run button to run the experiment. 3-13

68 Operation of the MicroMax 384 Constant-wavelength analysis with the MicroMax 384 Introduction Constant-wavelength analysis, or the Single Point scan-type, allows acquisition of excitation and emission pairs. Results may be plotted or retained as a table. Up to 32 wavelength-pairs and multiple acquisition modes may be selected. Sample positions may be tagged as blanks, standards, or unknowns. Data manipulation and correction includes: Blank subtraction Concentration curves Correction files for inhomogeneities in lamps and spectrofluorometers Variable-time kinetics, i.e., acquire scans at different time-intervals, and use different integration times Photobleach mode, i.e., protect light-sensitive samples by closing the shutter during dead time Method Note: The MicroMax 384 cannot be used with the AutoTitrator or temperature-bath accessories. 1 In the main FluorEssence window, click the Experiment Menu button. 3-14

69 Operation of the MicroMax 384 The Select Hardware Configuration window appears. 2 Choose the appropriate instrument configuration with a MicroMax 384 attached. Note: If you have only one instrument configuration, this window does not appear. The FluorEssence Main Experiment Menu opens. 3 Choose Single Point. Note: Grayed-out buttons are not available on your instrument configuration. The Experiment Setup window appears. 4 Activate the MicroMax

70 Operation of the MicroMax 384 g Click the Accessories icon. FluorEssence considers the MicroMax 384 an accessory. The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. 3-16

71 Operation of the MicroMax 384 h Choose the MicroMax tab. The grayed-out parameters of the MicroMax tab appear: 3-17

72 Operation of the MicroMax 384 i Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. 3-18

73 Operation of the MicroMax 384 j Choose the radio button for the type of sample in each well: Radio button Color code on Type of sample diagram Empty radio button gray No sample is in the well, or unscanned Blank radio blue Blank sample button Standard radio green Standard sample button Unknown radio button red Unknown sample to be determined by curve fitting or other means To choose a microwell, click on a corner of the diagram s area to be denoted as a particular sample type and drag through the area, or click on each individual microwell. To select all microwells, click the Select All button. To clear all microwells, click the Clear All button. As microwells are designated, their position and type appear in the table on the right side of the window. 3-19

74 Operation of the MicroMax 384 To delete a row from the table, click in the undesired row(s), and then click the Delete Row(s) button. Below is a sample window with many microwells designated for scanning: k The radio buttons in the Curve Fitting area are inactive in the current version of FluorEssence. l Enter the concentrations of the standards. 5 Set up the other experimental parameters. a Click the Monos icon. The parameters for the monochromators appear. b Set the parameters. 3-20

75 Operation of the MicroMax Click the Run button. The experiment starts. 3-21

76 Operation of the MicroMax 384 Changing the type of microwell plate The MicroMax 384 accepts common microwell plates with 384 microwells and 96 microwells, as well as custom arrays of microwells. To change from one type of plate to another, create a new instrument configuration for each extra type of plate as described in step 5 in the Requirements & Installation chapter, pp. 2-6 through

77 Operation of the MicroMax 384 Batch jobs The MicroMax 384 can perform batch jobs automatically using the FluorEssence software. 1 Set up the MicroMax 384 experiment to be repeated automatically. 2 In the Experiment area, give it a name in the File field. 3 Click the Save button. 4 In the FluorEssence toolbar, click the Run JY Batch Experiments button. The Setup batch experiments window appears. 3-23

78 Operation of the MicroMax Set up the file management. a Load a previously created batch job with the Load button, or browse for.xml (experiment) files with the Browse for experiment files to >> Add button. b Add each desired experiment file to the Execution List. c Reorder or remove the files as necessary using the Delete button, the Up button, and the Down button. d Add comments about the batch file in the Comments: field. e Save the new batch job in the correct path, i.e., in the File Name: field, and click the Save button. 6 Set up the various individual experiments. 3-24

