PerkinElmer LS 55 User Manual Download Page 170

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170 .

LS 55 User’s Guide

Signal Handling Diagram

Gating

Reference

Integrator

Multiplexer

Gating

Sample

Integrator

Autoranging

Amplifier

Comparator

Controller

for 12-bit

Successive

Approximation

A-D Converter

12-bit

DAC

Dark Current

Subtraction

Excitation

Correction

Block

Averaging

Ratioing

Smoothing

Filter

Scaling

CPU

System

Analogue

Outputs

10 V
1 V
10 mV

Sample and

Hold

PMT Voltage Control

Thermostat
Control

Stepper

Motor

Control

Reference

Sample

Photomultiplier

Excitation Monochromator
Emission Monochromator
Filter Wheel

Polarizers

Zero Order
Mirror

Other
Accessories

Photodiode

Figure 114 The signal handling diagram

Summary of Contents for LS 55

Page 1: ...LS 55 User s Guide ...

Page 2: ...mer shall not be liable for errors contained herein for incidental consequential damages in connection with furnishing performance or use of this material Copyright Information This document contains proprietary information that is protected by copyright All rights are reserved No part of this publication may be reproduced in any form whatsoever or translated into any language without the prior wr...

Page 3: ...Connection to electrical mains supply 29 Accessory connectors 29 Rear panel connectors 30 Switching on the LS 55 34 Accessories Installation and Use 37 Single Position Cellholder standard 38 Description 38 Installation and maintenance 38 Stirred Single Position Cellholder 39 Description 39 Installation and maintenance 39 Operation 39 Stirred Four Position Cellchanger 40 Description 40 Installation...

Page 4: ...63 Description 63 Installation 63 Operation 65 Front Surface Accessory 66 Description 66 Installation and maintenance 67 Operation 70 Using Powders 71 Plate Reader 73 Description 73 Installation 73 Operation 80 The Well Plate Reader application 80 The TLC Scan application 81 Measuring TLC plates gels or other flat samples 82 Red sensitive Detector 83 Description 83 Installation 84 Total Emission A...

Page 5: ...r Accessory FFA 129 Section 2 Command Formats 131 Monochromator Scan Control Commands 133 Time Drive Commands 136 TLC Scan Commands 139 Data Collection Processing Set Up Commands 142 Data Output Control Commands 147 Motor Control 148 Calibration Commands 151 Miscellaneous Commands 152 Plug in Accessory Control Commands 155 Well Plate Reader WPR Commands 157 Fast Kinetics Application FKA Commands 1...

Page 6: ... 55 for transport 188 Inserting the clamps 188 Lifting the LS 55 Luminescence Spectrometer 191 Removing the clamps 191 Cleaning the LS 55 192 Cleaning the air filter 193 Changing the Instrument Operating Voltage 194 Changing the mains fuse 196 The mains voltage plate 197 Index 199 Index 200 ...

Page 7: ...Warnings and Safety Information ...

Page 8: ...ty 75 maximum non condensing Environmental conditions for safe operation The LS 55 has been designed to be safe under the following conditions Indoor use Altitude up to 2000 m Temperature range 5 C to 40 C Maximum relative humidity 80 for temperatures up to 31 C decreasing linearly to 50 relative humidity at 40 C Mains voltage fluctuations not to exceed 10 of the nominal voltage An IEC Installatio...

Page 9: ...fore it is opened for any adjustment replacement maintenance or repair Capacitors inside the instrument may still be charged even if the instrument has been disconnected from all voltage sources Only fuses with the required current and voltage rating and of the specified type are to be used for replacement Makeshift fuses must NOT be used and fuse holders must not be short circuited Whenever it is...

Page 10: ...the beam in the sample compartment Near the mains inlet fuse where the symbol denotes that the operator replaceable fuse see Fuses on page 171 is not an IEC fuse but a CSA approved fuse By the left hand screw that fixes the top cover at the front left of the instrument where the symbol denotes that removing this cover may expose hazardous voltages Any adjustment maintenance or repair of the opened...

Page 11: ...e labels mean that the instrument must be switched off and the mains supply lead removed from its socket before the cover is removed Any adjustment maintenance or repair of the opened operating instrument must be performed by a skilled person who is aware of the hazards involved a RESPONSIBLE BODY Further assistance For technical assistance please contact your local PerkinElmer office or agent or ...

Page 12: ...12 LS 55 User s Guide ...

Page 13: ...Introduction ...

Page 14: ...d to provide information and instructions Bold text refers to text that is displayed on the PC screen UPPERCASE text for example ENTER or ALT refers to keys on the PC keyboard is used to show that you have to press two keys at the same time for example ALT F All eight character numbers for example L2250140 are PerkinElmer part numbers unless stated otherwise Notes warnings and cautions Three terms...

Page 15: ...ecuadros como éste se proporciona información sobre este tipo de circunstancias F Warning Danger Nous utilisons la formule WARNING DANGER pour avertir des situations pouvant occasionner des dommages corporels à l utilisateur ou à d autres personnes Les détails sur ces circonstances sont données dans un encadré semblable à celui ci I Warning Pericolo Con il termine WARNING PERICOLO vengono segnalat...

Page 16: ...recuadros éste se proporciona información sobre este tipo de circunstancias F Caution Attention Nous utilisons le terme CAUTION ATTENTION pour signaler les situations susceptibles de provoquer de graves détériorations de l instrument ou d autre matériel Les détails sur ces circonstances figurent dans un encadré semblable à celui ci I Caution Attenzione Con il termine CAUTION ATTENZIONE vengono seg...

Page 17: ...tions OPERATOR Person operating the equipment for its intended purpose RESPONSIBLE BODY Individual or group responsible for the use and maintenance of the equipment and for ensuring that the OPERATORS are adequately trained ...

Page 18: ...table 200 900 nm with optional R928 photomultiplier Synchronous scanning is available with constant wavelength or constant energy difference Wavelength accuracy 1 0 nm Wavelength reproducibility 0 5 nm Spectral bandpass The excitation slits 2 5 15 0 nm and emission slits 2 5 20 0 nm can be varied and selected in 0 1 nm increments Phosphorescence mode Delay and gate times can be varied to measure p...

Page 19: ...Introduction 19 Supply Voltages 100 V 120 V 220 V 230 V Frequency 50 60 Hz Power 150 VA ...

Page 20: ...20 LS 55 User s Guide ...

Page 21: ...Unpacking and Installation ...

Page 22: ... The compartment can be opened by using the finger grip on top of the compartment as shown in Figure 3 to pull the cover down Figure 3 The LS 55 The sample cells are located in the sample compartment a range of sampling accessories can be installed in this area Also within the sampling area are two sockets used for controlling accessories that are electrically operated Figure 4 Inside the sample c...

Page 23: ...ctrical mains socket is located on the lower right hand side viewed from the back of the rear panel On the left hand side of the rear panel are an RS232C socket and a terminal block Figure 5 The rear of the LS 55 CAUTION Do not obstruct the air filter and cooling vents on the rear of the instrument ...

Page 24: ... Blo OC962348 Fuse 250 V 1 AT 04969185 Fuse 250 V T100 mA Slo Blo 04970839 Fuse 250 V 2 A Slo Blo 04967940 Fuse 250 V 500 mA Slo Blo 04960999 Hex key 5 64 AF for lamp change 04961071 Spare septa x3 for septum injector 04967778 Screwdriver 3 16 inch Blade 04969221 Screwdriver No 2 Posidriv L2251286 L2251287 Septum injector assembly L2251188 Mains Label 100 V 220 V ...

Page 25: ... codes of practice issued by safety advisors before attempting to lift the spectrometer As the LS 55 Luminescence Spectrometer weighs approximately 49 kg approximately 59 kg with packaging we recommend that the spectrometer is lifted by 2 adults and that it is lifted by the base of the instrument ...

Page 26: ...sed resulting in a wavelength failure of 14 nm This will have to be recalibrated by a service engineer and is not covered by warranty To remove the shipping clamps proceed as follows 1 Position the LS 55 on the front of the bench so that it overhangs the bench by approximately 8 cm 2 Undo the two captive screws positioned in recesses underneath the front of the instrument There is one on the left ...

