Raman Manual Measurement Plus E

Laser Raman Microscope
RAMANplus
Operation Manual
Nanohoton cororation
November 10, 2011
Contents
1 Regarding Your Own Safety 7
1.1 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2 Oerating recommendations for RAMANlus as a roduct with a
built-in laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3 Preliminary setu notes . . . . . . . . . . . . . . . . . . . . . . 11
1.4 What to do in case of a ower outage . . . . . . . . . . . . . . . 11
1.5 Preliminary notes regarding comuter oeration . . . . . . . . . 12
1.6 Preliminary notes regarding maintenance, safety checls and reairs 12
1.1 Warning label . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2 Introduction to RAMANplus 13
2.1 Ceneral descrition . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2 Primary Comonents . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 Main body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.1 Aellation . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.2 lnside of the otical system art . . . . . . . . . . . . . . 15
2.4 The LPO switch (Otional} . . . . . . . . . . . . . . . . . . . . 15
2.5 The ower sulying box and the laser ower sulying box . . . 16
2.6 The light shading box covering the samle stage and a interlocl
system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1 A uright microscoe (Nilon Lclise 90i} . . . . . . . . . . . . . 17
2.8 A comuter . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3 Starting Measurement 23
3.1 Starting RAMANlus . . . . . . . . . . . . . . . . . . . . . . . 23
3.1.1 Attention before start . . . . . . . . . . . . . . . . . . . 23
3.1.2 How to start RAMANlus . . . . . . . . . . . . . . . . . 24
3.2 Finding the region of interest of the samle . . . . . . . . . . . . 26
3
3.2.1 Setting the samle on the stage . . . . . . . . . . . . . . 26
3.2.2 Bringing the samle into focus . . . . . . . . . . . . . . 28
3.2.3 Finding the region of interest . . . . . . . . . . . . . . . 31
3.3 Setting the arameters of Raman measurement . . . . . . . . . . 36
3.3.1 Scan mode . . . . . . . . . . . . . . . . . . . . . . . . 37
3.3.2 Objective lens . . . . . . . . . . . . . . . . . . . . . . 39
3.3.3 Measurement Area . . . . . . . . . . . . . . . . . . . . . 41
3.3.4 Measurement time . . . . . . . . . . . . . . . . . . . . . 48
3.3.5 Laser wavelength . . . . . . . . . . . . . . . . . . . . . . 49
3.3.6 Laser ower . . . . . . . . . . . . . . . . . . . . . . . . 51
3.3.1 Sectral range . . . . . . . . . . . . . . . . . . . . . . . 52
3.4 Starting measurement, and viewing and analyzing data . . . . . . 54
3.5 Surface roling measurement . . . . . . . . . . . . . . . . . . . 56
3.6 Shutting down RAMANlus . . . . . . . . . . . . . . . . . . . . 58
4 Software RAMAN Imager 61
4.1 Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.1.1 lnterface . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.1.2 How to start RAMAN lmager . . . . . . . . . . . . . . . 62
4.1.3 How to quit RAMAN lmager . . . . . . . . . . . . . . . 63
4.2 Common control anel . . . . . . . . . . . . . . . . . . . . . . . 64
4.3 Measurement setting anel . . . . . . . . . . . . . . . . . . . . 66
4.3.1 Raman . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.3.2 Scan mode . . . . . . . . . . . . . . . . . . . . . . . . 68
4.3.3 Surface(Otional} . . . . . . . . . . . . . . . . . . . . . 73
4.3.4 Wide-eld Raman measurement WF Raman(Otional} . 73
4.3.5 Wideeld observation WF Observation (Otional} . . . 76
4.3.6 Auto well-late measurement(m n } Otional} . . . . 79
4.4 Functions in the Menu . . . . . . . . . . . . . . . . . . . . . 82
4.4.1 The outline of the Menu . . . . . . . . . . . . . . . . 82
4.4.2 File,Save Load Settings . . . . . . . . . . . . . . . . 83
4.4.3 The wavelength and the outut ower of the laser Func-
tion,Laser . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.4.4 lmage Quality & Sectral ROl Function,lmage Quality
& Sectral ROl . . . . . . . . . . . . . . . . . . . . . . 85
4.4.5 Auto Save Settings Function,Auto Save Settings . . . 87
4.4.6 Trigger function Function,Trigger . . . . . . . . . . . 88
4.4.1 Function,Data Calibration and Comensation . . . . . 90
4
4.4.8 Function,Assist . . . . . . . . . . . . . . . . . . . . . 91
4.4.9 Standard samle Function,Reference Samles . . . . . 94
4.4.10 Function,Device,Sectrograh . . . . . . . . . . . . . 94
4.4.11 Function,Device,CCD Detector . . . . . . . . . . . . . 95
4.4.12 Automatic on,o of microscoe illumination. Function,lllumination
for Otical Microscoe . . . . . . . . . . . . . . . . . . 97
4.4.13 Motorized z stage Function,Device,Microscoe Z stage 97
5 To understand Raman scanttering 99
5.1 Raman sectrum . . . . . . . . . . . . . . . . . . . . . . . . . . 99
5.2 Raman scattering . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.3 Raman imaging (distribution of materials} . . . . . . . . . . . . 103
5.4 Sectional Raman imaging (xy Raman image} . . . . . . . . . . . 106
5.5 Cross-sectional Raman imaging (xz Raman image} . . . . . . . . 107
6 Support 109
5
Chapter 1
Regarding Your Own Safety
Please note that while the RAMANlus was develoed with safety in mind it could
cause damage to users and its surroundings if misused or the basic regulations
and guidelines are not followed, and therefore the consequences of these actions
are not covered by warranty. Lnsure to carefully read this manual before use.