79 Operation of the MicroMax 384 a Select the desired experiment in the Execution List. b In the Total Repeats: field, enter the number of times that experiment should be repeated. c In the Delay before executing: field, enter the number of seconds to wait before executing. d In the Delay between each repeat list: field, enter the number of seconds to wait before repeating the experiment. e Repeat steps a d for the other experiments in the Execution List. 7 Set up the batch job that runs the various experiments. a In the Total Repeats: field, enter the number of times to repeat the batch job. b In the Delay before first: field, enter the number of seconds to wait before starting the batch job. c In the Delay between each: field, enter the number of seconds to wait before rerunning the batch job. 8 Click the Run button to start the batch job. 3-25

80 Operation of the MicroMax

81 Daily Calibration Verification 4: Daily Calibration Verification Introduction HORIBA Jobin Yvon recommends that the operator verify the calibration of the MicroMax 384 and its associated spectrofluorometer at the start of each day. Method 1 Calibrate the spectrofluorometer Perform the normal calibration recommended for the instrument as described in the Operation Manual. For example, check the excitation output with a xenonlamp scan, and then the emission using a water-raman signal in a cuvette. 2 Install the F-3000 or F adapter. Remove the existing sample holder for cuvettes, and replace it with the F-3000 or F adapter for fiber-optic bundles. 3 Calibrate the MicroMax 384. Verify the microwell-plate alignment of the MicroMax 384 as described in the Calibrate and Align the MicroMax 384 section of this manual. 4-1

82 Daily Calibration Verification 4-2

83 5: Tutorials Introduction Tutorials To acquaint you with operation of the MicroMax 384, two tutorials are provided in this chapter. The first tutorial shows how to scan blanks and unknown samples to obtain a blank-subtracted spectrum. The second tutorial demonstrates the use of Single Point scans to scan a large number of microwells in a microwell plate at a particular wavelength-pair. Both tutorials assume that the spectrofluorometer and MicroMax 384 already have been set up, calibrated, and run properly. 5-1

84 Tutorial 1: full-spectrum analysis Tutorials The sample used in this tutorial is fluorescein dissolved in 0.1 N NaOH. Concentrations of 1 µm, 2 µm, 5 µm, 8.3 µm, and 10 µm fluorescein were mixed, along with a blank (0.1 N NaOH). A 96-well microwell plate is used. The fluorescein is in microwells F1 F5, while the blank is in microwell B1. The remainder of the microwell plate is empty. Warning: Refer to your Material Safety Data Sheet (MSDS) for information on the hazards of fluorescein and sodium hydroxide. 1 Calibrate the instrument and the MicroMax. 2 Insert the microwell plate (with samples) into the MicroMax, and close the lid. 3 In the main FluorEssence window, click the Experiment Menu button. The Select Hardware Configuration window appears. 5-2

85 4 Choose the appropriate instrument configuration with a MicroMax 384 attached. Tutorials Note: If you have only one instrument configuration, this window does not appear. The FluorEssence Main Experiment Menu opens. 5 Choose Spectra. Note: Grayed-out buttons are not available on your instrument configuration. The Experiment Type window asks what subtype of experiment to set up. 6 Choose Emission, and click Next >>. The Experiment Setup window appears. 5-3

86 7 Enter test in the Data Identifier field, and give a new File name. 8 Add a Comment describing the experiment. Tutorials 9 Enter the monochromator parameters. a Park the excitation monochromator at 485 nm. b Set the excitation Slit to 1 nm. c Start the scan at 490 nm. d End the scan at 650 nm. e Increment the scan 1 nm by entering 1 in Inc. f Set the emission monochromator Slit to 1 nm. 10 Choose the detector parameters. a Click the Detectors icon. b Set the Integration time to 0.1 s 5-4

87 Tutorials c Enable detector S1. 11 Activate the MicroMax 384. a Click the Accessories icon. The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. b Choose the MicroMax or XY Stage tab. Note: The name of the tab may vary, depending on how the instrument configuration is set. 5-5

88 Tutorials c Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. 5-6

89 Tutorials d Choose the Unknown radio button. e Click on microwell F1, and drag the mouse to microwell F5. As microwells are designated, their position and type appear in the table on the right side of the window. f Choose the Blank radio button. g Click on microwell B1. The window above shows many microwells designated for scanning. 5-7