Page 27: ...re 7 Inside the LS 55 4 Remove the two screws securing one of the clamp plates as shown in Figure 8 Figure 8 Shipping clamp screws 5 Lift out the plate 6 Remove the hexagonal bolt as shown in Figure 9 Figure 9 Removing the shipping bolt ...

Page 28: ... Figure 10 Figure 10 Replacing the plate 8 Repeat steps 4 to 7 for the second clamp 9 Store the two hexagonal clamps in their fixing points on the left side of the instrument as shown in Figure 11 Figure 11 Storing the bolts 10 Lower the instrument cover close the sample compartment and secure the cover by retightening the two screws loosened in step 2 11 Slide the instrument back on the bench so ...

Page 29: ...r to Changing the Instrument Operating Voltage on page 194 Figure 12 Mains connection and voltage plate Accessory connectors Inside the sample area door are two electrical control sockets for accessories as shown in Figure 13 Note that the left hand socket has 12 pins and the right hand socket has 19 pins so it is not possible to incorrectly connect accessory control plugs Connecting specific acce...

Page 30: ...de Rear panel connectors On the left hand side of the rear panel viewed from the back is a socket labeled RS232C Controller and an eight tag terminal block as shown in Figure 14 Figure 14 Rear panel electrical connections ...

Page 31: ...ction in the terminal block 1 Insert a small screwdriver into the bottom of the terminal block and push upwards 2 Feed the connecting wire into the hole at the top of the terminal block 3 Remove the screwdriver The wire is now firmly clamped Analog signal outputs External signal integration devices chart recorders etc can be connected to the analog outputs One of the connecting wires in an unbalan...

Page 32: ...ther start signal until the Scan Busy line shows that data collection is complete Since this signal is a logic level devices requiring a contact closure can be connected via a small solid state relay with a switching diode for anti bounce Optimal connection to external autosamplers Some commercially available autosamplers can be programmed to send and receive contact closure signals Connecting suc...

Page 33: ...ode provides the autosampler with status information from the LS 55 Such a system if programmed properly in the autosampler is considered closed since the LS 55 will wait for the autosampler signal before performing a measurement then the autosampler will wait until the LS 55 has finished being busy before taking the next sample ...

Page 34: ...e and should be switched on before the PC The procedure for switching on is as follows 1 Before switching on any of the units ensure that the PC is connected to the LS 55 via the RS232C cable and that both the LS 55 and PC are connected to the electrical mains supply As a precautionary measure a mains surge filter can be used to protect the PC the LS 55 is very robust and not damaged by typical ma...

Page 35: ...ded start the FL WinLab software NOTE If the instrument has not yet finished initializing this will be shown on the bottom right side of FL WinLab Application dialogs This will automatically reset to Online when the system is ready At this point data collection can proceed ...

Page 36: ...36 LS 55 User s Guide ...

Page 37: ...Accessories Installation and Use ...

Page 38: ...ed using circulated water as detailed in Installing Thermostatting Tubing to Cellholders on page 45 Installation and maintenance This cellholder needs no alignment or setting up Maintenance consists of routine cleaning of the optical mirrors built into the cellholder block see Care of Optical Mirrors Inside Cellholder Accessories on page 55 and cleaning the base and pressure bar if spillages occur...

Page 39: ...urate temperature work Without stirring temperature gradients of 1 C can build up within the cuvette Inert polymer coated bars are delivered with the accessory see Using Stirrer Bars on page 50 Installation and maintenance This cellholder needs no alignment or setting up Maintenance consists of routine cleaning of the optical mirrors built into the cellholder block see Care of Optical Mirrors Insi...

Page 40: ... Stirring is also essential for accurate temperature work Without stirring temperature gradients of 1 C can build up within the cuvette Inert polymer coated bars are delivered with the accessory see Using Stirrer Bars on page 50 Each position has its own stirrer so each cuvette can be stirred continuously Installation and maintenance This cellholder needs no alignment or setting up Maintenance con...

Page 41: ...re that cells proteins etc remain in suspension Stirring is also essential for accurate temperature work Without stirring temperature gradients of 1 C can build up within the cuvette Inert polymer coated bars are delivered with the accessory see Using Stirrer Bars on page 50 Installation and maintenance This cellholder needs no alignment or setting up Maintenance consists of routine cleaning of th...

Page 42: ...data collection in the result dataset header The Read application collects intensity polarization or anisotropy data and saves this as a spreadsheet If the Biokinetics accessory is fitted then the temperature is saved simultaneously Another important feature is the event marker button on the front plate of the accessory This is intended for identification of times when reagents are added to the cu...

Page 43: ...oughput requires high reliability due to the unattended nature of data collection Use the PerkinElmer Flange toolkit Part number B3000001 The internal volume of the flowcell the internal chamber has dimensions 3x3x10 mm a volume of 90 μL is almost perfectly matched to the light beam geometry of the LS 55 the measured volume is 3x3x9 mm a volume of 81μL This means that this flowcell does not suffer...

Page 44: ...d in the analysis If the system is to be left overnight or longer without being used then the system should ideally be cleaned with de ionized water then with ethanol then pumped to dryness Operation The inlet and outlet tubing should be installed with screw fittings using the PerkinElmer Flange tool B3000001 This enables you to fit a wide range of screw fittings to various diameter tubing Ideally...

Page 45: ...it with a suitable sealant with higher temperature specification At temperatures below 15 C condensation may occur on the surfaces of the cuvette If this is the case either purge the sample area with dry air or lead dry air onto the cuvette surfaces using tubing To fit the thermostatting tubing 1 Route the tubing into the sample area and onto the two tubing ports under the sample area door as show...

Page 46: ...6 LS 55 User s Guide Figure 24 Routing the tubing 3 Attach the tubing to the cellholder nozzles and secure with cable ties or clamps as shown in Figure 25 Figure 25 Attaching the tubing to the cellholder ...

Page 47: ...ally 2 Turn the cellchanger to appear as shown in Figure 26 Figure 26 Positioning the cellchanger 3 Install the tubing This will ensure that the tubing will only turn through a maximum of 135 degrees during operation CAUTION If the tubing is installed with the cellchanger at position 1 the tubing turns through 270 degrees putting greater strain on the stepper motor leading to potential step failur...

Page 48: ... injector accessory This locates on top of the sample area door and guides a syringe needle into the cuvette To install the septum injector 1 Place a septum into the injector barrel L2251286 and push the cap L2251287 into the barrel trapping the septum as shown in Figure 28 Figure 28 Assembling the septum injector ...

Page 49: ...re 29 The blanking plug 4 Insert the septum injector NOTE The distance between the top of the cuvette and the bottom of the injector barrel avoids the thermostatting tubing fouling the injector when the door is closed For this reason a short syringe needle will not reach the cuvette and there is no guarantee that injected reagents will enter the cuvette A minimum syringe needle length of 70 mm is ...

Page 50: ...ross or vane built into the top This type of stirrer is more stable and can rotate on an uneven surface PerkinElmer cuvette Polymer cuvette Special stirrer Figure 30 Stirrers in cuvettes The stirrer is controlled using the FL WinLab LS 55 Status application by clicking the accessory icon Three speeds can be set Off no stirring Low intended for keeping sensitive cells in suspension without physical...

Page 51: ... The excitation light beam is 9 mm high in the center of the cuvette The center of the excitation beam is 13 5 mm from the base of the cuvette Figure 31 Position of the beam through a cuvette In a 1 cm2 cuvette for example the liquid has to fill the cuvette to a level of 2 cm 2 ml of liquid in order to avoid light scattering by the meniscus Some users place a spacer block up to 8 mm thick at this ...

Page 52: ...ompromised since the LS 55 has a measured volume of 3 x 3 x 9 mm which is smaller than the semi micro cuvette s internal 5 mm pathlength Installation The holder is simply inserted into a standard cellholder as shown in Figure 33 NOTE The lip at the top for removal of the holder must be aligned away from you so that the lip does not contact the thermostatting nozzles which would stop the holder fro...