The rules described with the yellow signs such as the Warnings and Cautions in
articular 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 roerty could occur if
messages indicated by this sign are not followed.
7
1.1 Regulation
!
Warning
LNSURL to ress the emergency sto button, disconnect the lug from the
outlet, and immediately contact us if any of the secial situations described
below arise.
Lmergency Power o
The ower cord, lug, wall adater, extension cord or ower suly device
has cracled, is brolen, or not worling.
Any art of the instrument is too hot to touch or there are traces of
smole, sarls or re being emitted by the instrument.
Faint noises such as snas, craclles or hissing can be heard from the
instrument, or it is emitting an unusual odor.
There is a trace of something having fallen on the instrument, ower cord
or ower suly device.
There is a trace of something having fallen on the instrument, ower cord
or ower suly device.
The instrument has fallen or been damaged in some way.
8
The system doesn t worl in site of the correct rocedure having been
followed.
!
Warning
DO NOT tale the instrument aart as it could lead to electric shocl, failure of
the system or eole being exosed to the laser. Any roblems or damage
caused by the instrument having been talen aart are not covered by warranty.
!
Warning
LNSURL only the ower cord and ower suly device sulied by Nanohoton
Cororation are used. DO NOT use them for any other instrument as they
were secically designed for use with RAMANlus.
!
Warning
DO NOT use an outlet that has been damaged or corroded. DO NOT bend or
refurbish the lug. LNSURL to contact Nanohoton Cororation for a
relacement if the lug has been damaged. A 3 in ower lug is used with the
RAMANlus to ensure it is connected to a ground outlet. DO NOT connect
the ower lug to non-grounded outlet.
DO NOT share an outlet with RAMANlus and another electric aliance that
consumes a lot of electricity as any unstable voltage could ossibly damage
RAMANlus.
DO NOT overload the outlet used with RAMANlus. LNSURL to conrm that
the outlet to which RAMANlus is connected has been correctly wired, is within
easy reach, and near RAMANlus. DO NOT stretch a ower cord so taut that
it becomes overloaded. LNSURL to conrm that the current and voltage of an
outlet connected to RAMANlus are of the correct values. LNSURL to exercise
extreme caution when connecting or disconnecting a ower cord from an outlet.
!
Caution
Please note that as there are some arts in the roduct that could fail in the
event of a surge or sile in electricity from an outlet. Lnsure the system is o
and remove the lug from the outlet if excess voltage is exected to occur.
!
Caution
DO NOT insert or remove any other cables than the LAN connection to a PC.
Parts of the roduct are extremely suscetible to static electricity and could be
9
damaged their connectors are touched with no cable in them. LNSURL to
contact us when you need to insert or remove any cable other than a LAN
cable.
!
Caution
DO NOT allow any water over the roduct and DO NOT use RAMANlus
anywhere it may come in contact with water as it may result in it short
circuiting. Should the roduct come into contact with water LNSURL to
immediately switch the system o, remove the lug, and then dry with a cloth.
LNSURL to immediately contact us if any water has ossibly entered the
roduct.
!
Caution
DO NOT turn the RAMANlus on if it is covered with a case or a cloth as it
can lead to heat building u and result in roblems.
1.2 Operating recommendations for RAMANplus as a
product with a built-in laser
!
Warning
RAMANlus was labeled a Class 1 1lS code laser roduct. LNSURL to exercise
the following in reventing any accidents from occurring.
DO NOT lool directly at the laser beam as it can harm your vision.
LNSURL to avoid any exosure to the laser regardless of whether directly
or indirectly as it could result burns to yourself or your clothing.
RAMANlus is designed with no laser irradiation to human body excet
for maintenance time and checling time. However, for any reason, if
there is a ossibility to be exosed to laser, LNSURL to wear laser roof
safety glasses and clothing.
LNSURL to osition an aroriate cautionary sign at the entrance of
anywhere RAMANlus is being used.
10
DO NOT use large amounts of burnable, exlosive or volatile solvents
such as alcohol or ether anywhere near a laser roduct as it could result
in an exlosion.
LNSURL use of the roduct under the suervision of ersonnel
conversant with laser safety services and that understands the risls.
LNSURL all users of RAMANlus understand the above oerating
recommendations.
LNSURL to follow the safety recautions and control standards for laser
roduct users rovided by lLC60825-1(2001}, 1lS C 6802(2005} and
others.
1.3 Preliminary setup notes
The roduct will be assembled and aligned by Nanohoton sta. After
conrming the installation conditions our stu will ensure the aroriate
installation of RAMANlus.
!
Caution
LNSURL the roduct is at least 0.3m away from any wall to allow alignment
and maintenance to tale lace.
!
Caution
LNSURL to avoid any direct sunlight onto the roduct.
!
Caution
LNSURL the roduct is let away from any heat sources.
1.4 What to do in case of a power outage
ln the case of ower outage ensure switch the system o and remove the lug
from the outlet.
