Laser Raman Microscope

RAMANplus　　Operation Manual

Nanophoton corporation

November 10, 2011

Contents

1 Regarding Your Own Safety 71.1 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.2 Operating recommendations for RAMANplus as a product with a

built-in laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.3 Preliminary setup notes . . . . . . . . . . . . . . . . . . . . . . 111.4 What to do in case of a power outage . . . . . . . . . . . . . . . 111.5 Preliminary notes regarding computer operation . . . . . . . . . 121.6 Preliminary notes regarding maintenance, safety checks and repairs 121.7 Warning label . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2 Introduction to RAMANplus 132.1 General description . . . . . . . . . . . . . . . . . . . . . . . . . 132.2 Primary Components . . . . . . . . . . . . . . . . . . . . . . . . 142.3 Main body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.3.1 Appellation . . . . . . . . . . . . . . . . . . . . . . . . . 142.3.2 Inside of the optical system part . . . . . . . . . . . . . . 15

2.4 The EPO switch (Optional) . . . . . . . . . . . . . . . . . . . . 152.5 The power supplying box and the laser power supplying box . . . 162.6 The light shading box covering the sample stage and a interlock

system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.7 A upright microscope (Nikon Eclipse 90i) . . . . . . . . . . . . . 172.8 A computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3 Starting Measurement 233.1 Starting RAMANplus . . . . . . . . . . . . . . . . . . . . . . . 23

3.1.1 Attention before start . . . . . . . . . . . . . . . . . . . 233.1.2 How to start RAMANplus . . . . . . . . . . . . . . . . . 24

3.2 Finding the region of interest of the sample . . . . . . . . . . . . 26

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3.2.1 Setting the sample on the stage . . . . . . . . . . . . . . 263.2.2 Bringing the sample into focus . . . . . . . . . . . . . . 283.2.3 Finding the region of interest . . . . . . . . . . . . . . . 31

3.3 Setting the parameters of Raman measurement . . . . . . . . . . 363.3.1 ”Scan mode” . . . . . . . . . . . . . . . . . . . . . . . . 373.3.2 ”Objective lens” . . . . . . . . . . . . . . . . . . . . . . 393.3.3 Measurement Area . . . . . . . . . . . . . . . . . . . . . 413.3.4 Measurement time . . . . . . . . . . . . . . . . . . . . . 483.3.5 Laser wavelength . . . . . . . . . . . . . . . . . . . . . . 493.3.6 Laser power . . . . . . . . . . . . . . . . . . . . . . . . 513.3.7 Spectral range . . . . . . . . . . . . . . . . . . . . . . . 52

3.4 Starting measurement, and viewing and analyzing data . . . . . . 543.5 Surface profiling measurement . . . . . . . . . . . . . . . . . . . 563.6 Shutting down RAMANplus . . . . . . . . . . . . . . . . . . . . 58

4 Software ”RAMAN Imager” 614.1 Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

4.1.1 Interface . . . . . . . . . . . . . . . . . . . . . . . . . . 614.1.2 How to start RAMAN Imager . . . . . . . . . . . . . . . 624.1.3 How to quit RAMAN Imager . . . . . . . . . . . . . . . 63

4.2 Common control panel . . . . . . . . . . . . . . . . . . . . . . . 644.3 Measurement setting panel . . . . . . . . . . . . . . . . . . . . 66

4.3.1 Raman . . . . . . . . . . . . . . . . . . . . . . . . . . . 674.3.2 ”Scan mode” . . . . . . . . . . . . . . . . . . . . . . . . 684.3.3 Surface(Optional) . . . . . . . . . . . . . . . . . . . . . 734.3.4 Wide-field Raman measurement ”WF Raman”(Optional) . 734.3.5 Widefield observation ”WF Observation” (Optional) . . . 764.3.6 Auto well-plate measurement(m × n )（Optional) . . . . 79

4.4 Functions in the ”Menu” . . . . . . . . . . . . . . . . . . . . . 824.4.1 The outline of the ”Menu” . . . . . . . . . . . . . . . . 824.4.2 ”File/Save・Load Settings” . . . . . . . . . . . . . . . . 834.4.3 The wavelength and the output power of the laser ”Func-

tion/Laser” . . . . . . . . . . . . . . . . . . . . . . . . . 844.4.4 Image Quality & Spectral ROI ”Function/Image Quality

& Spectral ROI” . . . . . . . . . . . . . . . . . . . . . . 854.4.5 Auto Save Settings ”Function/Auto Save Settings” . . . 874.4.6 Trigger function ”Function/Trigger” . . . . . . . . . . . 884.4.7 ”Function/Data Calibration and Compensation” . . . . . 90

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4.4.8 ”Function/Assist” . . . . . . . . . . . . . . . . . . . . . 914.4.9 Standard sample ”Function/Reference Samples” . . . . . 944.4.10 ”Function/Device/Spectrograph” . . . . . . . . . . . . . 944.4.11 ”Function/Device/CCD Detector” . . . . . . . . . . . . . 954.4.12 Automatic on/off of microscope illumination. ”Function/Illumination

for Optical Microscope” . . . . . . . . . . . . . . . . . . 974.4.13 Motorized z stage ”Function/Device/Microscope Z stage” 97

5 To understand Raman scanttering 995.1 Raman spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . 995.2 Raman scattering . . . . . . . . . . . . . . . . . . . . . . . . . 1005.3 Raman imaging (distribution of materials) . . . . . . . . . . . . 1035.4 Sectional Raman imaging (xy Raman image) . . . . . . . . . . . 1065.5 Cross-sectional Raman imaging (xz Raman image) . . . . . . . . 107

6 Support 109

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Chapter 1

Regarding Your Own Safety

Please note that while the RAMANplus was developed with safety in mind it couldcause damage to users and its surroundings if misused or the basic regulationsand guidelines are not followed, and therefore the consequences of these actionsare not covered by warranty. Ensure to carefully read this manual before use.The rules described with the yellow signs such as the Warnings and Cautions inparticular should be strictly followed.

!Warning

Death or serious injury could occur if messages indi-cated by this sign are not followed.

!Caution

Damage to both humans and property could occur ifmessages indicated by this sign are not followed.

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1.1 Regulation

!Warning

ENSURE to press the emergency stop button, disconnect the plug from theoutlet, and immediately contact us if any of the special situations describedbelow arise.

Emergency Power off

• The power cord, plug, wall adapter, extension cord or power supply devicehas cracked, is broken, or not working.

• Any part of the instrument is too hot to touch or there are traces ofsmoke, sparks or fire being emitted by the instrument.

• Faint noises such as snaps, crackles or hissing can be heard from theinstrument, or it is emitting an unusual odor.

• There is a trace of something having fallen on the instrument, power cordor power supply device.

• There is a trace of something having fallen on the instrument, power cordor power supply device.

• The instrument has fallen or been damaged in some way.

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• The system doesn ’t work in spite of the correct procedure having beenfollowed.

!Warning

DO NOT take the instrument apart as it could lead to electric shock, failure ofthe system or people being exposed to the laser. Any problems or damagecaused by the instrument having been taken apart are not covered by warranty.

!Warning

ENSURE only the power cord and power supply device supplied by NanophotonCorporation are used. DO NOT use them for any other instrument as theywere specifically designed for use with RAMANplus.

!Warning

DO NOT use an outlet that has been damaged or corroded. DO NOT bend orrefurbish the plug. ENSURE to contact Nanophoton Corporation for areplacement if the plug has been damaged. A 3 pin power plug is used with theRAMANplus to ensure it is connected to a ground outlet. DO NOT connectthe power plug to non-grounded outlet.DO NOT share an outlet with RAMANplus and another electric appliance thatconsumes a lot of electricity as any unstable voltage could possibly damageRAMANplus.DO NOT overload the outlet used with RAMANplus. ENSURE to confirm thatthe outlet to which RAMANplus is connected has been correctly wired, is withineasy reach, and near RAMANplus. DO NOT stretch a power cord so taut thatit becomes overloaded. ENSURE to confirm that the current and voltage of anoutlet connected to RAMANplus are of the correct values. ENSURE to exerciseextreme caution when connecting or disconnecting a power cord from an outlet.

!Caution

Please note that as there are some parts in the product that could fail in theevent of a surge or spike in electricity from an outlet. Ensure the system is offand remove the plug from the outlet if excess voltage is expected to occur.

!Caution

DO NOT insert or remove any other cables than the LAN connection to a PC.Parts of the product are extremely susceptible to static electricity and could be

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damaged their connectors are touched with no cable in them. ENSURE tocontact us when you need to insert or remove any cable other than a LANcable.

!Caution

DO NOT allow any water over the product and DO NOT use RAMANplusanywhere it may come in contact with water as it may result in it shortcircuiting. Should the product come into contact with water ENSURE toimmediately switch the system off, remove the plug, and then dry with a cloth.ENSURE to immediately contact us if any water has possibly entered theproduct.

!Caution

DO NOT turn the RAMANplus on if it is covered with a case or a cloth as itcan lead to heat building up and result in problems.

1.2 Operating recommendations for RAMANplus as a

product with a built-in laser

!Warning　

RAMANplus was labeled a Class 1 JIS code laser product. ENSURE to exercisethe following in preventing any accidents from occurring.

• DO NOT look directly at the laser beam as it can harm your vision.

• ENSURE to avoid any exposure to the laser regardless of whether directlyor indirectly as it could result burns to yourself or your clothing.

• RAMANplus is designed with no laser irradiation to human body exceptfor maintenance time and checking time. However, for any reason, ifthere is a possibility to be exposed to laser, ENSURE to wear laser proofsafety glasses and clothing.

• ENSURE to position an appropriate cautionary sign at the entrance ofanywhere RAMANplus is being used.

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• DO NOT use large amounts of burnable, explosive or volatile solventssuch as alcohol or ether anywhere near a laser product as it could resultin an explosion.

• ENSURE use of the product under the supervision of personnelconversant with laser safety services and that understands the risks.

• ENSURE all users of RAMANplus understand the above operatingrecommendations.

• ENSURE to follow the safety precautions and control standards for laserproduct users provided by IEC60825-1(2001), JIS C 6802(2005) andothers.

1.3 Preliminary setup notes

The product will be assembled and aligned by Nanophoton staff. Afterconfirming the installation conditions our stuff will ensure the appropriateinstallation of RAMANplus.

!Caution

ENSURE the product is at least 0.3m away from any wall to allow alignmentand maintenance to take place.

!Caution

ENSURE to avoid any direct sunlight onto the product.

!Caution

ENSURE the product is kept away from any heat sources.

1.4 What to do in case of a power outage

In the case of power outage ensure switch the system off and remove the plugfrom the outlet.

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1.5 Preliminary notes regarding computer operation

Almost all RAMANplus features are computer-operated. The computer systemruns Windows 7, which users will need to know how to operate.