Page 53: ...hlength do not function efficiently Thermostatting is difficult due to the use of a semi micro cuvette adaptor which fits into the standard cuvette holder and accepts the smaller cuvette These adaptors are normally manufactured from polymer material which is an effective thermal insulator The stirred semi micro cuvette B0631132 overcomes both of these problems The outside dimensions are the same a...

Page 54: ...ser s Guide Figure 35 Cross section of the stirred semi micro cuvette Operation The stirrer is controlled using the LS 55 Status application in the FL WinLab software as discussed in Using Stirrer Bars on page 50 ...

Page 55: ...ed away from the instrument and reflects it back into the instrument Both mirrors together provide a 2 5 fold increase in signal so in the worst case if both mirrors are totally corroded they will not reflect light and the signal will decrease by a factor of 2 5 fold Additionally if fluorescent material is allowed to build up on the mirrors then background levels will increase significantly To pre...

Page 56: ... perchloric acid The pressure bar which presses the cuvette back into the mirrored corner of the cuvette is also manufactured from Delrin To prevent damage spillages should be washed with non corrosive solvent or methanol If the base becomes attacked the consequences are not severe unless it is totally dissolved in which case the cuvette can fall through the base of the holder onto the metal basep...

Page 57: ...r The Sipper consists of a flow cell coupled to a peristaltic pump When used without an autosampler sampling is initiated by putting the sample container under the tube lifting the container so that the tube goes into the sample and then raising it further until a microswitch is activated which starts the pump as shown in Figure 37 The sample is then pumped into the flowcell and measured Figure 37...

Page 58: ...der where sensitivity would be at least a factor of 20 less sensitive even for dedicated plate reader instruments Installation and maintenance The Sipper accessory must be aligned before use to ensure that the center of the flowcell sits correctly at the optical focus of the LS 55 Installation is carried out as follows 1 If the tubing is clamped in the switch lever loosen the tubing clamp screw as...

Page 59: ...at the peristaltic pump tubing is correctly held in the pump cam roller assembly 7 Turn the pressure block pin carrier until the pressure pin latches into its bracket as shown in Figure 40 Figure 40 Pressure pin and adjuster screw 8 Tighten the adjuster screw as shown in Figure 40 until sufficient pressure is applied to the pump tubing to enable the sipper to pump efficiently NOTE When the sipper ...

Page 60: ...hown in Figure 39 13 Re fit the front plate as shown in Figure 38 14 Re insert and re clamp the tubing using the tubing clamp screw as shown in Figure 38 Operation The Sipper accessory can be controlled manually using the LS 55 Status application The Scan and Concentration applications include full sipper control The Sipper parameters pump time wait time purge time and direction are set in the app...

Page 61: ... 2 Click the Sipper icon in the LS 55 Status application 3 Select whether to Pump Forwards or to Pump Reverse enter the required Pump Time in seconds and click Sip Scanning spectra 1 In the Scan application select the kinetic scan option ...

Page 62: ...which can be viewed by selecting 3D Viewer on the Data Handling menu in FL WinLab Routine quantitation with the Concentration application 1 In the Concentration application select Setup parameters and set up the sipper panel as required The example here will sample for 6 seconds wait 1 second for de bubbling measure the intensity then return the sample for 9 seconds 3 Select User info and set up t...

Page 63: ...material FEP which ensures biocompatibility NOTE FEP tubing has low structural strength and thus poor resistance to back pressure so care should be taken when placing the LS 55 with LC Flowcell into a series of detectors The LS 55 should be located at the end of the chain to reduce back pressure Installation The LC Flowcell accessory must be aligned before use to ensure that the center of the flow...

Page 64: ...ick release fasteners 4 Connect the syringe supplied to the inlet tube 5 Pump a fluorescent sample into the flowcell until the liquid emerges from the outlet tube checking by eye that there are no air bubbles trapped in the LC Flowcell 6 Start a TimeDrive using suitable wavelengths for the fluorescent dye used using slit widths of 10 nm excitation and 5 nm emission Duration should be 2 3 minutes 7...

Page 65: ...let and outlet tubes through the grommets on the front cover plate then re secure the front cover plate using the two screws as shown in Figure 43 Operation Data collection for the LC Flowcell is made using the TimeDrive application Data collection can be synchronized with a signal from an HPLC pump using contacts on the LS 55 s rear panel see Remote starts and event marking on page 31 ...

Page 66: ... etc It is also used in the life sciences for turbid samples where the sample cannot be diluted but gives severe quantitative problems due to light scattering The Front Surface accessory must be aligned to ensure correct sensitivity Failure to do this could lead to a large loss of sensitivity Furthermore the process of alignment optimizes sensitivity to luminescence signals and minimizes the effec...

Page 67: ...1 Remove the two screws that hold the fascia and front panel as shown in Figure 47 Figure 47 Front bolts 2 Refit the front plate without the fascia as shown in Figure 48 Figure 48 Front plate without fascia 3 Loosen the two upper clamping screws as shown in Figure 49 ...

Page 68: ...teners 6 Mount a flat fluorescent sample in the Front Surface accessory see Operation on page 70 A business card is useful for this purpose as most are highly fluorescent and the card is strong enough to remain flat during clamping 7 Start a TimeDrive from the FL WinLab software using suitable parameters For a business card these would be Excitation wavelength 300 nm Emission wavelength 550 nm Sli...

Page 69: ...screws NOTE If the signal exceeds 999 999 then the run will have to be stopped and more attenuation or narrower emission slit width used 10 When the maximal signal has been reached stop the run and remove the accessory from the LS 55 11 Tighten the two upper locking screws as shown in Figure 49 and the three locking screws underneath the accessory as shown in Figure 50 The alignment will now remai...

Page 70: ...low insertion of the sample Figure 52 The plunger Coarse movement of the plunger plate is achieved by moving the plunger base itself This moves linearly and is locked by a large vertical screw 1 Unlock the vertical screw as shown in Figure 52 and pull the plunger plate back 2 Insert the sample so that it rests against the measurement baffle 3 Push the plunger plate forwards so that some pressure i...

Page 71: ...a visible wavelength such as 550 nm and observe the relative locations of the light area and the sample For the measurement of oils or very viscous samples the sample can be sandwiched between two silica windows 1 Unscrew the base plate from the Powder holder and add the sample onto the silica window as shown in Figure 53 Figure 53 The powder accessory 2 Replace the cap and screw it home until the...

Page 72: ...72 LS 55 User s Guide Figure 54 Using the powder accessory ...

Page 73: ...cs Microplate measurement results in the creation of a data spreadsheet Flat sample measurement creates a 3D datafile that can be viewed using the 3D Viewer included with FL WinLab Installation Before use the Plate Reader must be correctly installed This involves optical alignment of the Plate Reader accessory and the creation of a Plate Format file so that the FL WinLab software can measure in th...

Page 74: ...n Figure 56 Figure 56 HT switch for the photomultiplier 3 Open the sample area accessory cover and remove the existing sampling accessory by pressing on the 2 quick release fasteners 4 Undo the three screws which attach the sample accessory bracket to the LS 55 as shown in Figure 57 ...

Page 75: ...t screws 5 Unscrew the two fixing screws and remove the optical assembly as shown in Figure 58 This is so that the sharp square edges at the front of the optical assembly cannot accidentally break the LS 55 s optical windows during fitting Figure 58 Optical assembly screws ...

Page 76: ...wels as shown in Figure 59 Figure 59 Locating dowels 7 Tighten the three screws that secure the Plate Reader to the LS 55 as shown in Figure 60 Figure 60 Plate Reader screws 8 Attach the optical assembly to the Plate Reader and tighten the two screws which secure it as shown in Figure 61 ...

Page 77: ...hite light to assist in alignment 10 Insert the dentist s mirror between the LS 55 front plate and the optical assembly The idea is to observe the excitation fibre to ensure that the excitation light falls directly on the fibre bundle optimising light throughput 11 Loosen the excitation fibre locking screw as shown in Figure 62 Figure 62 Excitation fibre screws ...