11
1.5 Preliminary notes regarding computer operation
Almost all RAMANlus features are comuter-oerated. The comuter system
runs Windows 1, which users will need to lnow how to oerate.
!
Warning
Before using the system ensure to carefully read the comuter manual and
follow any aroriate safety recautions.
1.6 Preliminary notes regarding maintenance, safety
checks and repairs
LNSURL to lee the worl lace and roduct clean and tidy. When cleaning
the roduct rub its surfaces with a dam mildly soay cloth.
!
Warning
The roduct contains no user maintainable arts. Maintenance, reair,
alignment, disosal or exchange of arts or any other oeration not included in
this manual will need to be carried out by Nanohoton sta. RAMANlus is a
recision otical instrument, and oeration of it by anyone not used to it could
ossibly result in roblems. Oening the cover of the instrument could lead to
blindness, being burnt or re from the laser being exosed. Any damage to the
instrument caused by oerations not included in this manual or use and
installation of inaroriate roducts rovided by other comanies are not
covered by warranty.
1.7 Warning label
LNSURL that the warning and caution labels are on the roduct.
12
Chapter 2
Introduction to RAMANplus
lt is exlained about aellations and functions that are imortant for the oer-
ation of a Laser Raman Microscoe RAMANlus in this chater.
2.1 General description
Laser Raman Microscoe RAMANlus is an instrument to observe and analyze
your samles.
The analysis is oerated in the area which ranges from sub-micron to millimeter
scale while the samle is observed under an otical microscoe. As an rincile
of the analysis, Raman sectroscoy is utilized.
For Raman sectroscoy, laser beam is irradiated on to the samle to excite
Raman scattering light at the irradiation osition on the samle. Raman scat-
tering light from the samle is collected and analyzed with the instrument.
Measured data is so-called Raman sectrum. Because Raman sectrum
is identical to molecules or crystalline form, the analysis is done as molecular
identication or evaluation of the crystalline in many cases.
Furthermore laser Raman microscoe RAMANlus has an rominent ro-
erty. lt is fast imaging function of Raman sectrum. The sectral imaging func-
tion rovides distribution information of molecules of crystallinity.
ln addition, RAMANlus has the surface roling function. With the com-
13
bination of surface roling and distribution analysis, it rovides multilateral
solutions.
2.2 Primary Components
Fig. 2.1 shows a whole setu of a laser Raman microscoe RAMANlus . The
RAMANlus adots a uright tye microscoe.
The main body of the instrument is installed on an anti-vibration table at the
left side in the gure. Under the table, a ower sulying box for the main body,
another sulying box for lasers and a ersonal comuter condoling instruments
are set. A monitor for the ersonal comuter, a ley board, a mouse and a stage
controller are set on another table at the right side in the gure. An emergency
ower o (LPO} switch is sulied otionally and set on the left side table.
Emergency Power O (Optional)
Computer
Laser Power Supply
Controller (LPSC)
Motorized Z stage controller
Motorized XY stage controller (Optional)
Anti-Vibration table
PC Table
Mouse &
Keyboard
Main body
Power supply for
a motorized XY stage
Monitor
Power Supply
Controller (PSC)
Figure 2.1: Comonents of RAMANlus
2.3 Main body
2.3.1 Aellation
Part names of the main body are shown in Fig. 2.2. A main body comoses a
microscoe art with a light shading box located in a front half and an otical
system art behind the microscoe art. ln the gure, a door of the shading box
14
is oened so that a oerator can access the microscoe stage to set a samle.
The microscoe comoses the microscoe body, a samle stage, a revolver with
objective lenses, a condenser lens, and so on. On the uer side of the micro-
scoe, a CCD camera for observing microscoic samle images is attached. A
thermo-electrically cooled CCD detector for recording Raman scattering light is
attached at the left side of the main body.
2.3.2 lnside of the otical system art
ln the otical system art, laser(s}, laser scanning otics, slit confocal otics
and sectroscoic otics are embedded. lt is no need for the oerator to access
these otics arts. All controls of otics, such as laser selection, laser ower
adjustment and sectroscoy 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 icture of a LPO switch is shown g. 2.3. ln the g, the LPO is set on the
table for switch o the instrument at a emergency case. Pushing red button
stos ower sulying to the instruments. Once the red button is ushed, it is
let ushed. You need to turn right the red button to recover ower suly.
15
Figure 2.3: A icture of the LPO switch
2.5 The power supplying box and the laser power sup-
plying box
A icture of a ower sulying box and a laser ower sulying box is shown in
g. 3.1. These are set under the anti-vibration table.
The ower sulying box sulies electrical ower to the instrument but
lasers. The laser ower suling box sulies electrical ower to the lasers inside
instruments.
On the front anel of the ower sulying box, a ley switch for ower on,o
and a indicator for the instrument oeration. On the front anel of the laser
ower suling box, two indicators are shown. One indicator shows the status of
laser emission of 532 nm wavelength. The other shows for 185 nm wavelength.
Turing the ley-switch right start to suly the ower. Turning left stos to
suly the ower. Administrator can remove the ley-switch to taling control of
the instruments.
16
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 icture of a ower sulying box and a laser ower sulying box.