!Warning

Before using the system ensure to carefully read the computer manual andfollow any appropriate safety precautions.

1.6 Preliminary notes regarding maintenance, safety

checks and repairs

ENSURE to keep the work place and product clean and tidy. When cleaningthe product rub its surfaces with a damp mildly soapy cloth.

!Warning

The product contains no user maintainable parts. Maintenance, repair,alignment, disposal or exchange of parts or any other operation not included inthis manual will need to be carried out by Nanophoton staff. RAMANplus is aprecision optical instrument, and operation of it by anyone not used to it couldpossibly result in problems. Opening the cover of the instrument could lead toblindness, being burnt or fire from the laser being exposed. Any damage to theinstrument caused by operations not included in this manual or use andinstallation of inappropriate products provided by other companies are notcovered by warranty.。

1.7 Warning label

ENSURE that the warning and caution labels are on the product.

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Chapter 2

Introduction to RAMANplus

It is explained about appellations and functions that are important for the oper-ation of a Laser Raman Microscope RAMANplus in this chapter.

2.1 General description

Laser Raman Microscope ”RAMANplus ” is an instrument to observe and analyzeyour samples.The analysis is operated in the area which ranges from sub-micron to millimeterscale while the sample is observed under an optical microscope. As an principleof the analysis, Raman spectroscopy is utilized.

For Raman spectroscopy, laser beam is irradiated on to the sample to exciteRaman scattering light at the irradiation position on the sample. Raman scat-tering light from the sample is collected and analyzed with the instrument.

Measured data is so-called ”Raman spectrum”. Because Raman spectrumis identical to molecules or crystalline form, the analysis is done as molecularidentification or evaluation of the crystalline in many cases.

Furthermore laser Raman microscope ”RAMANplus ” has an prominent prop-erty. It is fast imaging function of Raman spectrum. The spectral imaging func-tion provides distribution information of molecules of crystallinity.

In addition, RAMANplus has the surface profiling function. With the com-

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bination of surface profiling and distribution analysis, it provides multilateralsolutions.

2.2 Primary Components

Fig. 2.1 shows a whole setup of a laser Raman microscope ”RAMANplus ”. The”RAMANplus ” adopts a upright type microscope.

The main body of the instrument is installed on an anti-vibration table at theleft side in the figure. Under the table, a power supplying box for the main body,another supplying box for lasers and a personal computer condoling instrumentsare set. A monitor for the personal computer, a key board, a mouse and a stagecontroller are set on another table at the right side in the figure. An emergencypower off (EPO) switch is supplied optionally and set on the left side table.

Emergency Power Off (Optional)

Computer

Laser Power SupplyController (LPSC)

Motorized Z stage controller

Motorized XY stage controller (Optional)

Anti-Vibration table

PC Table

Mouse &Keyboard

Main body

Power supply fora motorized XY stage

Monitor

Power SupplyController (PSC)

Figure 2.1: Components of RAMANplus

2.3 Main body

2.3.1 Appellation

Part names of the main body are shown in Fig. 2.2. A main body composes amicroscope part with a light shading box located in a front half and an opticalsystem part behind the microscope part. In the figure, a door of the shading box

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is opened so that a operator can access the microscope stage to set a sample.The microscope composes the microscope body, a sample stage, a revolver withobjective lenses, a condenser lens, and so on. On the upper side of the micro-scope, a CCD camera for observing microscopic sample images is attached. Athermo-electrically cooled CCD detector for recording Raman scattering light isattached at the left side of the main body.

2.3.2 Inside of the optical system part

In the optical system part, laser(s), laser scanning optics, slit confocal opticsand spectroscopic optics are embedded. It is no need for the operator to accessthese optics parts. All controls of optics, such as laser selection, laser poweradjustment and spectroscopy setting

Door of a shading box

Cooled CCD detector

Shading box

CCD camera for a microscope

Optical system part

Microscope (Nikon 90i)

Figure 2.2: Parts of the main body

2.4 The EPO switch (Optional)

A picture of a EPO switch is shown fig. 2.3. In the fig, the EPO is set on thetable for switch off the instrument at a emergency case. Pushing red buttonstops power supplying to the instruments. Once the red button is pushed, it iskept pushed. You need to turn right the red button to recover power supply.

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Figure 2.3: A picture of the EPO switch

2.5 The power supplying box and the laser power sup-

plying box

A picture of a power supplying box and a laser power supplying box is shown infig. 3.1. These are set under the anti-vibration table.

The power supplying box supplies electrical power to the instrument butlasers. The laser power suppling box supplies electrical power to the lasers insideinstruments.

On the front panel of the power supplying box, a key switch for power on/offand a indicator for the instrument operation. On the front panel of the laserpower suppling box, two indicators are shown. One indicator shows the status oflaser emission of 532 nm wavelength. The other shows for 785 nm wavelength.

Turing the key-switch right start to supply the power. Turning left stops tosupply the power. Administrator can remove the key-switch to taking control ofthe instruments.

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Power Supplying Controller

Laser Power Supplying Controller

Key switch

Indicator (Blue) for AC power

Indicator (Red) for lasing

of the 785-nm laser

Indicator (Geen) for lasing

of the 532-nm laser

Figure 2.4: A picture of a power supplying box and a laser power supplying box.

2.6 The light shading box covering the sample stage and

a interlock system

A light shading box covering a sample stage makes it possible to measure thesample under the room light is kept on. It is very important because Ramanscattering light is very weak and is easily disturbed by the room light. Furthermore the shading box blocks laser light going out and protect the operator fromthe irradiation.

A interlock system is equipped to the shading box so that the laser beamnever comes out when the door of the shading box is opened.

2.7 A upright microscope (Nikon Eclipse 90i)

The upright microscope is set in the light shading box. The part names areshown in fig. 2.5.

It is very important to master how to operate the optical microscope. Be-cause, too measure Raman spectrum, the operator should put the sample onthe stage, observe the sample surface with the microscope, finding the region ofinterest and set the focus. It is very important to master how to operate theoptical microscope.

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The CCD camera at the front part of the microscope takes optical microscopeimages of the sample through the objective lens.

Left side Right side

Revolver

Objective lens

Stage

Obj. switch

Condensor lens

Escape switch

Filter IN/OUT switch

Z resolution switch

Aperture stop switch

Fiedl stop switch

Focus handle

XY stage handle

Figure 2.5: Part names of a upright microscope

The sample stageThe stage is place on which the sample is put. The operator moves sampleposition with controlling a xy-handle manually.

An electrical xy stage is provided optionally.

The revolverThe revolver is a part where objective lenses are attached. By being revolved,the objective lens which are used for observation can be selected. Six ( or Sevenoptionally) objective lenses can be equipped.

Electrical revolver is provided optionally.

For polarized Raman measurement, an optional revolver for polarization mea-surement is necessary.

Objective lenses.A standard set of five objective lenses are listed below.

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Name Magnification N.A. W.D. (mm)LU Plan FLUOR EPI ×5 0.15 23.5LU Plan FLUOR EPI ×10 0.3 17.5LU Plan FLUOR EPI ×20 0.45 4.5LU Plan FLUOR EPI ×50 0.8 1LU Plan FLUOR EPI ×100 0.9 1

N.A. is ”Numerical Aperture”.

W.D.is ”Working Distance” that is the distance between the bottom of theobjective and the focus.

In addition to the standard objective lenses, various objective lenses are pro-vided optionally.

A condenser lens and a focusing handleA condenser lens is used for focusing illumination light for microscope observa-tion. Condenser position can be adjusted withe the focusing handle.

Filter　 IN/OUT switchTwo filters of front side are ND filters of transmission illumination. ND filtersare used to adjust brightness of the illumination. The bigger number has lowertransmission and decrease brightness more.

• ND8: 1/8 decrement

• ND32: 1/32 decrement

• ND8 and ND32: 1/256 decrement

The focus handleRotating the focus handle moves the sample stage up and down to bring thesample surface in the focus. The resolution of the objective lens movement ofup and down can be selected with the Z resolution switch.

• Move the stage downward： Rotating the handle toward the front.

• Move the lens upward： Rotating the handle Derward the back.

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Z resolution switchThe mount of the stage movement depends on the rate of movement/rotationwhen the focus handle is rotated. The rate is changed in the sequence of”Coarse”, ”Fine” and ”Ultra fine” when the Z resolution switch. When theswitch is pushed, buzzer sounds and the indicator status also changes.

Resolution Amount of movement Indicator BuzzerCoarse 2500∼10µm/sec lighting One long buzzerFine 100µm/rotation extinction One short buzzer

Ultra fine 25µm/rotation blinking Two short buzzerObj. switchIn the case of an electrical revolver is equipped (option), this switch revolves therevolver and changes the objective lens.Escape switchThis switch moves the stage to the escape position with the indicator lighting.The escape position is 5 mm downward from the current position. You need topush the escape switch again to move the stage back to the former position withthe escape indicator lighting off.

Note that the escape function does not work when the distance between thecurrent stage and the bottom position is less than 5.5 mm.

Note that the focusing handle is disable at the escape position and while thestage is escaping.

2.8 A computer

All operation about controlling instruments, measurement, viewing data andanalysis is done with the computer. The operating system of the computer isWindows7.

In the computer, the RAMANplus controlling software, ”RAMAN Imager”and the data viewing and analyzing software, ”RAMAN Viewer” are installed.

With the controlling software ”RAMAN Imager”, the operator set the mea-surement parameters. The measured data is viewed in the ”RAMAN Viewer”.Various kind of analysis method is equipped in the ”RAMAN Viewer”

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Chapter 3

Starting Measurement

In this chapter, practical procedures of measurement are explained for your start.In the first step, it is explained about how to start the RAMANplus. In thesecond step, it is explained about how to set samples to stage and then how toset measurement parameters for measurement modes.The procedure from the start to the end is listed as below.

1. Starting RAMANplus.

2. Finding the region of interest in the sample.

3. (Knowing the items necessary for measurement.)

4. Setting the measurement parameters and Measuring.

5. Viewing Data and analyzing.

6. EndingRAMANplus.

3.1 Starting RAMANplus

3.1.1 Attention before start

Please confirm that all cables are connected correctly and that the EPO switchis correctly connected correctly and is located within close reach.

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3.1.2 How to start RAMANplus

1. Turning a key switch right on the power supplying box to switchpower on.

Please confirm the AC power indicator lighting with blue on the powersupplying box and also laser power supplying box shown in fig. 3.1.

Power Supplying Controller

Laser Power Supplying Controller

Key switch

Indicator (Blue) for AC power

Indicator (Red) for lasing

of the 785-nm laser

Indicator (Geen) for lasing

of the 532-nm laser

Figure 3.1: A power supplying box

2. Turning on the computer.

Note that there is no particular order between turning on the power sup-plying box and the computer.