Page 78: ... 15 Remove the main optical cover as detailed in Removing the Main Optical Cover on page 176 16 Switch off the room lighting This enables you to see the emission entrance slit easier 17 Set the excitation wavelength to zero order This produces white light 18 Set the emission slit width to 10 nm 19 Place a plane mirror beneath the fibre optic sampling end This reflects the white light back into the...

Page 79: ...n of the emission entrance slit 21 Loosen the emission fibre locking screw as shown in Figure 65 Figure 65 Emission fibre screws 22 Turn the adjustment screw until the white light is centralized on the emission slit inside the LS 55 as shown in Figure 66 ...

Page 80: ...t surface generating a 3D plot of intensity vs Distance mm vs Distance mm This is done using the FL WinLab TLC Scan application By manually driving the fibre optic to a discrete position followed by running any FL WinLab application for example Scan This is done within the FL WinLab Well Plate Reader or TLC Scan applications depending on the type of sample The Well Plate Reader application The Wel...

Page 81: ...e Reader application where the accessory is sent to one of a series of discrete positions the TLC Scan application enables you to send the Plate Reader to any position This is done from the Setup parameters tab by moving the mouse over the sample area image until the desired position shown as X and Y in millimeters on the top left of the tab and clicking with the right mouse button on the sample a...

Page 82: ...ue consistency as shown in Figure 69 Figure 69 Flat sample holder This is inserted in place of the microplate in the Plate Reader the clips are positioned so that they hold the sample securely but do not restrict the movement of the plate reader during measurement NOTE Position the clips so that the fibre optic probe does not come into contact with the clips during movement or a stepper motor erro...

Page 83: ... Red sensitive detector Description The red sensitive photomultiplier 52124966 is fitted for the collection of emission data above 630 nm above which point the standard photomultiplier has little or no sensitivity as shown in Figure 71 Figure 71 Photomultiplier sensitivity ...

Page 84: ... must be performed by a PerkinElmer Service Engineer NOTE In order to obtain corrected emission spectra the correction factor spectrum will have to be collected specifically for the red sensitive photomultiplier Data obtained using the standard photomultiplier is not applicable ...

Page 85: ...he zero order position where the grating transmits white light gives only small increases in sensitivity The Total Emission accessory L2250101 is a mirror which replaces the emission grating in the beam giving a 10 to 15 fold increase in sensitivity Installation The accessory must be installed in the factory or by a service engineer since alignment is required CAUTION Incorrect installation of the...

Page 86: ...g The emission grating is then driven out of the beam and is replaced by the mirror Select an emission filter to avoid excitation light being scattered directly onto the sample photomultiplier If this not done a warning will be displayed NOTE The use of the Total Emission accessory is strongly recommended for bio and chemi luminescence measurements ...

Page 87: ...n be used for remote sampling of luminescent samples The transmission characteristics of silica allow excitation down to approximately 260 nm At the sampling end the two fibre bundles are hemispherical as shown in Figure 74 Figure 74 The end of the fibre optic accessory The numerical aperture around 0 2 of the material produces an emergence acceptance angle of between 22 25 degrees as shown in Fig...

Page 88: ...nto the emission monochromator CAUTION Take care not to bend the fibre to a radius of less than 10 cm as this may cause damage to the fibres 1 Remove the top cover as described in Removing the Main Cover on page 173 2 Turn off the voltage to the photomultiplier as shown in Figure 76 Figure 76 Photomultiplier switch 3 Loosen the two knurled screws underneath the accessory as shown in Figure 77 ...

Page 89: ...lder grub screw 6 Using the FL WinLab LS 55 Status application set the excitation wavelength to 0 white light to give good visibility during alignment 7 Position the right side holder so that white light enters the fibre and emerges from the two free ends of the fibre optic 8 Tighten the knurled locking screw underneath the accessory so the right hand fibre holder is locked in place 9 Insert one o...

Page 90: ... vertical and tighten the grub screw as shown in Figure 80 Figure 80 Left holder grub screw 11 Open the central optical cover inside the LS 55 and observe the emission entrance slit through the open position of the emission filter wheel as shown in Figure 81 Figure 81 Viewing the emission slit ...

Page 91: ... the knurled screw under the left fibre holder to lock it 14 Remove the sampling fibre end from the right fibre holder 15 Loosen the grub screw in the right fibre holder and insert the free fibre end as shown in Figure 83 Figure 83 Inserting the fibre end 16 Rotate the fibre until it is vertical then tighten the grub screw Figure 84 Figure 84 Right holder grub screw ...

Page 92: ...voltage 22 Close the main cover Operation The 22o angle of emergence of light from the sampling end means that the further the sampling end is from the sample the larger the excitation image will be as shown in Figure 85 Figure 85 Sampling positions However if the sampling end contacts the sample very little light will be accepted by the emission fibre Optimal sensitivity is achieved with a 1 2 mm...

Page 93: ...Accessories Installation and Use 93 Figure 86 Masking the outer areas ...

Page 94: ...robes that bind to intracellular ions enables cell biologists to study the role of these ions as cellular messengers Changes in the fluorescent properties of these probes when they chelate to intracellular ions reflect increasing or decreasing concentrations of free ions inside the cell The spectral properties of some common probes are shown in the table ...

Page 95: ... 355 nm 405 nm 480 nm Mag FURA 2 Mg 340 nm 380 nm 510 nm 510 nm Mag INDO 1 Mg 355 nm 355 nm 405 nm 480 nm BCECF pH 495 nm 440 nm 530 nm 530 nm SNARF 1 pH 514 nm 514 nm 580 nm 640 nm An example of the spectral dependence on ion binding of a fluorescent probe is given by the binding of calcium ions to FURA 2 as shown in Figure 88 Figure 88 Binding of calcium ions to FURA 2 ...

Page 96: ...lter operation and standard polarizers the accessories must be exchanged It is this process that is described here There are two parts to the installation of the Fast Filter accessory these are the physical installation and the software installation Installing filters into the filter wheels 1 Lay the Fast Filter motor unit carefully onto a flat surface 2 Undo the central screw as shown in Figure 8...

Page 97: ...ing of the filters is predefined For polarizer filters the vertical filter MUST be located in position 1 or 2 For ratio filters the top part of the ratio for example the 340 nm filter for FURA 2 MUST be located in position 1 or 2 The positions for the excitation wheel are shown in Figure 91 ...

Page 98: ...xcitation wheel The positions for the emission wheel are shown in Figure 92 Figure 92 Positions for the emission wheel Installing the excitation Fast Filter into the LS 55 1 Remove the LS 55 main cover as described in Removing the Main Cover on page 173 ...

Page 99: ...ical Cover on page 176 3 Unplug the excitation filter wheel cable as shown in Figure 93 Figure 93 Unplugging the cable 4 Undo the two screws and remove the excitation filter wheel as shown in Figure 94 NOTE Take care not to touch any of the optical surfaces or mirrors Figure 94 Excitation filter wheel screws ...

Page 100: ...th the hole in the plinth 7 Secure the plinth using the two long hex screws 8 Fit the Fast Filter wheel to the plinth first aligning the pin on the plinth with the hole on the Fast Filter wheel motor assembly 9 Secure using the 2 long screws fixed to the motor assembly as shown in Figure 95 Figure 95 Fixing the filter wheel to the plinth 10 Attach the ribbon cable to the Fast Filter motor assembly...

Page 101: ... LS 55 The procedure for the emission Fast Filter is the same as the excitation Fast Filter with the final attaching screws shown in Figure 96 Figure 96 Installing the emission Fast Filter Cabling and bitswitches Cables should be arranged as shown in Figure 97 Figure 97 The cabling ...

Page 102: ...ved the other switches are shown here for example purposes but must not be moved from their factory settings Both Emission only Excitation only Software installation After physical installation of the Fast Filter wheels the FL WinLab software must be configured so that it knows where the filters are 1 Choose Configuration from the Utilities menu The Configuration dialog is dsiplayed 2 Ensure that ...

Page 103: ...rizers the entries would be made similar to those shown above note that the vertical polariser must be in position 1 or 2 while for ratio measurement filters the entries would be similar to those shown below note here that the top numerator part of the filter pair must be in position 1 or 2 as shown 5 When the positions of the filters have been entered click OK The settings will then be saved ...