2.6 The light shading box covering the sample stage and
a interlock system
A light shading box covering a samle stage males it ossible to measure the
samle under the room light is let on. lt is very imortant because Raman
scattering light is very weal and is easily disturbed by the room light. Further
more the shading box blocls laser light going out and rotect the oerator from
the irradiation.
A interlocl system is equied to the shading box so that the laser beam
never comes out when the door of the shading box is oened.
2.7 A upright microscope (Nikon Eclipse 90i)
The uright microscoe is set in the light shading box. The art names are
shown in g. 2.5.
lt is very imortant to master how to oerate the otical microscoe. Be-
cause, too measure Raman sectrum, the oerator should ut the samle on
the stage, observe the samle surface with the microscoe, nding the region of
interest and set the focus. lt is very imortant to master how to oerate the
otical microscoe.
17
The CCD camera at the front art of the microscoe tales otical microscoe
images of the samle 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 uright microscoe
The sample stage
The stage is lace on which the samle is ut. The oerator moves samle
osition with controlling a xy-handle manually.
An electrical xy stage is rovided otionally.
The revolver
The revolver is a art where objective lenses are attached. By being revolved,
the objective lens which are used for observation can be selected. Six ( or Seven
otionally} objective lenses can be equied.
Llectrical revolver is rovided otionally.
For olarized Raman measurement, an otional revolver for olarization mea-
surement is necessary.
Objective lenses.
A standard set of ve objective lenses are listed below.
18
Name Magnication N.A. W.D. (mm}
LU Plan FLUOR LPl 5 0.15 23.5
LU Plan FLUOR LPl 50 0.8 1
LU Plan FLUOR LPl 100 0.9 1
N.A. is Numerical Aerture.
W.D.is Worling Distance that is the distance between the bottom of the
objective and the focus.
ln addition to the standard objective lenses, various objective lenses are ro-
vided otionally.
A condenser lens and a focusing handle
A condenser lens is used for focusing illumination light for microscoe observa-
tion. Condenser osition can be adjusted withe the focusing handle.
Filter IN/OUT switch
Two lters of front side are ND lters of transmission illumination. ND lters
are used to adjust brightness of the illumination. The bigger number has lower
transmission and decrease brightness more.
ND8: 1,8 decrement
ND32: 1,32 decrement
ND8 and ND32: 1,256 decrement
The focus handle
Rotating the focus handle moves the samle stage u and down to bring the
samle surface in the focus. The resolution of the objective lens movement of
u and down can be selected with the Z resolution switch.
Move the stage downward Rotating the handle toward the front.
Move the lens uward Rotating the handle Derward the bacl.
19
Z resolution switch
The mount of the stage movement deends on the rate of movement,rotation
when the focus handle is rotated. The rate is changed in the sequence of
Coarse, Fine and Ultra ne when the Z resolution switch. When the
switch is ushed, buzzer sounds and the indicator status also changes.
Resolution Amount of movement lndicator Buzzer
Coarse 250010m,sec lighting One long buzzer
Fine 100m,rotation extinction One short buzzer
Ultra ne 25m,rotation blinling Two short buzzer
Obj. switch
ln the case of an electrical revolver is equied (otion}, this switch revolves the
revolver and changes the objective lens.
Escape switch
This switch moves the stage to the escae osition with the indicator lighting.
The escae osition is 5 mm downward from the current osition. You need to
ush the escae switch again to move the stage bacl to the former osition with
the escae indicator lighting o.
Note that the escae function does not worl when the distance between the
current stage and the bottom osition is less than 5.5 mm.
Note that the focusing handle is disable at the escae osition and while the
stage is escaing.
2.8 A computer
All oeration about controlling instruments, measurement, viewing data and
analysis is done with the comuter. The oerating system of the comuter is
Windows1.
ln the comuter, the RAMANlus controlling software, RAMAN Imager
and the data viewing and analyzing software, RAMAN Viewer are installed.
With the controlling software RAMAN lmager, the oerator set the mea-
surement arameters. The measured data is viewed in the RAMAN Viewer.
Various lind of analysis method is equied in the RAMAN Viewer
20
21
Chapter 3
Starting Measurement
ln this chater, ractical rocedures of measurement are exlained for your start.
ln the rst ste, it is exlained about how to start the RAMANlus. ln the
second ste, it is exlained about how to set samles to stage and then how to
set measurement arameters for measurement modes.
The rocedure from the start to the end is listed as below.
1. Starting RAMANlus.
2. Finding the region of interest in the samle.
3. (Knowing the items necessary for measurement.}
4. Setting the measurement arameters and Measuring.
5. Viewing Data and analyzing.
6. LndingRAMANlus.
3.1 Starting RAMANplus
3.1.1 Attention before start
Please conrm that all cables are connected correctly and that the LPO switch
is correctly connected correctly and is located within close reach.
23
3.1.2 How to start RAMANlus
1. Turning a key switch right on the power supplying box to switch
power on.
Please conrm the AC ower indicator lighting with blue on the ower
sulying box and also laser ower sulying box shown in g. 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 ower sulying box
2. Turning on the computer.
Note that there is no articular order between turning on the ower su-
lying box and the comuter.
3. Double-clicking the icon of RAMAN Imager to start RAMAN
Imager
After double-clicling the icon, the RAMAN lmager starts to initializing all
devices of RAMANlus ste by ste. While initializing the devices, the
rogress bar is shown. After nishing initializing, the main window turns
out. lt may tales several minutes after the double-clicling to nishing the
initializing.