3. Double-clicking the icon of ”RAMAN Imager” to start ”RAMANImager”

After double-clicking the icon, the RAMAN Imager starts to initializing alldevices of RAMANplus step by step. While initializing the devices, theprogress bar is shown. After finishing initializing, the main window turnsout. It may takes several minutes after the double-clicking to finishing theinitializing.

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Figure 3.2: The icon of ”RAMAN Imager”

Figure 3.3: A Main window of ”RAMAN Imager” just after initializingRAMANplus

4. Waiting for the thermo-electrically cooled CCD detector reaching-70 ℃

The thermo-electrically cooled CCD detector starts to be cooled automati-cally with the start of the RAMAN Imager. The warming up of RAMANplusis done when the temperature of the cooled CCD detector reaches atで-70℃. To check the temperature, you can click ”Detector temperature” fromthe menu of ”Window ”.

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Note that you can start measurement ever before reaching -70℃. In thiscase, the dark current noise of the cooled CCD detector may be larger.

Figure 3.4: Temperature of the thermo-electrically cooled CCD detector

5. Set the sample to the stage and start measurement

3.2 Finding the region of interest of the sample

3.2.1 Setting the sample on the stage

1. Opening the door of the shading box.

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1.Open the door.

2. Move stage down.

3.Put your sample on the stage.

Figure 3.5:

2. Taking the stage down with rotating the focusing handle.

It is necessary to taking the stage down enough to prevent the contact ofthe objective lens and the sample. The height of the stage can be adjustedwith the focusing handle.

⋆ Hint) The speed of the stage movement depends on the travellingrate. The rate can be switched with the ”Z resolution switch”. With the”Coarse” status of the switch, the stage is moved quickly.

⋆ Hint) The ”Escape switch” moves the stage down 5mm quickly (Escapeposition). The pushing the ”Escape switch” at the escape position, thestage comes back to the initial position.

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Escape switch

Z resolution switch Focus handle

Figure 3.6:

Amount of the movement Indicator BuzzerCoarse 2500∼10µm/sec Lighting A long buzzerFine 100µm/rotation Extinction A short buzzer

Ultra fine 25µm/rotation Blinking Two short buzzer

3. Putting the sample with the slide glass on the stage and clippingthe slide with the sample holder.

4. Moving the sample position so that the region of interest is justright below the objective lens.

3.2.2 Bringing the sample into focus

1. Select the objective lens of the proper magnification for observingthe region of interest of the sample.

For the selection of the objective lens, the revolver should be revolvedmanually.

If the motorized revolver equipped, the ”Objective Selector Switch” shouldbe pushed. Or the Objective lens can be selected through the software”RAMAN Imager”.

⋆Hint) The field of views of the microscope objective lenses are listedbelow.

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Magnification Optical Microscope　 Raman measurement5x About2.4mm× 1.8mm About0.9mm× 0.9mm10x About1.2mm× 0.9mm About800µm× 800µm20x About600µm× 450µm About400µm× 400µm　50x About240µm× 180µm About160µm× 160µm　100x About120µm× 90µm About80µm× 980µm　

⋆Hint) For beginners of a microscope, it is recommended that start tobringing the sample into the focus with the objective lens of lower magnifi-cation. It is easier to bring the sample into focus with higher magnificationafter focusing with the lower magnification.

Attention) The view field of Raman is smaller than that of optical micro-scope.

Revolve manually

Obj. switch(Motorized revolver)

Revolver

Figure 3.7:

2. Lighting on the illumination for the microscope observation withthe ”Light control slider” in the RAMAN Imager.

There two ”Illumination slider”. One is ”Reflection lamp” for ”Reflec-tion illumination” and the other is ”Transmission lamp” for ”Transmissionillumination”

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Figure 3.8: Light control slider for the microscope illumination

3. Moving the sample stage upward where the sample surface comesin the focus.

⋆Hint) It makes bringing the sample the into focus easier to observe thelaser illumination pattern.To observe the laser illumination pattern, a mechanical shutter stoppingthe laser beam should be opened and , furthermore it is important to adjustthe laser power at the same time. For these controls, please refer to thechapter 3.6.To open the mechanical shutter, The Check box ”Check Laser Spot” shouldbe checked.

When the sample is in the focus, the laser illumination looks like the small-est spot. When the sample is out of the focus, the laser illumination lookslike defocus spot. It is good way to find the sample stage height with thesmallest laser spot.　The laser spot observation can effective even for laser line illumination.

⋆Hint）When bringing the sample into focus, the sample height can becontrolled more precisely with the ”Fine” or ”Ultra Fine” mode of the ”Zresolution”. the status of ”Z resolution” is

⋆Hint)　 The sample stage height can also be controlled with the ”Up”and ”Down” button of the RAMAN Imager. The step distance is deter-mined with the ”Step” value.

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Figure 3.9: Checking laser spot

■ Attention) When bringing the sample into focus with the laser illumina-tion, decrease the laser power enough before the laser illumination.

■ Attention）The door of the shading box has the interlock system. It isnecessary to close the door to illuminate laser beam on the sample.

3.2.3 Finding the region of interest

For the finding the region of interest, XY stage is moved manually.

If the motorized stage is quipped (provided optionally), the xy stage is con-trolled with the joystick and/or the RAMAN Imager.

Manual control (Standard)

The stage X handle moves the sample holder in X direction on the stage(from side to side). The stage Y handle moves the stage in Y direction (backand forth).

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X handle

Y handle

X

Y

Figure 3.10: Manual XY stage

A motorized XY stage is provided with a joystick optionally. The motorizedstage moves in the direction where the joystick pushed. The speed of the motor-ized stage is selected with the ”Speed” button. There are four speeds and thespeeds are indicated as the number of the indicator lamps. Four indicator lampsshows the fastest speed. By pushing the ”Speed” button, the speed is changedto the next speed.

• ”Speed” Button: Changing the speed.

• ”M-ORG” Button: Finding the Hardware origin.

• ”ZERO” Button: Setting the current position as the home position.

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• ”E-ORG” Button: Moving to the home position.

Speeds are listed below.

The number of lamps Speed　1 0.001 mm/s2 0.05 mm/s3 0.3 mm/s 　4 1 mm/s 　

　Note) The position when the power is on is set as the home position.

Stick

X

Y

Current position display

Speed button

Speed indicator

Figure 3.11: Joystick controller (optional)

The control of the motorized stage with RAMAN Imager.

1. Select the ”Stage Controller” from the ”Window” menu on theRAMAN Imager

Microscope Stage Controller widow is opened.

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Figure 3.12:

⋆Hint）　The stage controller on the ”Wide Field” setting panel can bealso used. Please click the ”WF-Raman” tab to open the ”Wide Field”setting panel.

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Figure 3.13: Wide Field Stage Controller

2. Set the region of interest at the center of the view field by mov-ing the stage. For moving the stage, ”UP”, ”DOWN”, ”LEFT”,”RIGHT” buttons are pushed.

⋆Hint）The speed of the stage is selected from the pull-down menu ofthe ”Speed” box.

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3. If necessary, the sample surface is confirmed in the focus withmoving the stage height.

3.3 Setting the parameters of Raman measurement

For measuring Raman spectrum, measurement parameters should be set on theRAMAN Imager.

The measurement parameters are listed below. After setting the parameters,start the measurement with pushing the 0. ”Measurement” button,

1. ”Scan mode”

2. ”Objective lens”

3. ”Measurement area”

4. ”Measurement time”

5. ”Laser wavelength” and ”Laser power”

6. ”Spectral range”

0.

1.

2.

4.3.

6.

5.

Note）Setting controls for measurement area changes

accoriding to the scan mode.

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Figure 3.14:

3.3.1 ”Scan mode”

There are eight scan modes as below (including two optional scan modes.)

1. ”Point”・・・　 A spectrum at a point is measured.

2. ”x-Line”・・・Plural spectra at plural points along a line are measured.

3. ”xy-imaging”・・・Two dimensional xy spectrum imaging.

4. ”xz-imaging”・・・Two dimensional xz spectrum cross-sectional imaging.

5. ”Point-z”・・・Plural spectra at a point with different height are measured.

6. ”xy-mapping”・・・Two dimensional xy spectrum mapping.

7. ”xy-z-imaging”(Optional)・・・Plural xy-spectra imaging are measured withz scanning.

8. ”xy-z-mapping”(Optional)・・・Plural xy-spectra mapping are measured withz scanning.

Note) ”xz-imaging”, ”Point-z”, ”xy-z-imaging”, ”xy-z-mapping” measurementsare possible for the transparent samples where the laser light and Raman scatteredlight can travel.

Note) ”xz-imaging”, ”Point-z”, ”xy-z-imaging”, ”xy-z-mapping” measure-ments are not provided for the Product which does not equipped with a motor-ized Z stage.

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z:Stage Scanx

z

y

x

x

x

xz-imaging

xy-imaging

x-line

Point

Point-z

z:Stage Scan

z

x

y

Figure 3.15:

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xy-mapping

xy-z-imaging xy-z-mapping

y

x

xy-images

x

y

z

z=1.0

z=2.0

z=4.0

z=5.0

z=3.0

xy-maps

x

y

z

z=1.0

z=2.0

z=4.0

z=5.0

z=3.0

z:Stage Scan

z

z:Stage Scan

z

Figure 3.16:

3.3.2 ”Objective lens”

The objective lens which are used currently should be selected. The correctsetting of the objective lens give correct information about the laser power andthe scale (size).

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Figure 3.17:

■ Attention）The measurement can star with the wrong objective lens selection.In the case, the scale information and laser power information recorded in themeasurement data are different from the one of the true objective lens.

♣ Tips 1. Parfocality and Working distance（W.D.）

The focal points of the microscope are the almost same among the differentobjective lenses. For Nikon microscope, the focal point is designed at the 60 mmfrom the mounting position of the objective lens (Parfocal length). For Olympusand Zeiss microscope, the parfocal length is designed as 45mm. Objective lenseshas parfocal design

Sample

Parfocal length60mm

W.D.

Figure 3.18:

♣ Tips 2. Numerical aperture (N.A.) and the spatial resolution of microscope.

The performance to observe small structure is expresses as spatial resolution.The resolution is determined not by magnification but the Numerical aperture ofobjective lenses.

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Spatialresolution = 0.61× λ/NA （λ：wavelength）Magnification, W.D. and N.A. of standard objective lenses are listed below.

Name Magnification N.A. W.D. (mm)LU Plan FLUOR EPI x5 0.15 23.5LU Plan FLUOR EPI x10 0.3 17.5LU Plan FLUOR EPI x20 0.45 4.5LU Plan FLUOR EPI x50 0.8 1LU Plan FLUOR EPI x100 0.9 1

3.3.3 Measurement Area

The Setting of ”Measurement Area” depends on the ”Scan mode”

• Point mode

A measurement point is specified with click-and-drag on the microscopemonitor which has 640 pix in X axis and 480 pix in Y axis. The pixcoordinate of the point is shown on the RAMAN Imager. The width ”w”and the height ”h” should be ”1”.