Page 104: ... application interrogates the filter position information entered via the LS 55 Status application If polarizers are present then options for time dependent collection of polarization and anisotropy will be offered If the filters are standard band pass type then ratiometric options are offered A description of the Fast Filter application is given in the FL WinLab Software manual ...

Page 105: ... applications include the investigation of membrane structure protein structure and function and polarization immunoassays NOTE The use of plastic cuvettes will give severe corruption of polarization and anisotropy measurements since the plastic material strongly polarizes light Glass or better synthetic fused silica cuvettes should be used When using the polarizer the temperature of the sample sh...

Page 106: ... main cover as described in Removing the Main Cover on page 173 2 Remove the central optical cover as described in Removing the Main Optical Cover on page 176 3 Unplug the excitation filter wheel cable as shown in Figure 99 Figure 99 Excitation filter wheel cable 4 Undo the two screws and remove the excitation filter wheel as shown in Figure 100 NOTE Take care not to touch any of the optical surfa...

Page 107: ...xcitation polarizer wheel into the excitation filter wheel location by first locating the hole on the bottom of the filter bracket with the pin in the baseplate then by tightening the two fixing screws as shown in Figure 101 Figure 101 Fixing the excitation polarizer wheel in position ...

Page 108: ...tial location of the wheel as shown in Figure 103 Figure 103 Fixing the emission polarizer wheel in position 8 Before tightening the fixing screws fully check that the emission filter wheel and emission polarizer wheel are correctly set and do not touch one another during rotation as shown in Figure 104 Do this by manually rotating the two wheels in opposite directions If the wheels touch gently t...

Page 109: ...se 109 Figure 104 Checking the clearance 9 Tighten the polariser wheel fixing screws 10 Attach the emission filter wheel control cable as shown in Figure 105 Figure 105 Attaching the emission control cable 11 Refit the LS 55 covers ...

Page 110: ...formed using the FL WinLab software where movement of the polarizers is done completely automatically 1 In the FL WinLab software select Read from the Applications menu 2 Choose polarization or anisotropy The polarisation anisotropy value will be saved together with instrument parameters in the file specified in the Destination filename textbox The format of the data file is as follows Mode Polari...

Page 111: ... be taken not to touch the metal surfaces of the accessory with unprotected hands Description The Low Temperature Accessory can be used for measurement of fluorescence and phosphorescence from liquid samples at liquid nitrogen temperature 77 K The sample is located in a silica capillary tube of 4 mm outer diameter 2 mm inner diameter and 75 mm length This tube is held in contact with the top of a ...

Page 112: ... for improved sample throughput Fewer reflective and absorptive surfaces are located within the optical path Installation Before installing the accessory any filters or attenuators that need to be inserted into the sample area brackets should be installed Figure 108 shows the parts of the Low Temperature Accessory Figure 108 Cross section through the Low Temperature Accessory Liquid nitrogen Nitro...

Page 113: ...ple accessory 3 Remove the sample area cover and accessory bracket by undoing the three fixing screws as shown in Figure 109 Figure 109 Accessory bracket screws 4 Place the accessory on a stable surface and remove the shipping tape 5 Lift the cover vertically from the accessory The black foam block around the sample tube holder will now be visible Screws ...

Page 114: ...ment Alignment Before samples can be measured with the Low Temperature Accessory you must align the sample tube position to centralize it in the focus point where the excitation and emission beams cross This must be performed at room temperature 1 Remove the two screws that secure the black foam block and remove the foam block and the black metal securing plate 2 Loosen but do not remove the four ...

Page 115: ...o look down into the sample tube The fluorescence emission is in the visible region allowing you to to see when the sample is being excited optimally NOTE There are two positions that produce high signals The first is that which correctly locates the center of the sample tube in the crossover point of the excitation and emission light beams This is the desired position The second position is that ...

Page 116: ...removed previously 12 Replace the top cover of the accessory Purging the instrument Before measurement at liquid nitrogen temperatures can be performed the instrument must be purged with dry gas for example helium or nitrogen This gas must be free of carbon dioxide and moisture The purpose of purging is to avoid the build up of ice on optical surfaces inside the instrument in the sample area and o...

Page 117: ...ecure the tube turn the tube if necessary to overcome this resistance 2 Remove the funnel cover and carefully pour liquid nitrogen into the funnel This will require approximately 3 L of liquid nitrogen Pour slowly until the tank overflows When the copper finger has almost reached temperature the liquid nitrogen will boil for several seconds after this the system settles down 3 When the boiling has...

Page 118: ...ab TimeDrive application The sample should require about 1 minute to reach 77 K 6 Measure the sample fluorescence or phosphorescence as desired If the measurement takes more than ten minutes the sample tube should be rotated through 360 degrees to avoid liquid carbon dioxide building up in the sample holder area NOTE A potential problem is snowing of the solvent In this case the solvent does not f...

Page 119: ...der to avoid this if necessary The empty sample position can accumulate CO2 ice making it impossible to insert a sample This could require allowing the accessory to return to room temperature and repeating the cooling down process Removing the accessory WARNING Always allow the accessory to return to room temperature before removal and handling 1 Turn off the purge supply to conserve gas 2 Remove ...

Page 120: ...t Surface Powder holder complete with silica window 52123164 Front Surface Silica window for powder holder 52123814 Septum injector Silicone rubber septa pack Any cuvette holder Semi micro cuvette adapter 1cm Square top cuvettes Coverslip accessory Pulsed xenon source L2251157 Red sensitive photomultiplier 52124966 Festoon filament lamp 04969486 Plate reader Hi white 96 well microplates pack of 10...

Page 121: ...RS232 Commands ...

Page 122: ...ed Remote Integrate Both monochromators may be driven to any valid wavelength 200 800 nm for excitation and 200 900 nm for emission They may be scanned either individually or synchronously When synchronously scanning either a constant wavelength separation or a constant frequency separation can be selected All these operations can be aborted The source is automatically turned off 30 minutes after ...

Page 123: ...t of other instrument conditions The excitation filter is selected automatically according to the current excitation monochromator position unless a polarizer or blank position has been selected A manual override to the automatic excitation filter selection is provided A separate emission polarizer wheel accessory is provided If fitted a total emission accessory emission mirror can be driven in or...

Page 124: ...ies being plugged in or removed For more complex plug in accessories for example the autosampling accessory some initialization in addition to plugging in may be required before the software can control the device correctly The software will respond to control commands for an incorrectly initialized accessory as if the accessory was not fitted ...

Page 125: ...free running or prompted modes In free running mode output can be stopped restarted by conventional DC1 DC3 protocol DC3 pauses transmission at the end of the current record being transmitted The interface defaults to prompted mode on power up The 25 way connector on the instrument is configured as DCE pin 1 chassis ground pin 6 DSR pin 2 data from instrument pin 7 signal ground pin 3 data to inst...

Page 126: ...record consisting of a 4 digit error code On some commands other output records may follow The last transmission in response to a command is an error code output record Any output records that do not contain only an error code have a DEL character in the first position In some cases the user may start an operation which requires some interaction with the user for example calibration procedures In ...

Page 127: ...n an abort request In GOTO mode either monochromator is driven as fast as possible in the specified direction Destination wavelengths must either be 0 nm or within the scan limits above Unless an excitation polariser or blank position has been selected or a fast filter excitation unit is fitted the excitation filter is selected according to the excitation monochromator destination For excitation w...

Page 128: ...alibrated An emission mirror is provided as an option When the mirror is moved into place the emission monochromator is driven to 800 nm and emission wavelength subsequently assumed to be 0 An emission filter with 8 positions is provided The positions are cut off filter 1 to 5 blank 1 attenuator and no filter An excitation cutoff filter at 390 nm is provided This is switched in to the beam at 410 ...

Page 129: ...he instrument Explicit initialization commands are available The instrument software automatically initializes accessories if a command to move a fitted accessory is issued and the accessory has not been initialized since it was last plugged in An error is reported from accessory control commands if the accessory becomes unplugged at any time before or during the command or if the software initial...