24
Figure 3.2: The icon of RAMAN lmager
Figure 3.3: A Main window of RAMAN lmager just after initializing
RAMANlus
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 lmager. The warming u of RAMANlus
is done when the temerature of the cooled CCD detector reaches at -10
. To checl the temerature, you can clicl Detector temerature from
the menu of Window .
25
Note that you can start measurement ever before reaching -10 . ln this
case, the darl current noise of the cooled CCD detector may be larger.
Figure 3.4: Temerature 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 samle on the stage
1. Opening the door of the shading box.
26
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.
lt is necessary to taling the stage down enough to revent the contact of
the objective lens and the samle. The height of the stage can be adjusted
with the focusing handle.
Hint} The seed of the stage movement deends on the travelling
rate. The rate can be switched with the Z resolution switch. With the
Coarse status of the switch, the stage is moved quiclly.
Hint} The Lscae switch moves the stage down 5mm quiclly (Lscae
osition}. The ushing the Lscae switch at the escae osition, the
stage comes bacl to the initial osition.
27
Escape switch
Z resolution switch
Focus handle
Figure 3.6:
Amount of the movement lndicator Buzzer
Coarse 250010m,sec Lighting A long buzzer
Fine 100m,rotation Lxtinction A short buzzer
Ultra ne 25m,rotation Blinling Two short buzzer
3. Putting the sample with the slide glass on the stage and clipping
the slide with the sample holder.
4. Moving the sample position so that the region of interest is just
right below the objective lens.
3.2.2 Bringing the samle into focus
1. Select the objective lens of the proper magnication for observing
the region of interest of the sample.
For the selection of the objective lens, the revolver should be revolved
manually.
lf the motorized revolver equied, the Objective Selector Switch should
be ushed. Or the Objective lens can be selected through the software
RAMAN lmager.
Hint} The eld of views of the microscoe objective lenses are listed
below.
28
Magnication Otical Microscoe Raman measurement
5x About2.4mm1.8mm About0.9mm0.9mm
10x About1.2mm0.9mm About800m800m
20x About600m450m About400m400m
Hint} For beginners of a microscoe, it is recommended that start to
bringing the samle into the focus with the objective lens of lower magni-
cation. lt is easier to bring the samle into focus with higher magnication
after focusing with the lower magnication.
Attention} The view eld of Raman is smaller than that of otical micro-
scoe.
Revolve manually
Obj. switch
(Motorized revolver)
Revolver
Figure 3.1:
2. Lighting on the illumination for the microscope observation with
the Light control slider in the RAMAN Imager.
There two lllumination slider. One is Reection lam for Reec-
tion illumination and the other is Transmission lam for Transmission
illumination
29
Figure 3.8: Light control slider for the microscoe illumination
3. Moving the sample stage upward where the sample surface comes
in the focus.
Hint} lt males bringing the samle the into focus easier to observe the
laser illumination attern.
To observe the laser illumination attern, a mechanical shutter stoing
the laser beam should be oened and , furthermore it is imortant to adjust
the laser ower at the same time. For these controls, lease refer to the
chater 3.6.
To oen the mechanical shutter, The Checl box Checl Laser Sot should
be checled.
When the samle is in the focus, the laser illumination lools lile the small-
est sot. When the samle is out of the focus, the laser illumination lools
lile defocus sot. lt is good way to nd the samle stage height with the
smallest laser sot.
The laser sot observation can eective even for laser line illumination.
Hint When bringing the samle into focus, the samle height can be
controlled more recisely with the Fine or Ultra Fine mode of the Z
resolution. the status of Z resolution is
Hint} The samle stage height can also be controlled with the U
and Down button of the RAMAN lmager. The ste distance is deter-
mined with the Ste value.
30
Figure 3.9: Checling laser sot
Attention} When bringing the samle into focus with the laser illumina-
tion, decrease the laser ower enough before the laser illumination.
Attention The door of the shading box has the interlocl system. lt is
necessary to close the door to illuminate laser beam on the samle.
3.2.3 Finding the region of interest
For the nding the region of interest, XY stage is moved manually.
lf the motorized stage is quied (rovided otionally}, the xy stage is con-
trolled with the joysticl and,or the RAMAN lmager.
Manual control (Standard)
The stage X handle moves the samle holder in X direction on the stage
(from side to side}. The stage Y handle moves the stage in Y direction (bacl
and forth}.
31
X handle
Y handle
X
Y
Figure 3.10: Manual XY stage
A motorized XY stage is rovided with a joysticl otionally. The motorized
stage moves in the direction where the joysticl ushed. The seed of the motor-
ized stage is selected with the Seed button. There are four seeds and the
seeds are indicated as the number of the indicator lams. Four indicator lams
shows the fastest seed. By ushing the Seed button, the seed is changed
to the next seed.
Seed Button: Changing the seed.
M-ORC Button: Finding the Hardware origin.
ZLRO Button: Setting the current osition as the home osition.
32
L-ORC Button: Moving to the home osition.
Seeds are listed below.
The number of lams Seed
1 0.001 mm,s
2 0.05 mm,s
3 0.3 mm,s
4 1 mm,s
Note} The osition when the ower is on is set as the home osition.