1.Click

Coordinate of

Measurement Area

Origin X: 640

Y: 480

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Figure 3.19: Setting the Measurement point of ”Point mode”

It is possible to input numerical values (an integer) of the pix coordinate.

• XY-imaging mode

A measurement area is specified as a rectangle area with click-and-drag

The laser line is illuminated along the x axis and scanned in y axis so thatthe whole rectangle area are scanned with laser beam.The length of laser line or width of the rectangle area is fixed. The lengthof the height of the rectangle can be set arbitrarily. With determining theheight, the scanning number ”Y step” is automatically determined so thatthe pixel sizes are the same in X axis and Y axis. The operator can alsospecify the scanning number by inputing the integer value.

Click and drag

Measurement Area

Scanning setting

Coordinate X: 640

Y: 480

W (Wdith, fixed)

H

(Height, arbitrary)

Figure 3.20: Setting a XY imaging area

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Figure 3.21: Scanning direction of line laser. Scanning step width ”dy”,Scanning step number ”Y step”.

• XZ-imaging mode

1. Determining a line area on the microscope monitor.The laser line will be scanned inside the sample at the determinedline area in xy coordinate as the result of Z stage movement.

2. Reseting the coordinate in z axis so that the current position is 0.

3. Determining the ”Start position (Z Start)” and ”End position (Zend)” of the stage movement.”Start position (Z Start)” and ”End position (Z end)” can be inputwith the ”Read” button that inputs current position to ”Start position(Z Start)” or ”End position (Z end)”. Operator can also input thenumber directory with a keyboard.

4. Determining the ”Scanning number (Z step)””Step width (dz)” is automatically calculated following the equationbelow.(”Startposition”− ”Endposition”)/(Scanningnumber + 1)

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1. Click and drag

Z scanning setting

2.Current position in

Z and reset the origin.

1.Position of laser

3.

4.

Figure 3.22: Setting a XZ imaging area

• Point-Z mode

A measurement point is be specified with a mouse click on the microscopemonitor. Spectra at the point are measured while the stage is scanned inthe same manner with ”XZ-imaging”.”Start position (Z Start)”, ”End position (Z end)” and ”Scanning number(Z step)” are set in the same manner with ”XZ-imaging”.”Start position (Z Start)”, ”End position (Z end)” and ”Scanning number(Z step)” are set in the same manner with ”XZ-imaging”.

• X-line mode

Determining a line area on the microscope monitor. Spectra along the lineare measured simultaneously.

• XY-mapping mode

A measurement area is specified as a rectangle area with click-and-drag

A point laser is scanned in the rectangle area with the raster scan andwhole spectra in the rectangle are are measured.

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The operator should determines ”Width” and ”Height” of the rectanglearea, and ”Scanning step distance (nm)” before the XY-mapping measure-ment. The scanning number in X and Y direction are automatically calcu-lated from the ”Width”, ”Height” , and ”Scanning step distance (nm)”

Delta

Figure 3.23: The direction of the raster scanning and Scanning step dis-tance (nm) ”Delta”

• XY-Z-imaging

A measurement area is specified as a rectangle area with click-and-drag inthe same manner with ”XY-imaging”. The scanning number ”Y step” isautomatically calculated. Afterwards, ”Start position (Z Start)”, ”End po-sition (Z end)” and ”Scanning number (Z step)” are should be determinedin the same manner with ”XZ-imaging”.

■ Note）The folder for automatic data-save function should be set beforeXY-Z-imaging measurement. (Refer to the section, ”Automatic Data-SaveFunction”)。A number of XY-image data, that is the same number of Z scanning

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number, are generated and automatically saved and closed at every XY-Z-imaging measurement.

⋆Hint) The number of XY-image data of XY-Z-imaging measurementcan be reconstructed as 3D (XYZ) data of Raman image via ”3D DataGenerator” function. (Refer to the Manual for Data Viewing and Analysis)

Scan Settings in Z

Scan Setting in YScan Setting in Y

XY Measurement Area

Figure 3.24: Scanning setting of XY-Z-imaging

• XY-Z-mapping

A measurement area is specified as a rectangle are with click-and-drag inthe same manner with the ”XY-mapping”. ”Width”,”Height” and ”Scan-ning step distance (nm)” should be determined. Afterwards, ”Start posi-tion (Z Start)”, ”End position (Z end)” and ”Scanning number (Z step)”are should be determined in the same manner with ”XZ-imaging”.

■ Note）The folder for automatic data-save function should be set beforeXY-Z-mapping measurement. (Refer to the section, ”Automatic Data-Save Function”)。A number of XY-mapping data, that is the same number of Z scanning

46

number, are generated and automatically saved and closed at every XY-Z-imaging measurement.

⋆Hint) The number of XY-mapping data of XY-Z-imaging measurementcan be reconstructed as 3D (XYZ) data of Raman image via ”3D DataGenerator” function. (Refer to the Manual for Data Viewing and Analysis)

XY measurement area

Scanning step

Scanning settings in z

Figure 3.25: Scanning setting of XY-Z-mapping

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Figure 3.26: 3D (XYZ) Data reconstruction with ”3D Data Generator” ofRAMAN Viewer

3.3.4 Measurement time

Measurement time is automatically calculated from the ”Scanning number” and”Exposure time/shot”.

Scanninng number n

Total measurement time

Exposure time

Figure 3.27: Measurement time

The value between 0.01 sec and 1800 sec can be input for ”Exposure time/shot”

”Scanning number n” is automatically calculated when the measurement areis set. The scanning number of ”Point” and ”X-line” is one.The scanning number is ”Y step” for ”XY-imaging”, ”Z step” for ”XZ-imaging”and ”Point-Z”.

The measurement time is calculated according to the equation below. (Measurementtime) =(Exposuretime/shot) + (Datatransfertime)× n

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”Data transfer time” is the time in which the signal of cooled CCD detectoris transfered to the computer.

■ Note）”Data transfer time” depends the pixel number of the detector, ADconversion speed and the spec of the computer. As indication, it takes about 0.5second for the standard detector format (400x1340pix), standard AD conversionsetting (2 MHz) and the standard computer for RAMANplus.

■ Note）The equation for the measurement time should be modified whenthe setting of averaging, cosmic ray filter, binning and spectral ROI.

⋆Hint) For the beginner, the measurement time between 1 sec and 10 sec isrecommended for the start. However the measurement time over 1000 sec canstill be set for some cases.

3.3.5 Laser wavelength

Laser wavelength can be selected in the ”Laser Setting Window”. The windowis shown by clicking the menu, ”Menu/Function/Laser.”

Laser Setting Window

Selection of wavelength

Current wavelength

49

Figure 3.28: Laser setting window

The shortest wavelength should be selected from two reasons.

1. 　 Raman scattering efficiency

Raman scattered light is excited more efficiently with the laser of shortwavelength than long wavelength. The efficiency is proportional to the 4thpower of inverse wavelength.

Raman scattering efficiency ∝ 1/λ4

For example, the efficiency for 532nm-laser excitation is 4.8 times for785nm-laser excitation.

2. 　 Spatial resolution

Laser focusing ability depends on the laser wavelengths. Shorter wave-length, smaller focusing spot and higher resolution.

Spatial resolution = 0.61× λ/NA (NA:Numerical aperture)

The reason of the selection of longer wavelength is to avoid fluorescence ordecrease

Because it can not be known if the fluorescence signal is dominant or notbefore measurement, it is reasonable to start with the laser of short wavelength.After judging that the fluorescence signal should be decreased, the longer wave-length may be selected.In general, samples colored tend to emit the fluorescence rather than Ramanscattered light.The laser of longer wavelength does not have to avoid the fluorescence.

When Raman Imager starts, the laser of shortest is selected.

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3.3.6 Laser power

Laser power at a laser focus spot is shown at ”Laser power” monitor. In general,laser power should be between 0.5 mW and 10 mW. For samples tough to laserdamage, the laser power can be adjust more than 10 mW to obtain better datawith shorter measurement time.

The laser power on the monitor is shown after calibrated with the transmissionof objective lenses.

There are two ways to adjust the laser power.

1. Laser output power from a laser headLaser output power from a laser head can be adjusted by changing theinjection current to the laser diode. The injection current is adjusted be-tween 0 % to 100 % on the laser setting window.

■ Note）Laser output power is not proportional to the injection current.And there is a threshold of the injection current to lase.

■ Note) The injection current of Standard 785nm laser can not be ad-justed. Only 100 % current is available.

2. Neutral density filterThe transmission is adjusted with 255 variable steps by ND filter.

■ Note）Please pay attention the laser adjustment when the scan mode ischanged.

There is much difference about the laser power between point illuminationmeasurement such as ”Point” mode and ”Point-Z” mode, and line illuminationmeasurement such as ”X-line”, ”XY-imaging” and ”XZ-imaging”. There is morethan double digits difference between point/line illumination under the same NDfilter and the laser injection current. It is because the laser poser is distributedalong the line and the laser power is shown as the calibrated power at at point.

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Laser power at sample

Injection current

ND filter

Figure 3.29: Laser setting window

x

Power is focused on one point.

Power is distributedalong a line.

Figure 3.30: The difference of the power at the point between point/line illumi-nation

3.3.7 Spectral range

Spectral range where Raman spectrum is measured is determined with groovenumber of ”Grating” and grating angle determined with ”Center wavenumber”.

The bigger number of the groove means higher spectral resolution but nar-rower spectral range.

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Spectral Range

Gratingto be selected

Center of Wavenumberto be set

Figure 3.31: Spectral range setting

Three gratings are installed with RAMANplus and can be selected from RA-MAN Imager. Gratings listed below are available.

Groove number Range (532nm ex.) Resolution Range(785nm ex.) Resolution2400gr/mm 450cm-1 1.5cm-1 NA NA1800gr/mm 1000cm-1 1.7cm-1 400cm-1 1cm-11200gr/mm 1300cm-1 2cm-1 500cm-1 1.2cm-1600gr/mm 2500cm-1 4cm-1 1300cm-1 2cm-1300gr/mm 4500cm-1 7 cm-1 2200cm-1 3.5cm-1150gr/mm 8000cm-1 14cm-1 4000cm-1 7 cm-1

The spectral range between 100cm−1 and 5000cm−1 will be determined ac-cording to the samples.

Note)Spectral ranges between excitation lasers differ because the wavenumber, wave-length and laser wavelength has the relation as below.

”Wavenumber (cm−1)” = 1/λex − 1/λ

lambdaex is the wavelength of the excitation laser. lambda is the wavelengthof Raman scattering light. The spectral range for the 785nm laser excitation is

53

one half of the range for 532nm laser excitation with the same grating.