Page 130: ...software Pulses must occur exactly once per filter unit rotation be at least 1 7 ms in duration and start in a window between 7 0 and 11 0 ms after the source flash 50 Hz and 60 Hz operation If these conditions are not observed data collection is terminated immediately and an error reported via the RS232C data output ...

Page 131: ... Input buffer overflow 0022 Parity framing or overrun error 0099 Abort termination of command 0100 Accessory not fitted 0103 Invalid phosphorescence data collection parameters 0104 Calibration peak not found 0105 Calibration operation not successful user message gives reason 0106 Non volatile memory error fatal 0107 PMT over range warning 0108 Non volatile memory error recovered from backup copies...

Page 132: ...to follow 1002 Spectral data records to follow The following sections list commands their associated parameters and output Unless otherwise stated all commands that have associated parameters will return the current value of the parameter s if the command is sent with no parameters and there is no error The format of the response is 0000c r l f parameter values c r l f 0000c r l f Parameter values...

Page 133: ...its are 800 for excitation and 900 for emission Returns high limit for selected monochromator if no parameter is specified NOTE Excitation limit is set if monochromator selection is both Refer to command MX Command ABSCISSA LOW LIMIT Syntax AL abscissa limit Description As for high limit Power on defaults for low limits are 200 for both Returns low limit for selected monochromator if parameter is ...

Page 134: ...ns of the value 9999 and contains at least one of these A data record is transmitted either in ASCII or binary In ASCII mode points are separated with commas In binary mode a 4 byte representation is sent with the least significant bytes foremost Each real output value is an integer in the range 5000 to 999999 sign suppressed and is derived from the data available in the data processing chain mult...

Page 135: ... of the scan in rim speed is a 4 digit integer representing speed in nm min A leading minus is given for negative speeds response is a 3 digit integer representing current filter response number ex slit width is fixed format xx x representing the excitation slit width in nm em slit width is fixed format xx x representing the emission slit width in nm max points is a 5 digit integer representing th...

Page 136: ...ion Returns error 0005 if no parameter specified A time drive status record consists of the following 55 rev ms mq dc fi ex wavelength em wavelength speed response ex slitwidth em slitwidth max points data int c r l f 0000 c r l f Fields within the record separated by commas are of fixed length and are left filled with spaces Parameters in angle brackets are substituted as follows x ASCH digit spa...

Page 137: ...ts that may be returned in the time drive data int is fixed format xx xx representing the abscissa interval between points in seconds Command RESPONSE TIME Syntax RT time Description Set the time drive response time to the specified value 0 to 9 99 s rounded down to nearest 0 02 s Positive values turn filter and block average priming off hence a propagation delay equal to the response time will oc...

Page 138: ...aw data are smoothed according to current RT setting Command REMOTE TRIGGERED TIMEDRIVE Syntax DS points Description Starts TimeDrive when START signal is detected Specified number of points output Syntax and output format exactly as for TD command Raw data are block averaged according to current RT setting AB aborts command before or after remote signal is detected Command REMOTE TRIGGERED PHOSPH...

Page 139: ...er than TLC x low limit Power on default is 63 0 mm Command TLC X AXIS LOW LIMIT Syntax XL limit Description Sets low limit for TLC x scan Range 0 to 98 9 mm Low limit may not be higher than TLC x high limit Power on default is 0 mm Command TLC Y AXIS HIGH Syntax YH limit Description Set high limit for TLC y scan Range 0 1 to 63 0 mm High limit may not be lower than TLC y low limit Power on defaul...

Page 140: ...arated by commas are of fixed length and are left filled with spaces Parameters in angle brackets are substituted as follows x ASCII digit space or minus rev is two character code representing software revision ms is a single character integer code representing current monochromator selection see MX command mq is a single character integer code representing current monochromator selection qualifie...

Page 141: ...ng the abscissa interval between points in mm Command TLC Y SCAN Syntax TY Description Starts TLC y scan See TLC X SCAN command for further information Command MOVE TO TLC X POSITION Syntax XM position Description Moves plate reader x axis motor to the position specified The position may be in the range 0 to 99 0 mm and is rounded to the nearest multiple of 0 1 mm Power on default is 0 Command MOV...

Page 142: ...turn error code 0111 if no valid correction data in non volatile memory and the code is 1 Command PHOSPHORESCENCE DARK CURRENT SELECT Syntax DC code Description Selects whether a dark current of zero or one generated by the SET DARK CURRENT command is used Code is 0 for dark current 0 1 otherwise Power on default is 0 Dark current value is subtracted from the emission main signal on the fly before...

Page 143: ...ust be an odd integer in range 3 to 101 Power on default is response 19 Returns response of currently active filter if no parameter is specified Command PHOSPHORESCENCE CYCLE Syntax PC time Description Sets phosphorescence mode cycle time Time is an integer in the range 0 to 9999 and has units of ms If a time of zero is specified then the cycle time will be set to the minimum value required given ...

Page 144: ...SCENCE FLASH COUNT Syntax PF count Description Sets the number of flashes per phosphorescence data collection cycle Count is integer in range 1 to 10 Returns error 0103 if flash count requested cannot be accommodated with current phosphorescence data collection parameters Power on default is 1 Command PHOSPHORESCENCE GATE Syntax PG time Description Sets specified phosphorescence gate time Time is ...

Page 145: ...uccessful a no error response is sent immediately then a single point data record followed by a final error code Single point data record format DEL ex wavelength em wavelength temp intensity wavelength format xxx x temp format xxx x C if biokinetic accessory is fitted else intensity format xxxxxxx EVENT detected during data collection Final error 0107 is returned if a PMT error was detected durin...

Page 146: ...ing to be subtracted from all subsequent data before output Data collection cycles must be integer and in the range 1 to 1000 Returns current autozero offset if no parameter given AZ 0 sets offset zero autozero off Power on default is autozero off Command SET AUTOCONC Syntax AC concentration value Description Uses current data collection parameters to average 100 data collection cycles to produce ...

Page 147: ...itation main integration data 1 Excitation dark integration data 2 Emission main integration data 3 Emission dark integration data 4 Excitation dark subtracted data 5 Emission dark subtracted data 6 Excitation corrected data 7 Emission corrected data 8 Excitation block averaged data 9 Emission block averaged data 10 Ratioed data 11 Normalized data 12 Filtered data NOTE Selections 0 10 cannot be gu...

Page 148: ... 1 to 10 000 Units are microvolts at the 0 10 V output per intensity unit as output from the RS232C channel Power on default scaling factor is 10 Command SPECTRAL DATA TRANSMISSION MODE Syntax DT mode Description Sets data transmission mode for scan and time drive data output 0 ASCII 1 binary Power on default is 0 Motor Control Command EXCITATION SHUTTER Syntax ES position Description Causes the s...

Page 149: ...sa value Description Drives both monochromators simultaneously to the specified wavelengths Abscissa values must be zero or in the range 200 0 to 800 0 excitation and 200 0 to 900 0 emission and are rounded to the nearest 0 1 Units are nm zero corresponds to zero order position Returns current wavelengths if no parameters specified If either parameter is null the corresponding monochromator does n...

Page 150: ...al polarizer 2 horizontal polarizer 3 blank 9 clear fixed no automatic cutoff filter Power on default is 0 Returns current position if no parameter specified NOTE Cutoff filter position 5 returned if selected Command EMISSION SLIT Syntax SM position Description Sets the position of the emission slit Position is in range 0 to 20 nm and is rounded to the nearest multiple of 0 1 nm Power on default i...

Page 151: ...tion during calibration Command EMISSION MONOCHROMATOR CALIBRATION Syntax MC Description Starts automatic emission monochromator calibration Command EMISSION SLIT CALIBRATION Syntax MS Description Starts automatic calibration of the emission slit Command EXCITATION MONOCHROMATOR CALIBRATION Syntax XC Description Starts automatic calibration of the excitation monochromator Command EXCITATION SLIT C...