Stick
X
Y
C
urrent position display
Speed button
Speed indicator
Figure 3.11: 1oysticl controller (otional}
The control of the motorized stage with RAMAN Imager.
1. Select the Stage Controller from the Window menu on the
RAMAN Imager
Microscoe Stage Controller widow is oened.
33

Figure 3.12:
Hint The stage controller on the Wide Field setting anel can be
also used. Please clicl the WF-Raman tab to oen the Wide Field
setting anel.
34

Figure 3.13: Wide Field Stage Controller
2. Set the region of interest at the center of the view eld by mov-
ing the stage. For moving the stage, UP, DOWN, LEFT,
RIGHT buttons are pushed.
Hint The seed of the stage is selected from the ull-down menu of
the Seed box.
35
3. If necessary, the sample surface is conrmed in the focus with
moving the stage height.
3.3 Setting the parameters of Raman measurement
For measuring Raman sectrum, measurement arameters should be set on the
RAMAN lmager.
The measurement arameters are listed below. After setting the arameters,
start the measurement with ushing the 0. Measurement button,
1. Scan mode
2. Objective lens
3. Measurement area
4. Measurement time
5. Laser wavelength and Laser ower
6. Sectral range
0.
1.
2.
4. 3.
6.
5.
Note
Setting controls for measurement area changes
accoriding to the scan mode.
36
Figure 3.14:
3.3.1 Scan mode
There are eight scan modes as below (including two otional scan modes.}
1. Point A sectrum at a oint is measured.
2. x-Line Plural sectra at lural oints along a line are measured.
3. xy-imaging Two dimensional xy sectrum imaging.
4. xz-imaging Two dimensional xz sectrum cross-sectional imaging.
5. Point-z Plural sectra at a oint with dierent height are measured.
6. xy-maing Two dimensional xy sectrum maing.
1. xy-z-imaging(Otional} Plural xy-sectra imaging are measured with
z scanning.
8. xy-z-maing(Otional} Plural xy-sectra maing are measured with
z scanning.
Note} xz-imaging, Point-z, xy-z-imaging, xy-z-maing measurements
are ossible for the transarent samles where the laser light and Raman scattered
light can travel.
Note} xz-imaging, Point-z, xy-z-imaging, xy-z-maing measure-
ments are not rovided for the Product which does not equied with a motor-
ized Z stage.
37
Figure 3.15:
38
Figure 3.16:
3.3.2 Objective lens
The objective lens which are used currently should be selected. The correct
setting of the objective lens give correct information about the laser ower and
the scale (size}.
39
Figure 3.11:
Attention The measurement can star with the wrong objective lens selection.
ln the case, the scale information and laser ower information recorded in the
measurement data are dierent from the one of the true objective lens.
Tis 1. Parfocality and Worling distance W.D.
The focal oints of the microscoe are the almost same among the dierent
objective lenses. For Nilon microscoe, the focal oint is designed at the 60 mm
from the mounting osition of the objective lens (Parfocal length}. For Olymus
and Zeiss microscoe, the arfocal length is designed as 45mm. Objective lenses
has arfocal design
Sample
Parfocal length
60mm
W.D.
Figure 3.18:
Tis 2. Numerical aerture (N.A.} and the satial resolution of microscoe.
The erformance to observe small structure is exresses as satial resolution.
The resolution is determined not by magnication but the Numerical aerture of
objective lenses.
40
Spatialresolution = 0.61 /NA wavelength
Magnication, W.D. and N.A. of standard objective lenses are listed below.
Name Magnication N.A. W.D. (mm}
LU Plan FLUOR LPl x5 0.15 23.5
LU Plan FLUOR LPl x50 0.8 1
LU Plan FLUOR LPl x100 0.9 1
3.3.3 Measurement Area
The Setting of Measurement Area deends on the Scan mode
Point mode
A measurement oint is secied with clicl-and-drag on the microscoe
monitor which has 640 ix in X axis and 480 ix in Y axis. The ix
coordinate of the oint is shown on the RAMAN lmager. The width w
and the height h should be 1.
1.Click
Coordinate of
Measurement Area
Origin
X: 640
Y: 480
41
Figure 3.19: Setting the Measurement oint of Point mode
lt is ossible to inut numerical values (an integer} of the ix coordinate.
XY-imaging mode
A measurement area is secied as a rectangle area with clicl-and-drag
The laser line is illuminated along the x axis and scanned in y axis so that
the whole rectangle area are scanned with laser beam.
The length of laser line or width of the rectangle area is xed. The length
of the height of the rectangle can be set arbitrarily. With determining the
height, the scanning number Y ste is automatically determined so that
the ixel sizes are the same in X axis and Y axis. The oerator can also
secify the scanning number by inuting 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
42
6WDUWRI/LQHVFDQQLQJ
(QGRI/LQHVFDQQLQJ
/DVHUOLQHEHDP
G\
Figure 3.21: Scanning direction of line laser. Scanning ste width dy,
Scanning ste number Y ste.
XZ-imaging mode
1. Determining a line area on the microscoe monitor.
The laser line will be scanned inside the samle at the determined
line area in xy coordinate as the result of Z stage movement.