■ Attention)The gratings of 1800gr/mm and 2400gr/mm can not be used for the laser of785nm.

Rayleigh light and the the cut off of spectral range at the lower side.

Spectrum lower than 80cm−1 can not be measured because the laser blockingfilter blocks the light of the wavenumber lower than 80cm−1. But the peakof Rayleigh light is observed at 0cm−1 when the range is set including 0cm−1

because the Rayleigh light is so strong so that a fraction of Rayleigh light comesthrough the filter partially. It is recommended that the measurement rangeexcludes the 0cm−1.

ZoomZoom

(A) Normarized at

Rayleigh light(B) Zoom in (B) Cut of in the

lower range

Cut of range

Figure 3.32: Rayleigh light and the the cut off of spectral range at the lowerside.

3.4 Starting measurement, and viewing and analyzing

data

After setting the measurement parameters, ”Measurement” button will be pushedto start measurement.

The procedures listed below are done automatically just after the start.

1. 　 Saving an optical microscope image.

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2. 　 Saving the measurement parameters.

3. 　 Switching off the transmission/reflection illumination light.

4. 　Starting ”RAMAN Viewer” software with which measured data is shownand analyzed.

5. 　 Laser irradiation of the sample and recording the signal.

6. 　Switching on the transmission/reflection illumination light after finishingthe measurement.

The window type of RAMAN Viewer shown with the measurement start de-pends on the ”Scanning mode”. The data with RAMAN Viewer can be operatedeven while the measurement is proceeding.Please refer to a Manual for RAMAN Viewer.

λ

n

1

Window for point mode measurement

X-ling/Point-Z XY-imaging/XZ imaging

Figure 3.33: ”RAMAN Viewer” software with which measured data is shown andanalyzed.

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3.5 Surface profiling measurement

1. 　Clicking the ”Surface” tab to show the ”Surface profiling setting panel”.

Click

Surface plot panel

Figure 3.34: Surface profiling setting panel

2. 　 Setting the scanning range of the surface profile in z axis.

The upper limit and lower limit in z scanning should be determined fromthe microscope image with moving the sample stage in z axis. At theupper limit of the stage, ”Read” button should be pushed to input theupper limit position to ”Z:Start”. At the lower limit of the stage, ”Read”button should be pushed to input the lower limit position to ”Z:End”.Afterwards, ”Z Scanning Pitch” should be selected. Scanning step numberis automatically determined.

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0. Initial status

Microscope image

1. Setting start

position of Z scan

2. Setting end

position of Z scan3.Setting step width

Read Z start

Read Z EndZ Scanning Pitch

Figure 3.35: Setting the scanning range of the surface profile in z axis

3. 　Switch to ”Real-Time Frame” mode. On the ”Real-Time Frame” mode,”ND filter” and ”Gain” should be adjusted so that the laser reflection lightfrom the sample is strong enough but is not saturated at any stage height.

1. Select “Real t-Time Frame”

2. Adjust “ND filter” and “Gain”

to maxmize intensity of refection image

and its dyanamic range without saturation

while manual z scannig.

3. Select averaging number.

Figure 3.36: Adjustment of ”ND filter” and the ”Gain”

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4. 　 Showing the surface profile data after the measurement.”Menu/Window/Surface Profile” should be selected.

Click

Menu/Window/Surface Profile

3D Surface Plot Window

Figure 3.37: The surface profile data

Note)The surface profile data with other data format can be done with the ”Menu/File/”.

⋆Hint)

Automatic focusing is possible after the surface profiling measurement. Onthe microscope monitor, jut right-click and select ”Set Focus here” so that thethe point at the cursor is brought into the focus.

3.6 Shutting down RAMANplus

1. Closing RAMAN Imager with clicking ”Close” button at the topright of the window.

2. Switching off the power supplying unit with turning the key switchleft.

3. Shutting down the computer.

■ Attention）The order of switching off should be 1. RAMAN Imager and2. the power supply unit.

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Figure 3.38: The shut down of RAMANplus

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Chapter 4

Software ”RAMAN Imager”

RAMAN Imager is the software controlling RAMANplus˙This chapter explains RAMAN Imager version 1.2 in detail.

4.1 Outline

4.1.1 Interface

There are three parts in the interface of RAMAN Imager. Each part with sectionsin detail will be explained as follows.

• Common control panelThe common control that is mainly for optical microscope is done in thispart.

• Measurement setting panelThe measurement parameters are set. The Measurement type is dividedwith the tab panel.

– Standard Raman Measurement,”RAMAN”

– Surface profiling measurement,”Surface”

– Raman imaging in an wide-field area, ”WF-Raman (Optional)”

– Optical microscope observation in an wide-field area,”WF-Observation(Optional)”

– Multi-well (m x n) automatic measurement, ”m x n(Optional)”

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• Menu”Menu” provides with other settings and functions.

– File

– Function

– Window

Menu

Common control panel

Button for quit

Measurement

setting panel

(tab select)

Figure 4.1: The interface of RAMAN Imager

4.1.2 How to start RAMAN Imager

1. Turning on the Computer.

2. Turning on the power supply unit.note) The sequence of turning on the computer and the power supply unitcan be converse.

3. Starting RAMAN Imager with double-clicking the icon of RAMANImager.

RAMAN Imager starts to initialize devices in RAMANplusWhile initializing,only the progress bar of the initialize is shown. After finishing the initialize,

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the main window will show up. It takes several minutes by the end of theinitialize and main window showing up.

■Attention）Because RAMAN Imager communicates the devices, it is nec-essary to turn on the Power supply unit first.

Figure 4.2: The icon of RAMAN Imager

Figure 4.3: The main window of RAMAN Imager just after showing up

4.1.3 How to quit RAMAN Imager

1. Closing RAMAN Imager with clicking ”Close” button at the topright of the window.

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Attention）It is necessary to quit RAMAN Imager before turning off thepower supply unit. Otherwise the computer will hung up because of theinterception between RAMAN Imager and devices.

Figure 4.4: How to quit RAMAN Imager

4.2 Common control panel

Appellations of parts in the ”Common control panel” are as below.

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1. Miroscope monitor

2.Illumination contoroller for microscope

3. Z stagecontroller4. Memorandum

to be saved

5.Display assist

Figure 4.5: Common control panel

1. Optical microscope monitorThe optical microscope monitor shows the microscope image of samplestaken with a CCD camera.

2. Illumination controller for microscope observation

• ”Transmission lamp”The controller for illumination of transmission observation. This iscontrolled with 255 steps.

• ”Reflection lamp”The controller for illumination of reflection observation. This is con-trolled with 255 steps.

3. MemorandumThis is the field that an operator write down memorandum about mea-surement condition. The memorandum with measured data is transferedto data of RAMAN Viewer at every measurement.

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• ”Operator Name”

• ”Sample Description”

• ”Comments”

4. Z stage controller

• ”Up” and ”Down” buttonsThese buttons move the stage with the distance of ”Step”.

• ”Step” boxTravel distance of one push of ”Up” and ”Down” buttons. The unitis µm.

• ”Pos”This box shows the relative height of the stage.

• ”Z Rest” buttonThis button resets the origin of the stage height and inputs ”0” µmin ”Pos”

• ”Focus Handle” buttonThis button switches the speed of the z stage movement with threesteps.

5. Display assistant

• ”Scale”This check-box controls the show/hide of the scale bar on the micro-scope monitor.

• ”Cursor”This check-box controls the show/hide of the cursor indicating laserirradiation position on the microscope monitor.

6. XY stage controller（Optional）If a motorized XY stage is equipped, the xy position is controlled.

4.3 Measurement setting panel

The panel of Standard Raman measurement ”Raman” is main panel. In addi-tion, There are Surface profiling measurement ”Surface”, Raman imaging in an

66

wide-field area ”WF Raman”, microscope observation of an wide-field area ”WFObservation” and automatic multi well (m × n) measurement ”m × n (Automulti-well measurement)”. These panels are selected by clicking the tab locatedat the top of the panel.

Figure 4.6: Switch of the measurement panel with the tab

4.3.1 Raman

Measurement parameters for Raman measurement are set on the ”Raman” panel.

1. Measure button

2. Scan mode

3. Obj.lens

4. Measurement time

5. Laser

6. Spectral setting

7. Cursor position

8. Scan setting

Figure 4.7: The ”Raman” panel

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1. ”Measure” buttonThis button starts the measurement according the measurement parame-ters.

■ Note)There are cases when the text on the button is not ”Measure”.The text is ”Start for trigger” or ”Wait for trigger” when trigger functionis used. (Please refer to the section of ”Trigger measurement”) ）The text is ”Measure Ne Spectrum”, ”Measure Si Spectrum” and so onwhen the ”Reference sample” function is used. (Please refer to the sectionof ”Reference sample”）

2. ”Scan mode”Please select the Scan mode.

4.3.2 ”Scan mode”

There are eight scan modes as below (including two optional scan modes.)

(a) ”Point”・・・　 A spectrum at a point is measured.

(b) ”x-Line”・・・Plural spectra at plural points along a line are measured.

(c) ”xy-imaging”・・・Two dimensional xy spectrum imaging.

(d) ”xz-imaging”・・・Two dimensional xz spectrum cross-sectional imag-ing.

(e) ”Point-z”・・・Plural spectra at a point with different height are mea-sured.

(f) ”xy-mapping”・・・Two dimensional xy spectrum mapping.

(g) ”xy-z-imaging”(Optional)・・・Plural xy-spectra imaging are measuredwith z scanning.

(h) ”xy-z-mapping”(Optional)・・・Plural xy-spectra mapping are measuredwith z scanning.

3. Objective lens selection box　”Obj. lens”The objective lens which are used currently should be selected. The correct

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setting of the objective lens give correct information about the las

4. Measurement time

• Exposure time ”Exposure s/shot”The exposure time that is equivalent to laser irradiation time at everyscan should be input.

• Total measurement time　”Total” Total measurement time is shown.”h”,”m” and ”s” mean the unit of hour, minute, second.For ”Point” mode measurement, the total measurement time is equalto the exposure time.

For ”XY-imaging” mode, the total measurement time is equal to theexposure time multiplied with the scanning number.

5. Laser

• Laser power monitorLaser power irradiated to the sample is shown with ”mW” unit.The laser power is measured with the power meter equipped insidethe instrument and is shown in real time. The displayed laser power isthe value after calibration with the transmission of the optical systemand the objective lens.

♦ The laser power when the laser line beam is used is the value ateach point along the line. The calibration coefficient is derived fromthe ratio of the spectral peak intensity of Silicon crystal measuredwith the line and point focus of the laser.

• Laser wavelength displayThe wavelength of the laser used to excite Raman scattering currentlyis displayed.

• Laser injection current ”Current”The lasing power can be controlled with the laser injection current.The unit of the current is %.