Page 152: ...ady for use Code format is a comma separated list of ASCII digits with 0 not fitted 1 fitted Order of codes Emission mirror plate reader excitation polarizer emission polarizer cell holder well plate reader stirrer temperature controller peristaltic pump autosampler FFA excitation unit FFA emission unit Command NON VOLATILE MEMORY ERROR STATUS Syntax ME Description Returns error code 0000 0108 or ...

Page 153: ... sent If this response indicates no error then the following status record is sent followed by another no error response A status record consists of the following 55 rev ms mq dc fi ex wavelength em wavelength speed response ex slitwidth em slitwidth gate delay flashcount cyc time Fields within the record separated by commas are of fixed length and are left filled with spaces Parameters in angle b...

Page 154: ...ting the emission slit width in nm gate is a 5 digit integer representing current phosphorescence gate time units 0 01 ms delay is 6 digit integer representing current phosphorescence delay time units 0 01 ms flashcount is a 2 digit integer representing current number of flashes per phosphorescence data collection cycle cyc time is a 5 digit integer representing current data collection cycle lengt...

Page 155: ...e mains supply to the instrument Command SERIAL NUMBER Syntax SN Description Returns the serial number of the instrument Plug in Accessory Control Commands Command RETURN TEMPERATURE Syntax TP Description Returns temperature XX X C as measured at sensor Returns error 0100 if temperature sensor accessory not fitted Command TEMPERATURE OFFSET Syntax TO offset Description Returns temperature offset i...

Page 156: ...on Position is an integer in the range 1 to 4 Initialization default is 1 Command PERISTALTIC PUMP BLOW Syntax PB time Description Causes the peristaltic pump to blow for the specified time Time is in the range 0 1 to 100 s and is rounded to 0 1 s Command PERISTALTIC PUMP SUCK Syntax PS time Description Causes peristaltic pump to suck for the specified time Time is in the range 0 1 to 100 seconds ...

Page 157: ...mmand FIND DATUM Syntax FD Description Locates the datum point of the well plate reader or returns an error Command MOVE PLATE READER Syntax MP x coordinate y coordinate Description Moves plate reader to position corresponding to the coordinates given where x can be 0 to 128 and y can be 0 to 86 Command PARK Syntax PP Description Moves the plate reader to the parked position and returns 0000 when ...

Page 158: ...rrent setting power on default 340 and 510 nm If only one parameter given assumes other wavelength to be current monochromator position Command FKA CYCLE TIME Syntax FC time Description Sets FKA cycle time interval between successive points output for FKA runs Time format xxx x seconds Zero time sets minimum possible cycle time for current RD integration time data collection cycle length and wavel...

Page 159: ... current monochromator position Command FKA RUN Syntax FK points Description Starts FKA run to produce specified number of data points Syntax is as for TD command Output format is 0000 spectral data records as for time drive giving intensifies for wavelengths Wl W2 Wl terminating record containing at least one 9999 value final error code as for time drive NOTE No status line is output Command REMO...

Page 160: ...aracter echo from the instrument Echo must be turned off before control is returned to the PC Command ENTER EXIT SOFTWARE MONITOR MODE Syntax BE Description BE 6161 causes entry into software monitor mode A diagnostic software monitor task known as the multi tasking executive monitor MTEM is started The first action of MTEM is to output a record of the first few hundred commands sent to the instru...

Page 161: ...turned Command SET EMISSION UNIT STATE Syntax FM state Description Sets emission unit state if fitted Valid states are as for FX command If no parameter is specified the current excitation unit state is returned Error code 0100 means units are not fitted Error code 0112 signifies a synchronization error NOTE Excitation and emission units may not be run simultaneously Command SET FFA CYCLE LENGTH S...

Page 162: ...pecified whole number of cycles worth of data points are output range 1 99999 The cycle duration is as specified by the FI setting Cycle lengths 4 mains cycle data is block averaged No smoothing or normalization is applied to the data regardless of the 0 state The optional prompt parameter specifies one of EVENT 0 or START 1 input line triggers for data collection and output Returns error code 000...

Page 163: ...System Description and Maintenance ...

Page 164: ... the toroidal mirror M T 11 onto the entrance slit of the Emission monochromator The monochromator consists of the entrance slit a spherical mirror M S 31 a grating and the exit slit A narrow wavelength band emerges from the exit slit with the center wavelength being determined by the setting of the grating the angle of which is controlled by a stepper motor The Excitation and Emission monochromat...

Page 165: ...System Description and Maintenance 165 Figure 112 The optical layout ...

Page 166: ...is shown in the electronics diagram and in the signal handling diagram The electronics diagram is shown in Figure 113 CPU System PC Stepper Motor Control Reference Photodiode Sample Photomultiplier Excitation Monochromator Emission Monochromator Filter Wheel Accessories XE Source Excitation Monochromator Sample Beam Splitter Emission Monochromator Analogue Subsystem Photomultiplier PSU Emission Sl...

Page 167: ...om the main fluorescence signals to give a dark current corrected signal Phosphorescence When the instrument is operating in the phosphorescence mode the emission integration time is not fixed but can be selected by the user The integration can be started between 0 seconds and 9000 milliseconds after the start of the flash This is the delay time The time period over which the integration is perfor...

Page 168: ... D conversion The conversion is obtained by using a 12 bit DAC digital to analogue converter and a comparator where a 12 bit word is successively approximated to the analogue signal with the control loop being supervised by the on board microprocessor The dark current is digitally subtracted from the reference signal and likewise from the sample signal to give a true zero The DAC is also used to d...

Page 169: ...e to step signals over the specified response time The default response time is 0 5 seconds Ratioing and Correction After the sample and reference channels have been block averaged they are ratioed to improve the signal to noise and to remove source related artifacts This ratioed output is then scaled to either the wavelength range scanned or the time scale chosen to give the corrected result The ...

Page 170: ...DAC Dark Current Subtraction Excitation Correction Block Averaging Ratioing Smoothing Filter Scaling CPU System PC Analogue Outputs 10 V 1 V 10 mV Sample and Hold PMT Voltage Control Thermostat Control Stepper Motor Control Reference Sample Photomultiplier Excitation Monochromator Emission Monochromator Filter Wheel Polarizers Zero Order Mirror Other Accessories Photodiode Figure 114 The signal ha...

Page 171: ...use Location Current rating Voltage rating Type PerkinElmer part number Mains Input Mains input panel 2A 250V CSA Time lag 0C97 3134 For instruments set to 220 to 240 Volts supply voltage Fuse Location Current rating Voltage rating Type PerkinElmer part number Mains Input Mains input panel 1A 250V CSA Time lag 0C96 2348 For details on how to change the fuse see Changing the mains fuse on page 196 ...

Page 172: ...o is aware of the hazards involved Fuse Location Current rating Voltage rating PerkinElmer part number Source board 100mA T 250V 04969185 FS1 Motor Control board 500mA T 250V 04967940 FS2 Motor Control board 2A T 250V 04970839 WARNING Using the instrument in a manner not specified herein may impair the protection provided by the instrument ...

Page 173: ...ESPONSIBLE BODY The main cover is hinged along the rear edge of the instrument 1 Switch off the instrument and disconnect the electrical mains supply 2 Position the LS 55 on the front of the bench so that it overhangs the bench by approximately 8 cm 3 Loosen the two screws located on the left hand and right hand underside of the instrument as shown in Figure 115 They are captive so will remain in ...

Page 174: ... right hand side as shown in Figure 116 Figure 116 Cover stay 6 Unclip the earth lead from the cover as shown in Figure 117 Figure 117 Unclipping the earth lead 7 Open the cover to an angle greater than 90 degrees to the bench From this position the cover can be lifted off its hinges ...

Page 175: ...aintenance 175 Refitting the main cover To replace the cover proceed as follows 1 Place the cover on its hinges 2 Close the cover slightly and attach the earth lead 3 Close the cover and tighten the screws to secure the cover ...

Page 176: ...Main Optical Cover 1 Remove the main cover as described in Removing the Main Cover on page 173 2 Undo the 3 screws which secure the middle optical cover as shown in Figure 118 and Figure 119 Figure 118 Center screw Figure 119 Rear screws ...