2. Reseting the coordinate in z axis so that the current osition is 0.
3. Determining the Start osition (Z Start} and Lnd osition (Z
end} of the stage movement.
Start osition (Z Start} and Lnd osition (Z end} can be inut
with the Read button that inuts current osition to Start osition
(Z Start} or Lnd osition (Z end}. Oerator can also inut the
number directory with a leyboard.
4. Determining the Scanning number (Z ste}
Ste width (dz} is automatically calculated following the equation
below.
(Startposition Endposition)/(Scanningnumber + 1)
43
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 oint is be secied with a mouse clicl on the microscoe
monitor. Sectra at the oint are measured while the stage is scanned in
the same manner with XZ-imaging.
Start osition (Z Start}, Lnd osition (Z end} and Scanning number
(Z ste} are set in the same manner with XZ-imaging.
Start osition (Z Start}, Lnd osition (Z end} and Scanning number
(Z ste} are set in the same manner with XZ-imaging.
X-line mode
Determining a line area on the microscoe monitor. Sectra along the line
are measured simultaneously.
XY-maing mode
A measurement area is secied as a rectangle area with clicl-and-drag
A oint laser is scanned in the rectangle area with the raster scan and
whole sectra in the rectangle are are measured.
44
The oerator should determines Width and Height of the rectangle
area, and Scanning ste distance (nm} before the XY-maing measure-
ment. The scanning number in X and Y direction are automatically calcu-
lated from the Width, Height , and Scanning ste distance (nm}
Delta
Figure 3.23: The direction of the raster scanning and Scanning ste dis-
tance (nm} Delta
XY-Z-imaging
A measurement area is secied as a rectangle area with clicl-and-drag in
the same manner with XY-imaging. The scanning number Y ste is
automatically calculated. Afterwards, Start osition (Z Start}, Lnd o-
sition (Z end} and Scanning number (Z ste} are should be determined
in the same manner with XZ-imaging.
Note The folder for automatic data-save function should be set before
XY-Z-imaging measurement. (Refer to the section, Automatic Data-Save
Function}
A number of XY-image data, that is the same number of Z scanning
45
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 measurement
can be reconstructed as 3D (XYZ} data of Raman image via 3D Data
Cenerator function. (Refer to the Manual for Data Viewing and Analysis}
Scan Settings in Z
Scan Setting in Y Scan Setting in Y
XY Measurement Area
Figure 3.24: Scanning setting of XY-Z-imaging
XY-Z-maing
A measurement area is secied as a rectangle are with clicl-and-drag in
the same manner with the XY-maing. Width,Height and Scan-
ning ste distance (nm} should be determined. Afterwards, Start osi-
tion (Z Start}, Lnd osition (Z end} and Scanning number (Z ste}
are should be determined in the same manner with XZ-imaging.
Note The folder for automatic data-save function should be set before
XY-Z-maing measurement. (Refer to the section, Automatic Data-
Save Function}
A number of XY-maing 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-maing data of XY-Z-imaging measurement
can be reconstructed as 3D (XYZ} data of Raman image via 3D Data
Cenerator 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-maing
47
Figure 3.26: 3D (XYZ} Data reconstruction with 3D Data Cenerator of
RAMAN Viewer
3.3.4 Measurement time
Measurement time is automatically calculated from the Scanning number and
Lxosure time,shot.
Scanninng number n
Total measurement time
Exposure time
Figure 3.21: Measurement time
The value between 0.01 sec and 1800 sec can be inut for Lxosure time,shot
Scanning number n is automatically calculated when the measurement are
is set. The scanning number of Point and X-line is one.
The scanning number is Y ste for XY-imaging, Z ste for XZ-imaging
and Point-Z.
The measurement time is calculated according to the equation below. (Measurementtime) =
(Exposuretime/shot) + (Datatransfertime) n
48
Data transfer time is the time in which the signal of cooled CCD detector
is transfered to the comuter.
Note Data transfer time deends the ixel number of the detector, AD
conversion seed and the sec of the comuter. As indication, it tales about 0.5
second for the standard detector format (400x1340ix}, standard AD conversion
setting (2 MHz} and the standard comuter for RAMANlus.
Note The equation for the measurement time should be modied when
the setting of averaging, cosmic ray lter, binning and sectral ROl.
Hint} For the beginner, the measurement time between 1 sec and 10 sec is
recommended for the start. However the measurement time over 1000 sec can
still be set for some cases.
3.3.5 Laser wavelength
Laser wavelength can be selected in the Laser Setting Window. The window
is shown by clicling 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 eciency
Raman scattered light is excited more eciently with the laser of short
wavelength than long wavelength. The eciency is roortional to the 4th
ower of inverse wavelength.
Raman scattering efficiency 1/
4
For examle, the eciency for 532nm-laser excitation is 4.8 times for
185nm-laser excitation.
2. Satial resolution
Laser focusing ability deends on the laser wavelengths. Shorter wave-
length, smaller focusing sot and higher resolution.
Spatial resolution = 0.61 /NA (NA:Numerical aerture}
The reason of the selection of longer wavelength is to avoid uorescence or
decrease
Because it can not be lnown if the uorescence signal is dominant or not
before measurement, it is reasonable to start with the laser of short wavelength.
After judging that the uorescence signal should be decreased, the longer wave-
length may be selected.
ln general, samles colored tend to emit the uorescence rather than Raman
scattered light.