• ND filter controller ”ND filter”The laser irradiation power to the sample can be controlled with

69

variable ND filter. The power is determined with the transmission ofthe NF filter. The transmission is controlled wit the ”ND filter” sliderwith 255 steps.

• ND filter setting monitorThe ND filter setting is shown as the ”Transmission” and the positionindex of 255 steps.

• Check box ”Check Laser Spot”Checking this box opens the mechanical shutter stopping the laserbeam and irradiates laser beam on to the sample. This function maybe used to observe and confirm how the laser irradiation is made onthe sample surface.The checking laser beam is convenient when the operator brings thesample in the focus.

6. Spectroscopy setting

• ”Spectral Range”The range of measured Raman spectrum is shown.

• ”Center Wavenumber” and ”Set” buttonTo set the ”spectral range”, the center wavenumber of the ”spec-tral range” should be input in the ”Center wavenumber” box andafterward the ”Set” button should be pushed.

• ”Grating” Selection boxA grating among three gratings can be selected. By choosing thegrating, the motorized grating turret will rotate and the grating choseis set.

7. Cursor position ”Position (x: pix, y : pix)”

• x position ”x”, y position”y”The coordinate of the cursor position indicating laser focus spot isshown. The unit of the coordinate is pixel of the microscope monitor.The width and height of the monitor are 640 pixel and 480 pixelrespectively.The coordinate of the top and left is shown in the case that the cursoris rectangle area or line shape for the XY-imaging, X-line mode andso on.

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• Scanning area boxThe width and height of scanning area is shown in the coordinate ofthe microscope monitor.

8. ”Scan Setting”The scanning setting parameter panel changes according to the scanningmode.point

• No setting parameters.

• No setting parameters.

xy-image

• ”Y step”The scanning number in y direction is automatically input when thescanning area is changed by the click-and-drag on the microscopemonitor. The number is calculated so that the pixel size in x directionis equal to the size in y direction.The operator can also input any integer number manually.

• The scanning interval in y direction ”nm”The unit is ””nm”.

xz-image

• ”Z step”The scanning number in y direction is automatically input when thescanning area is changed by the click-and-drag on the microscope

71

monitor. The number is calculated so that the pixel size in x directionis equal to the size in y direction.The operator can also input any integer number manually.

• The scanning interval in z direction ”nm”The unit is ””nm”.

• ”Z Start” and ”Read” buttonThe beginning position of the z scanning should be input in the unitof µm. Pushing button inputs the current position to the ”Z Start”.

• ”Z End” and ”Read” buttonThe end position of the z scanning should be input in the unit of µm.Pushing button inputs the current position to the ”Z End”.

point-z

• ”Z step”The scanning number in y direction is automatically input when thescanning area is changed by the click-and-drag on the microscopemonitor. The number is calculated so that the pixel size in x directionis equal to the size in y direction.The operator can also input any integer number manually.

• The scanning interval in z direction ”nm”The unit is ””nm”.

• ”Z Start” and ”Read” buttonThe beginning position of the z scanning should be input in the unitof µm. Pushing button inputs the current position to the ”Z Start”.

• ”Z End” and ”Read” buttonThe end position of the z scanning should be input in the unit of µm.Pushing button inputs the current position to the ”Z End”.

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XY-mapping

• ”delta”The scanning interval of XY-mapping is input. The unit is ”nm”.

• ”x”,”y”The scanning numbers in x and y direction respectively are shown.With the input of the interval, the scanning numbers are calculatedfrom the interval and the area the width and height automatically.

4.3.3 Surface(Optional)

4.3.4 Wide-field Raman measurement ”WF Raman”(Optional)

The measurement parameter of wide-field Raman measurement is set in the”WF-Raman” measurement panel. This function is provided with the motorizedxy-stage option.The measurement procedure is explained in the chapter of the measurement.

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1. Measurement button

2. Measurement area

3. Obj. lens

4. Measurement time

5. Quality of WF-image

(resolution)

6. Information

of WF image

7. Buttons for checking the measurement area

8. Setting for

motorized XY stage

Figure 4.8: WF-Raman setting panel

1. ”Measure WF Raman” buttonThis button starts the wide-field Raman measurement according to thesettings.

2. ”Measurement Area”The measurement area of the wide-field is determined by setting the startposition and the end position. The start position should be top left andthe end position is the right bottom of the rectangle area.

• ”Read To Start” buttonThis button sets the current position as the start position.

• ”Read To End” buttonThis button sets the current position as the end position.

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• ”Start” boxThis box shows the start position in the coordinate. The unit is”mm”.

• ”End” boxThis box shows the end position in the coordinate. The unit is ”mm”.

3. ”Obj. lens”The objective lens used currently should be selected. By selecting theobjective lens, the scale bar on the monitor is updated. The laser powermonitor is also updated described later..

4. Measurement time

• ”Exposure s/shot”The signal recording time, equivalent to the laser irradiation time, atevery scanning is input.For the point mode, the exposure time is equal to the measurementtime.

• 　”Total” measurement timeThe total measurement time is calculated by the exposure time andthe scanning number. ”h”,”m” and ”s” are the units of hour, minuteand second respectively.

5. Image ”Quality” of the wide-field measurement (the resolution)The image quality is determined automatically not to exceed the memoryof the computer. When the are is large, the pixel of the wide-field image isbinned in x direction. The scanning number in Y direction is automaticallydetermined so that the pixel size in x and y direction are the same.

• FineThe total pixel number is 160,000.

• StandardThe total pixel number is 40,000.

• CoarseThe total pixel number is 10,000.

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• CustomThe operator can set the total pixel number.

6. The information of the wide-field image.The information of the wide-field image is shown.

• ”Pixel size”The ”Width” and the ”Height” are shown in the unit of µm.

• ”Pixel Number”The pixel number of the ”Width” and the ”Height” are shown.

7. Measurement area confirmation buttonThis button is used to confirm the measurement area.

• ”Move to Start” button

• ”Move to END” button

8. The setting of the motorized xy stage.

• ”Move Speed”The speed of the wide-field scanning is set.

– 1 mm/s

– 0.01 mm/s

– 0.05 mm/s

– 0.005 mm/s

• ”Move Stop”This button stops the motorized stage moving.

4.3.5 Widefield observation ”WF Observation” (Optional)

WF observation is used to observe and record the wide-field microscope image.The microscope images in the widefiled area recorded while the xy stage scanningand are stitched to one large image.On the stitched wide-field image, measurement area of WF-Raman can be de-teremined.

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1. Measurement buttons

2. Obj.lens

3. Measurement Area

4.WF-Raman Measurement Area

5.Button for showing

WF-microscope image

monitor

6.Setting formotorized XY stage

”WF Observation” panel

1. Buttons for measurement

• ”Measure Wide Field Picture”

• ”Measure WF Raman”

2. Obj. lens■ The correct objective lens should be selected for the stitching.

3. Setting measurement area. The wide-field microscope image are generatedfrom ”m” by ”n” microscope images. ”m” is the number of images in xaxis. ”n” is the number os images in y axis. The orgins of these ”m” by”n” images are defined as the software Origin (SO).

• ”Reset SO”This button sets current position as the software origin.

• Origin setting

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– CenterThe software origin is located at the centerof the wide-field mi-croscope image.

– TL (Top Left)The software origin is located at the top and left of the wide-fieldmicroscope image.

– TR (Top Right)The software origin is located at the top and right of the wide-field microscope image.

– BL (Bottom Left)The software origin is located at the bottom and left of the wide-field microscope image.

– BR (Bottom Right)The software origin is located at the bottom and right of thewide-field microscope image.

• 　 Setting unit

– 　 FrameThe unit is the number of microscope images.

– mmThe unit is the distance. The distance will be tranlated to thenumber of microscope images automatically.

4. Setting measurement area of WF-RamanThe measurement area of WF-Raman can be set by click-and-drag on thewide-field microscope iamge. The measurement area of WF-Raman will beshown as the coodinates of the ”Start” position and the ”End” position.

5. Displaying wide-field microscope image

• ”Display Wide Field” check boxAfter measuring the wide-field microscope image, the image is dis-played on the microscope monitor. Uncheking this box changes themiroscope monitor from the measured wide-field microscope imageto real-time microscope image.

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• ”Display realtime monitor” buttonThis button shows the another microscope monitor for real-time mi-croscope image while the measured wide-field microscope image isdisplayed on the microscope monitor.

• ”Display Image Zoom” sliderThis slider adjusts the zoom of the wide-field microscope image.

• ”Display position”The current field view position of real-time microscope is shown asthe red rectangle while the wide-field microscope image is shown.The click-and-drag of the red rectangle area moves the zoomed areashown in the microscope image.

• ”Display position zoom” sliderThis slider adjusts the zoom of ”Display position”.

6. The control of motorized stage

• ”Go To SO” buttonThis button moves to the software origin.

• ”Find HO” buttonThis button finds and moves to the hardware origin.

• ”Move Stop” buttonThis button stops the movement of the motorized stage.

• ”Move Speed” boxThe speed of the stage movement of WF-Raman and WF-observationcan be selected.

– 1 mm/s

– 0.01 mm/s

– 0.05 mm/s

– 0.005 mm/s

4.3.6 Auto well-plate measurement(m × n )（Optional)

Auto well-plate measurement if m × n wells is done in this panel. This functionis provided optionally. The number of m and n are set according to the stageand the well type.

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The procedure are explained later,

1.Button forstarting measurement

2. Setting statusof wells

3.Obj.lens

4.Scan mode

5. Measurement time

6. Sequence

7. Measurement area

8. Sample name

9. Buttonfor applying the setting to all

10. Button for stopping thestage movement

The panel of Auto well-plate measurement(m × n ).

1. ”Start mxn measurements” button

2. Overviews of wells.The overview of well plate and the status of each well setting are shown.

• The status of the wells The status of the wells are shown by thedifferent color. The well sell is selected by the click and turns tobe ready for setting the parameter. The double-click moves currentposition to the well.

– WhiteThe measurement parameter setting is not done.

– PinkThe selected well for the parameter setting.

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– 　 BlueThe measurement parameter setting is done.

• Select

• ”Apply” button

• ”Clear” button

3. Objective lens

4. Scan modeThe scan mode should be selected for the current selected well.

5. Measurement time

• ”Exposure (s/shot)” for the selected well.

• ”Time / a sample” for the selected well.

• ”Total time” for measurement of all wells.

6. Sequence of the wellThe Sequence of the well should be selected. The white well will be skipped.

7. Measurement areaThe measurement area for the selected well is shown.

8. ”Sample Name” boxThe name of the sample in the selected well can be input and is shown.The name will be saved with the data.

9. Setting at once

• ”Apply to all samples” buttonThis button applies the parameter in the selected button to all othersamples.