Page 177: ...ption and Maintenance 177 3 Remove the middle optical cover by lifting it vertically as shown in Figure 120 Figure 120 Lifting the cover CAUTION Be careful not to touch any of the optical surfaces mirrors or windows ...

Page 178: ...itting the optical cover 1 Replace the middle optical cover locating the central groove as shown Figure 121 Figure 121 Replacing the main optical cover 2 Secure the three screws 3 Replace the main cover including the earth lead ...

Page 179: ... high pass filters into these open positions if desired Furthermore the filters in the standard emission cut off filter wheel can be changed if desired In all of these cases FL WinLab must be informed of the changes This is done by entering information into the file LS55 INI that is located in the flwinlab directory Information about the updated filters is entered into the section headed ACCESSORY...

Page 180: ...ng of the relevant section of LS55 INI follows Open filter wheel positions are shown in bold and italic script ACCESSORY PMT_Type std ExFilter 1 clear auto cutoff on ExFilter 2 vertical polariser ExFilter 3 horizontal polariser ExFilter 4 blank ExFilter 5 custom filter 1 ExFilter 6 auto ex cut off filter ExFilter 7 custom filter 2 ExFilter 8 UG 5 ExFilter 9 clear auto cutoff off EmFilter 1 290nm C...

Page 181: ...ply switch of the LS 55 and remove the mains supply cable the xenon lamp uses high voltages which could lead to a lethal electric shock if the above precaution is not taken 1 Open the top cover of the LS 55 as described in Removing the Main Cover on page 173 2 Locate the metal lamp housing located rear left and remove the earth contact as shown in Figure 122 3 Undo the cover fixing screw as shown ...

Page 182: ...om the LS 55 as shown in Figure 123 and store it securely Figure 123 Lifting the cover 5 Locate the lamp and the two electrode connectors as shown in Figure 124 Figure 124 Locating the parts 6 Press each connector in turn and remove the cables as shown in Figure 125 ...

Page 183: ...sen the grub screw at the bottom of the lamp holder bracket as shown in Figure 126 Figure 126 Loosen the grub screw 8 When the screw is correctly loosened the lamp can be easily pulled vertically out of its bracket NOTE If force is required then the screw is still not loose enough ...

Page 184: ...contaminated leading to serious loss of sensitivity If the capillary is accidentally touched wipe immediately with spectroscopic grade ethanol or methanol and dry with a cuvette wipe tissue NOTE In order to seat the lamp correctly in the bracket it needs to be rotated before insertion into the bracket so that it is correctly aligned with the optical system 10 Turn the lamp so that the trigger stri...

Page 185: ...amp will most probably break it 12 When the lamp has been fully inserted into the bracket gently tighten the grub screw NOTE The grub screw does not need to be overtight Once the grub screw makes contact with the lamp post this is tight enough Overtightening could lead to scoring on the column of the lamp making future removal difficult 13 Connect both electrode cables by depressing the connector ...

Page 186: ...er into the slot around the optical cover and gently lower the metal cover taking care that no cables are trapped underneath as shown in Figure 130 Figure 130 Replacing the cover 15 Re connect the earth tag and replace the fixing screw as shown in Figure 131 Red cable Black cable ...

Page 187: ... the new lamp fitted can be tested using the FL WinLab validation routine A silica cuvette with deionized water is inserted into the cuvette holder and then the routine is started by selecting Validate LS 55 on the Applications menu in FL WinLab The routine will prompt you for the water filled cuvette then will measure and display the signal to noise value for the instrument and the wavelength acc...

Page 188: ...serting the clamps 1 Switch on the LS 55 and allow it to initialize fully If the LS 55 is already running then switch it off and on again This is because the instrument is automatically driven to lock down monochromator positions at the end of initialisation WARNING Switch off the mains supply switch of the LS 55 and remove the mains supply cable before continuing this procedure 2 Switch off the L...

Page 189: ...move the two shipping clamp screws from their storage positions on the left hand side of the LS 55 as shown in Figure 133 Figure 133 Shipping clamp screws storage positions 6 Screw the shipping clamp screws into the screw threads revealed when the plates were removed as shown in Figure 134 ...

Page 190: ... clamp screws 7 Replace the shipping clamp plates by turning them through 180 to align the hole with the shipping clamp screw as shown in Figure 135 Figure 135 Replacing the shipping clamp plates 8 Secure the plates with the two screws as shown in Figure 136 ...

Page 191: ...isors before attempting to lift the spectrometer As the LS 55 Luminescence Spectrometer weighs approximately 49 kg approximately 59 kg with packaging we recommend that the spectrometer is lifted by 2 adults and that it is lifted by the base of the instrument Removing the clamps Once the LS 55 has been moved to its new position remove the clamps as detailed in Removal of the shipping clamps on page...

Page 192: ... a damp cloth Mild detergent may be used if necessary Always perform a patch test on an inconspicuous area of the instrument before you clean the entire instrument Avoid spilling liquid into the instrument Clean all external spills immediately If anything that is spilled enters the main body of the LS 55 switch off the power and call a PerkinElmer Service Engineer ...

Page 193: ...lows WARNING Switch off the mains supply switch of the LS 55 and remove the mains supply cable before starting this procedure 1 Switch off the LS 55 and remove the mains supply plug 2 Place a flat bladed screwdriver in one of the two slots on the right side of the air filter holder and then lever off the air filter holder 3 Remove the air filter 4 Clean off dust from the filter grid 5 Replace the ...

Page 194: ...ged by altering the connections on the mains transformer located inside the instrument 1 Switch off the instrument and disconnect the electrical mains supply 2 Open the main cover as detailed in Removing the Main Cover on page 173 CAUTION Do not change any of the terminal connections on the right hand side of the transformer viewed from the front of the instrument The mains transformer is located ...

Page 195: ...s 3 Disconnect the terminal brown wire from the current terminal This will be connected to either the 240V 220V 120V or 100V terminal 4 Connect the brown wire to the terminal marked with the required operating voltage NOTE The 240V connection should be used for 230V supplies 5 Ensure that the operating voltage plate shows the correct operating voltage The appropriate operating voltage may be displ...

Page 196: ...ains fuse Having changed the operating voltage of the instrument the mains fuse must be changed to correspond with the operating voltage The table below gives the correct fuse rating for the operating voltage Operating Voltage Mains Fuse Rating PerkinElmer Part Number 220 240 V 50 60 Hz 1A Slo Blo OC97 3107 100 120 V 50 60 Hz 2A Slo Blo OC97 3134 The mains fuse is located on the mains input panel ...

Page 197: ...188 Mains Label 100 V 220 V The voltage plate is secured by two screws which also secure the mains socket To change the voltage plate proceed as follows 1 Remove the two screws from the mains socket 2 Carefully pull out the mains socket a small distance so that the voltage plate is released 3 Place the correct plate behind the mains socket with the appropriate side facing outwards 4 Push the mains...

Page 198: ...198 LS 55 User s Guide ...

Page 199: ...Index ...

Page 200: ...3 semi micro cuvette 52 septum injector 48 single position 38 stirred four position cellchanger 40 stirred semi micro cuvette 53 stirred single position 39 stirrer bars 50 thermostatting tubing 45 changing the lamp 181 changing the mains fuse 196 changing the operating voltage 194 cleaning cellholders 56 cleaning the air filter 193 cleaning the LS 55 192 connecting to a PC 31 connections 29 access...

Page 201: ...for transport 188 moving 188 O opening the main cover 173 operating voltage 194 operator 17 optical system 164 P peristaltic pump commands 129 photomultiplier red sensitive 83 photomultiplier control commands 128 plate reader commands 129 driver commands 127 plate reader accessory 73 polarizer accessory 105 power switch 34 preparing for transport 188 R red sensitive detector 83 refitting the main ...

Page 202: ...lter setup 102 polarization 110 sipper control 60 well plate reader 80 source control commands 128 spares for accessories 120 specifications 18 standard cellholder 38 start up 34 starting 34 stirred four position cellchanger 40 stirred semi micro cuvette 53 stirred single position cellholder 39 stirrer bars 50 stirring samples 50 switching on 34 T temperature control 45 terminal block 31 thermosta...

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