90 Tutorials 12 Click the Save button or Save As... button to save this experiment. The software asks for a filename. 13 Click the Run button to run the experiment. The Intermediate Display shows the data appearing in real time. Here the MicroMax scans the blank first, then the unknowns. 5-8

91 When the scanning is complete, the Project name window appears. 14 Enter a project name, and click the OK button. The final graphs appear in the window as a 3-D plot. Tutorials 15 Show the graphs as an overlay plot. a Click File, and choose New. b The New window appears. c Click Graph, and then the OK button. A new graph appears. 5-9

92 Tutorials d Right-click on the 1 in the upperleft corner. A menu appears. e Choose Layer Contents... The Layer 1 window opens. f In the left column, highlight the datafiles to display, then click the => after each datafile. The desired files appear in the right column. g Click the OK button. The Layer 1 window disappears. h Choose the Rescale button to rescale the graph so that all data appear. 5-10

93 i Tutorials Click on each dataset as desired, and choose Color to give it a different color. Note: For more information about plotting in Origin, see the Origin on-line help files. 5-11

94 Tutorial 2: constant-wavelength analysis Tutorials The sample used in this tutorial is fluorescein dissolved in 0.1 N NaOH. Concentrations of 1 µm, 2 µm, 5 µm, 8.3 µm, and 10 µm fluorescein were mixed, along with a blank (0.1 N NaOH). A 96-well microwell plate is used. The fluorescein is in microwells F1 F5, while the blank is in microwell B1. The remainder of the microwell plate is empty. Warning: Refer to your Material Safety Data Sheet (MSDS) for information on the hazards of fluorescein and sodium hydroxide. 1 In the main FluorEssence window, click the Experiment Menu button. The Select Hardware Configuration window appears. 2 Choose the appropriate instrument configuration with a MicroMax 384 attached. 5-12

95 Tutorials Note: If there is only one instrument configuration, this window does not appear. The FluorEssence Main Experiment Menu opens. 3 Choose Single Point. The Experiment Setup window appears. 4 Enter the wavelengths to monitor: 485 in the Excitation 1 column, then 518 in the Emission 1 column. Note: Grayed-out buttons are not available on your instrument configuration. 5-13

96 5 Set the detector parameters. Tutorials a Choose the Detectors icon. The detector parameters appear. b Enable S1. c Enter an integration time of 0.1 s. 6 Activate the MicroMax 384. a Click the Accessories icon. The accessories tabs appear: Note: The number of tabs varies depending on the accessories set up in your instrument s configuration. 5-14

97 Tutorials b Choose the MicroMax or XY Stage tab. The grayed-out parameters of the MicroMax tab appear: Note: The name of the tab may vary, depending on how the instrument configuration is set. 5-15

98 Tutorials c Click the Activate checkbox to activate the MicroMax 384. Several items in the MicroMax tab become active. d Choose the Blank radio button. e Click on microwell B1 in the chart. Microwell B1 turns blue, and it appears in the table on the right. f Choose the Unknown radio button. g Click on microwell F1, hold the mouse button down, and drag to microwell F5. Microwells F1 to F5 turn red, and they appear in the table on the right. h Enter the concentrations of the standards. In the table, click in the desired row, and enter the concentration in the Concentration column. Below is a sample window with many microwells designated for scanning: 5-16

99 Tutorials 7 Click the Run button. The experiment starts, and the Intermediate Display appears: When the scanning is complete, the Project name window appears. 5-17

100 8 Enter a project name, and click the OK button. The final spreadsheet appears:. Tutorials 9 Create a calibration curve. a In the FluorEssence TM toolbar, click the Create/Use Calibration Curve from CWA Data button. The Calibration Curve Parameters window appears. b Choose the Linear radio button for these samples, then click the OK button. The Save Curve window appears. c If you wish to save the calibration curve, click the Yes button. The Enter a FileName window appears. 5-18

101 Tutorials d Enter the desired filename for the calibration curve, and click the Save button. e The calibration curve appears. The correlation coefficent R 2 appears in this box. The standards appear as stars. Note: The unknowns do NOT appear on the graph, but are in the spreadsheet. The equation for the best fit appears here. Note: If you repeat the same experiment, the software will overwrite this graph. Save or rename the graph to keep it. 5-19