• ”Clear all samples” buttonThis button clear the setting of all wells.

10. ”Stop” buttonThis button stops the movement of the motorized stage.

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4.4 Functions in the ”Menu”

4.4.1 The outline of the ”Menu”

There are three functions in the ”Menu”. These are ”File”,”Function” and”Window”.

1. File

• Save Microscope ImageThis saves the microscope image on the microscope monitor to animage file.

• Save Real-Time FrameThis saves the real-time confocal reflection image (Plus) to an imagefile.

• Save Surface ProfileThis saves the surface profile image measured with the Plus functionto an image file.

• Save Extended depth image This saves the extended depth imageimage measured with the Plus function to an image file.

• Save/Load SettingsThis shows the ”Save/Load Settings” windows. The measurementparameters are saved or loaded with this function.

2. Function

• LaserThis shows the laser setting window. From the window, the wave-length of the laser can be selected. The laser output power can alsobe adjusted by the injection current.

• Image Quality & Spectral ROIImage binning and spectral ROI are set for the purpose of the com-pressing the data size, the improvement of the data quality and theshortening of measurement time.

• Auto Save SettingsThis functions determines the auto-saving setting after measurement.

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• Trigger SettingThis function enables time-course measurement (”Internal trigger”mode). This function also provides the measurement with combina-tion with external devices (”External trigger” mode).

• Data Calibration and CompensationThis function provide the setting of data calibration and compensa-tion.

• AssistThis includes the supplementary functions.

• Reference SamplesThe reference smaples equipped inside RAMANplus are utilized throughthis function.

• Device/SpectrographSpectrograph setting is provided.

• Device/CCD DetectorThermo-electrically cooled CCD detecotor setting is provided.

• Device/Illumination for Optical MicroscopeThe on/off controll setting of microscope transmission/reflection set-ting.

• Device/Microscope Z stageThe motorized z stage settinn is provided.

3. Window

• Detector TemperatureThis shows the ”Detector Temperture” widnow.

• Stage controllerThis shows the ”Motorized stage control” widnow (optinal).

4.4.2 ”File/Save・Load Settings”

”Menu/File/Save・Load Settings””Save・Load Settings” will be shown.

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Figure 4.9:

1. Name list of Measurement paramtersFor saving the current measurement parameter, the name of ther para-menter into the list. For loading the measurement paramter to the currentmeasurement parameter, select the parameter name from the list.

2. ”Load” buttonThis button loads the parameter on the list to the current measurementparameter.

3. ”Save” buttonThis button saves the current measurement parameter to the list with theparameter name.

4. ”Delete” buttonThis button deletes the selected parameter on the list.

4.4.3 The wavelength and the output power of the laser ”Func-tion/Laser”

”Menu/Function/Laser”The laser setting window is shown

1.Seelction of wavelength 2.Laser power at sample

3.Laser4.Injection current control

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Figure 4.10:

1. ”Selected wavelength” boxThe laser wavelegths that are quipped in RAMANplus are listed. Afterselecteing the wavelength, laser waveleght will start to be chaged. It taksea few ten seconds to finish changing the wavelength.

2. Laser power monitorLaser power on the sample is shown when the laser is irradiated.

3. Lasing laserThe check box of the laser under lasering shows the checked status. Check-ing the box of laser under no lasing makes the laser lasing.

■ Some of the lasers need a few minutes to lase after checked.(e.g.785nmlaser)

4. Injection current controlThe injection current can be contolled to adjust the laser outpout power.After moving the slider, the ”Set” button should be pushed to set thecurrent.

■ Some of the lasers have no injection current control.(e.g. 785nm laser)

4.4.4 Image Quality & Spectral ROI ”Function/Image Quality &Spectral ROI”

”Menu/Function/Image Quality & Spectral ROI””Image Quality & Spectral ROI” window is shown.

This function provides the setting about the data size, spectral and imagequality.

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Figure 4.11:

Image Quality

The image quality in ”xy-imaging”, ”xz-imaing” mode and so on, can beadjusted to decrease the data size and improve the spectral quality. 400 spectraalong a line illumination are measured simulataneously by default (”Fine”). Inthe case of ”Standard”, 200 spectra are measured by combining two neighborspectra to one spectrum.♣ It is called ”binning” that plural pixel are combined to one pixel.Combiningplural pixel increas the sigal at a pixel. Because the number of read-out (ADconversion) is decreased, the read-out noise is also decreased.

• Fine400 spectra per a line. No binning is done.

• Standard200 spectra per a line. 2 pixel binning is done.

• Coarse100 spectra per a line. 4 pixel binning is done.

• CustomThe number of the binning is set arbitrarily.

Spectral ROIThe spectral range is trimmed to decrease the data size.

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♣”ROI”とは”Region of Interest”の略です。

• Range input box, ”From to”Trimmed spectral range shown in ”From to”.

• ”Add” buttonThis button shows range input window.

• ”Delete”buttonThis button deletes the selected range.

4.4.5 Auto Save Settings ”Function/Auto Save Settings”

This function sets auto-save parameters.

”Menu/Function/Auto Save Settings””Auto Save Settings” is shown.

Figure 4.12:

Auto-save function works differently according to the scan mode.

• x-line, xy-imaging, xz-imaging, xy-mapping, point-zAll paramters for the auto-save function are available.

• point modeThe name setting is used for the measured spectral name. The Multi-Spectral View data is not saved automatically.

• xy-z-imaging, xy-z-mappingFor these measurements, the folder setting for auto-save is necessary. Themeasured data are always save and closed automatically.

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• The case with trigger function(refer to the next section)For the measurements with the trigger function, the folder setting for auto-save is necessary. The measured data are always save and closed automat-ically.

Setting items of auto-save

• ”Auto Save” check boxAuto-save fuction is enbale/disable.

• ”Auto Close” check boxAuto-close fuction is enbale/disable. Auto-close is available only when theauto-save function is enable.

• ”Save Folder”The folder for the save should be selected.

• ”File Name Format” The naming rule for the auto-save is selected. Thename is determiend with the combination of 4 paramters.

4 paramters for the naming

• ”Scan mode”

• ”Sample Name”

• ”Operator Name”

• ”Date Time”

4.4.6 Trigger function ”Function/Trigger”

This function enables time-course measurement (”Internal trigger” mode). Thisfunction also provides the measurement with combination with external devices(”External trigger” mode).

”Menu/Function/Trigger”Trigger settin window is shown

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Figure 4.13:

External trigger mode starts measurements with the trigger signal fromthe external device.The trigger signal is neglected while measurement. The next measurement startswith the trigger while the idling time.

The TTL singal is acceptable for the trigger through the BNC connector(Trigger port) behind the power supply unit.

Internal trigger mode starts to repeat measurements with the constantinterval ”Cycle(ms)” using the clock inside the computer.

• Trigger mode selection button

– ”Trigger off”

– ”External trigger”

– ”Internal Trirgger”

• Measurement interval ”Cycle time”This determines the interval of the internal trigger mode.

• ”Sampling count”The number of measurement of the trigger mode should be set. With thenumber of ”0”, the measurement is repeated endlessly till ”Stop measure-ment” is pushed.

Measurement procedure with trigger mode.

1. Setting

Setting of External trigger or internal trigger on the trigger setting window.

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2. Stopped state

Closing the trigger setting window. The description on the ”Measurement”button changes to ”Start Waiting Trigger”. With the status of ”StartWaiting Trigger”, the measurement will not start when internal/externalsingal is detected.

3. Idling stateChanging to idling state by pushing ”Start Waiting Trigger” button. Themeasurement will not start while the trigger signal does not come. Afterpushing the button, the description will change to ”Stop Waiting Trigger”.

4. Measurement stateAt the moment when the trigger signal comes, the measurement startsautomatically. While the measurement, the trigger signal is ignored.

5. Idling stateAfter the measurement, the idling state restarts. At the same time, themeasured data will be saved and closed automatically.

6. Termination of the trigger mode To terminate the trigger mode, The ”StopWaiting Trigger” button should be pushed. Or the trigger mode is finishedwhen the the number of ”Sampling count” measurements are done.The description on the button will be ”Start Waiting Trigger” button.

通常測定モード

トリガー停止状態

トリガー待機 or

トリガー測定状態

Figure 4.14:

4.4.7 ”Function/Data Calibration and Compensation”

This function sets calibration data and compensation data.”Menu/Function/Data Calibration and Compensation”

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Figure 4.15:

The following three calibration and compensation data are set. These datashould be prepared in advance. The data are utilized as the data transfered tothe RAMAN Viewer with measured data.

• Wavelength calibration dataThis data is used as the wavelength data of Raman spectrum. Without thesetting, the wavelength data calculated from the data of the grain and itsangle automatically is used. With this setting, the wavelength data, thatthe operator prepare, is used.

• Spectral intensity compensation dataThe spectral intensity pattern is distorted with QE curves of detector andgrating, and transmittance curve of optics. No compensation is proceededwithout this setting.

• Flat-field compensation data.This data compensates the sensitivity difference between pixels of the de-tector. No compensation is proceeded without this setting.

4.4.8 ”Function/Assist”

”Menu/Function/Assist”

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Figure 4.16:

• ”Averaging number”This set the averaging number of measurement.

• ”Cosmic Ray Removal Filter”With this function, the measurement is done with removing ”cosmic raynoise”. This function makes measurement time longer.

• ”Beep sound”This function set the ”beep sound” informing the end of measurement. Nobeep sound is set as default. Two kinds of sounds are equipped.

• ”Optical Line Shaper”

As default, an optical element ”optical line shaper” is used to form theline shaped laser illumination for xy-imaging measurement so on. Without

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the optical line shaper, the quasi-line illumination, so-called flying spotillumination is utilized for xy-imaging.

Line illumination method Line length　 power at a point at a momentOptical line shaper Fixed Low

Flying spot Arbitrary High

• ”Wobbling” for the line illuminationWobbling is used with optical line shaper. Generally the intensity patternof line illumination by the optical line shaper is not uniform along theline. The high intensity at the center and the lower intensity at the edges.To obtain uniform intensity pattern with the optical line shaper, the non-uniform pattern is wobbled in the line direction (patented). The length ofthe wobbling is selected.

– Long

– Medium

– Short

• ”Automatic Shutter OPEN/CLOSE”The mechanical shutter stops laser irradiated to the sample while no mea-surement (CLOSE state). To irradiate the laser to the sample, the shuttershould be removed from the laser path (OPEN state). While the measure-ment, the shutter repeats OPEN and CLOSE state synchronizing with theexposure time as default.

With this function disabled, the shutter keeps CLOSE while measurement.This function is uses to measure the background signal of this instrument.

• ”Auto ND Filter Adjustment at Shutter OPEN/Close”

This function is auto-adjustment of ND filter when the laser is irradiatedto the sample by ”Check Laser Spot” button. As default, this function isenable and the ND filter is set at the position of the minimum laser power.”Set” button inputs current ND filter position as the position when theshutter is opened.

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4.4.9 Standard sample ”Function/Reference Samples”

Silicon crystal is equipped inside the instrument as a standard sample. Thespectrum of the Si can be measured through this function. The neon lamp isalso quipped and its spectrum can be measured.Other standard samples may be provided optionally. In the case the optionalstandard sample is transparent such as quartz, the spectrum of the sample onthe stage can be measured with the spectrum of the standard sample. Thestandard spectrum of the standard sample gives the exact information of theunknown sample.

”Menu/Function/Reference Samples”

Figure 4.17:

• Silicon crystal

• Neon lamp for the wavelength calibration.

• Quartz (optional)

4.4.10 ”Function/Device/Spectrograph”

”Menu/Function/Device/Spectrograph”

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The following three parameters can be set. The unit of spectrum is ”nm”.

• ”Grating”

• ”Range of wavelength”

• ”Slit width”60 µm width is set as default. Narrower slit width, higher spectral andspatial resolution. Wider slit width, bigger signal.

4.4.11 ”Function/Device/CCD Detector”

The setting of a cooled CCD detector. ”Menu/Function/Device/CCD Detector”

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Figure 4.18:

The following three parameters can be set.

• ”ADC Speed”AD conversion speed of the pixel of the CCD is selected between 2MHz and100kHz. Higher ADC speed, the bigger readout noise. 2 MHz is selectedas default.

– 2MHzIt takes about 0.25 second to transfer the signal of 400× 1340 format.( 400× 1340÷ 2000000 = 0.25)

– 100kHz It takes about 5 second to transfer the signal of CCD of 400× 1340 format. ( 400× 1340÷ 100000 = 5)

• ”Readout port”There are two readout ports.

– Low Noise(High Sensitivity)300,000e- (typ.), 250,000e- (Min)

– High Capacity1,000,000e- (typ.), 750,000e- (Min)

• ”Gain”It is the gain of CCD. It determines the number of electron generation fora single photon. The high gain is set as default.

– High16e- with High Sensitivity port, 4e- with High Capacity port

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– Medium8e- with High Sensitivity port, 2e- with High Capacity port

– Low4e- with High Sensitivity port, 1e- with High Capacity port

4.4.12 Automatic on/off of microscope illumination. ”Function/Illuminationfor Optical Microscope”

The microscope illumination both for reflection and transmission is turned offautomatically while the measurement. This setting can disable this automaticon/off. This is enable as default.

”Menu/Function/Device/Illumination for Optical Microscope”

Figure 4.19:

• 　 EPI LEDLED for reflection.

• Transmission LEDLED for transmission.

4.4.13 Motorized z stage ”Function/Device/Microscope Z stage”

”Menu/Function/Device/Microscope Z stage”

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Figure 4.20: The window for the setting of motorized z stage

• 　”Step width”The step width when ”Up”/”Down” button is pushed.

• ”Tolerance”Motorized Z stage equipped with linear encoder and moves with closedfeedback system. The tolerance can be set.

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Chapter 5

To understand Raman scanttering

It is imporant to understand of basis of Raman scattering to utilize the manykinds of functions of RAMANpluseffectivellly. In this chapter, It is explainedabout Raman scattering including the relation between molecular vibration andRaman spectrum, a concept of a spectral image, and peak shift imaging.

5.1 Raman spectrum

The acquirable data of RAMANplusis the Raman spectrum. Figure below de-picts a Raman spectrum of particles observed with an optical microscope. Themeasured area is given by the yellow crossed-cursor in the microscopic image.The spectrum accords well with that of polystyrene, the material of which theparticles are in fact part of. The Raman spectrum of any point on the samplecan be measured by simply clicking the mouse while observing a microscopicfield-of-view on a monitor.

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Polystyrene

Microscope image

Figure 5.1: The spectrum of polystyrene

5.2 Raman scattering

Incidental light gets scattered by materials (Figure below). If the incidentallight is of a single wavelength the scattered light is also of a single wavelength(Ex. Green incidental light leads to green scattered light). However, preciselyanalyzing the color of scattered light revealed extremely weak light whose colorslightly differs from that of the incidental light. This phenomenon is called Ramanscattering and the scattered light is Raman light, both of which are named afterthe discoverer of them, C. V. Raman.

Raman scattering is caused by the interaction between the incidental light andmolecular vibrations of a material. When the incidental light gets scattered bythe molecular vibrations the light gains (or loses) amounts of energy correspondto the molecular vibrations and the wavelength of the scattered light shifts fromincidental. The relationship between the energy and wavelength of light can beexpressed with the following equation:

E = hν = hc/λ(E: energy of light, λ: wavelength, ν: frequency of light, c: light speed, h:

Planck’s constant)

To represent the change in Raman light color a graph called the Raman spec-trum (Figure 3.3) is used in which the horizontal axis plots the energy (color,

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wavelength) shift amounts of Raman light while the vertical axis plots the Ra-man light intensity with each shift in energy. Because the energy of molecularvibrations is discrete the amount of energy shifts of Raman light also becomesdiscrete, thus making the Raman spectrum discrete along the horizontal axis.Or that is to say, the Raman spectrum generically has a number of sharp peaks(called Raman peaks) within it. The energy of the molecular vibrations causedisruptions in a specific range of the spectrum through fluctuations caused bythermal vibrations, and the band width of the Raman peaks is finite.

Raman scattering light

Rayleigh scattering light

Rayleigh scattering light

Raman scattering light

Sample

Incident light

Figure 5.2: Raman scattering

Molecule consisting

of many bondings

(e.g. ethanol)

Figure 5.3: Molecular vibration and Raman spectrum

Abscissa of Raman spectrum is the dimension of envergy difference that isinverse of wavelength and is called ”wavenumber” or ”Raman shift” idiomatically.The unit is ”cm−1”

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Wavenumber(cm−1) = 1/λlaser − 1/λRaman scanttering light

The Raman scattering light which has higher energy (shorter wavelength) iscalled anti-Stokes Raman and the Raman scattering light which has lower energy(longer wavelength) is Stokes Raman. The peak positions of stokes/anti-stokesRaman scattering light from the Rayleigh light are the same while the intensityof Raman light with Stokes raman is bigger than that of anti-stokes.

Raman spectrum is identical for each molecule. The spectra below are ofribose and glucose. These spectra has many peaks at the different position. Itis possible to identify the material from Raman spectrum.

Different molecule has

different set of peaks.

Figure 5.4: Raman spectra of different molecules

Figure 5.5: Difference of structure also leads to different Raman peaks

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Not only the interaction with the energy of molecular vibrations but also theinteraction with the energy of the lattice vibrations induces Raman scattering.Figure 3.5 gives the Raman spectra of graphite, coal and diamonds. All of themconsist of carbon but each has its own individual Raman spectrum because thecrystal structure and energy of the lattice vibrations differ from each other. Thewidth of a Raman peak reflects the purity of the crystals (singularity of the latticevibration energy) while the position of a Raman peak reflects the energy of thecrystal vibrations, respectively. In addition, the stress within the crystals distortsthe crystal lattice, thereby resulting in a shift in the position of a Raman peak.This therefore allows positional shift analysis of Raman peaks to be utilized instress analysis.

5.3 Raman imaging (distribution of materials)

An image of the material distribution of a sample can be created by measuringthe Raman spectra from every point (pixel) in the field-of-view of an opticalmicroscope and identifying the type of material that exists at each point. Fig-ure below gives a Raman image of the material distribution within the yellowrectangular area selected in the field-of-view of an optical microscope. Opticalmicroscopic images do not reflect the material of an object but a Raman im-age reveals that it contains two kinds of material there. In this Raman image,the Raman peak at 1610cm-1 is colored green while at 1738cm−1 it is red, theformer being inherent with polystyrene while the latter PMMA from databaseinformation. The spectrum of the green area and the red area in this Ramanimage reveals the typical forms of Polystyrene and PMMA, respectively.

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Raman image

Raman spectrum

Optical microscope image

Figure 5.6: Optical microscopic image and Raman image

This Raman image is 400 pixels (in the x direction) x 400 pixels (in the ydirection), for a total of 160,000 spectra, with one spectrum displaying 1,340points along the horizontal axis (wave-number). The data structure of a Ramanimage, therefore, can be assumed to provide a data set of 400(x) x 400 (y) x1340(λ). As described above an image that contains spectrum information ateach pixel is called a spectrum image. To enable a material to be more intuitivelyunderstood and analyzed 3 x-y sections were selected at 1440cm-1, 2840cm-1and 2930cm-1, colored blue, green and red, respectively, and then overlaid in thecolor image given in Figure 3.7. In addition, RAMANplus includes the functionof Peak-shift imaging through use of which the amount a particular Raman peakshifts at each pixel can be visualized (Figure). It provides a particularly a goodmethod for analyzing the stress distribution of a crystal.

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Color image

Wavenumber (cm-1)

Figure 5.7: Data structure of Raman image

Distortion in a crystal induces a frequency shift in Raman light when comparedto a crystal without any distortion. The compressive stress in silicon shifts theRaman peak (520cm-1; Γ 2g optical mode) to a higher frequency while thetensile stress shifts it to a lower frequency. RAMANplusincludes other methodsof providing spectrum images, which are explained in the data display and analysismanual.

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Stress distribution of silicon substrate(Peak shift imaging)160,000 spectra (400 x 400 pix)

Raman shift [cm-1]

Ra

ma

n in

ten

sity [a

.u.]

Figure 5.8: Peak-shift image

5.4 Sectional Raman imaging (xy Raman image)

RAMANplusincorporates confocal optics, thus making internal sectional Ramanimages of transparent samples possible. As described above, optically gaininginternal sectional images of a sample, without actually having to slice a sampleinto sections, is called optically slicing or optically sectioning a sample. Figurebelow gives internal sectional Raman images of a cell. Four Raman images areobtained by displacing the focal plane in increments along the z axis.

Optical sectioning imagewith confocal optics

Raman imagesof living HeLa Cellsat different hieght.

Figure 5.9: Sectional Raman imaging of living cell

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5.5 Cross-sectional Raman imaging (xz Raman image)

The confocal optics mounted in RAMANplus enables cross-sectional Ramanimaging of transparent samples (Figure below), thus eliminating the need forsamples to be cut and their cross-sections exposed.

Multi-layer analysis

Green: Polyethylene (PE)Orange: Nylon (PA)Blue: Polypropylene (PP)

Figure 5.10: 400 × 120 = 480,000 spectra (480,000 pixels)

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Chapter 6

Support

Please contact with Nanophoton corporation when the problems about this prod-uct that is not solved within this operational manual happens.

Nanophoton corporationTel：+81-6-6878-9911Fax：+81-6-6878-9912Email: info@nanophoton.jp

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