The laser of longer wavelength does not have to avoid the uorescence.
When Raman lmager starts, the laser of shortest is selected.
50
3.3.6 Laser ower
Laser ower at a laser focus sot is shown at Laser ower monitor. ln general,
laser ower should be between 0.5 mW and 10 mW. For samles tough to laser
damage, the laser ower can be adjust more than 10 mW to obtain better data
with shorter measurement time.
The laser ower on the monitor is shown after calibrated with the transmission
of objective lenses.
There are two ways to adjust the laser ower.
1. Laser outut ower from a laser head
Laser outut ower from a laser head can be adjusted by changing the
injection current to the laser diode. The injection current is adjusted be-
tween 0 % to 100 % on the laser setting window.
Note Laser outut ower is not roortional to the injection current.
And there is a threshold of the injection current to lase.
Note} The injection current of Standard 185nm laser can not be ad-
justed. Only 100 % current is available.
2. Neutral density lter
The transmission is adjusted with 255 variable stes by ND lter.
Note Please ay attention the laser adjustment when the scan mode is
changed.
There is much dierence about the laser ower between oint illumination
measurement such as Point mode and Point-Z mode, and line illumination
measurement such as X-line, XY-imaging and XZ-imaging. There is more
than double digits dierence between oint,line illumination under the same ND
lter and the laser injection current. lt is because the laser oser is distributed
along the line and the laser ower is shown as the calibrated ower at at oint.
51
Laser power
at sample
Injection current
ND filter
Figure 3.29: Laser setting window
x
Power is focused
on one point.
Power is distributed
along a line.
Figure 3.30: The dierence of the ower at the oint between oint,line illumi-
nation
3.3.1 Sectral range
Sectral range where Raman sectrum is measured is determined with groove
number of Crating and grating angle determined with Center wavenumber.
The bigger number of the groove means higher sectral resolution but nar-
rower sectral range.
52
Spectral Range
Grating
to be selected
Center of Wavenumber
to be set
Figure 3.31: Sectral range setting
Three gratings are installed with RAMANlus and can be selected from RA-
MAN lmager. Cratings listed below are available.
Croove number Range (532nm ex.} Resolution Range(185nm ex.} Resolution
2400gr,mm 450cm-1 1.5cm-1 NA NA
1800gr,mm 1000cm-1 1.1cm-1 400cm-1 1cm-1
1200gr,mm 1300cm-1 2cm-1 500cm-1 1.2cm-1
600gr,mm 2500cm-1 4cm-1 1300cm-1 2cm-1
300gr,mm 4500cm-1 1 cm-1 2200cm-1 3.5cm-1
150gr,mm 8000cm-1 14cm-1 4000cm-1 1 cm-1
The sectral range between 100cm
1
and 5000cm
1
will be determined ac-
cording to the samles.
Note}
Sectral ranges between excitation lasers dier because the wavenumber, wave-
length and laser wavelength has the relation as below.
Wavenumber (cm
1
) = 1/
ex
1/
lambda
ex
is the wavelength of the excitation laser. lambda is the wavelength
of Raman scattering light. The sectral range for the 185nm 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 of
185nm.
Rayleigh light and the the cut o of spectral range at the lower side.
Sectrum lower than 80cm
1
can not be measured because the laser blocling
lter blocls the light of the wavenumber lower than 80cm
1
. But the eal
of 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 comes
through the lter artially. lt is recommended that the measurement range
excludes the 0cm
1
.
Zoom
Zoom
(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 o of sectral range at the lower
side.
3.4 Starting measurement, and viewing and analyzing
data
After setting the measurement arameters, Measurement button will be ushed
to start measurement.
The rocedures listed below are done automatically just after the start.
1. Saving an otical microscoe image.
54
2. Saving the measurement arameters.
3. Switching o the transmission,reection illumination light.
4. Starting RAMAN Viewer software with which measured data is shown
and analyzed.
5. Laser irradiation of the samle and recording the signal.
6. Switching on the transmission,reection illumination light after nishing
the measurement.
The window tye of RAMAN Viewer shown with the measurement start de-
ends on the Scanning mode. The data with RAMAN Viewer can be oerated
even while the measurement is roceeding.
Please refer to a Manual for RAMAN Viewer.
[
\
]
[
&&'
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 and
analyzed.
55
3.5 Surface proling measurement
1. Clicling the Surface tab to show the Surface roling setting anel.
Click
Surface plot panel
Figure 3.34: Surface roling setting anel
2. Setting the scanning range of the surface role in z axis.
The uer limit and lower limit in z scanning should be determined from
the microscoe image with moving the samle stage in z axis. At the
uer limit of the stage, Read button should be ushed to inut the
uer limit osition to Z:Start. At the lower limit of the stage, Read
button should be ushed to inut the lower limit osition to Z:Lnd.
Afterwards, Z Scanning Pitch should be selected. Scanning ste number
is automatically determined.
56
0. Initial status
Microscope image
1. Setting start
position of Z scan
2. Setting end
position of Z scan
3.Setting step width
Read Z start
Read Z End
Z Scanning Pitch
Figure 3.35: Setting the scanning range of the surface role in z axis
3. Switch to Real-Time Frame mode. On the Real-Time Frame mode,
ND lter and Cain should be adjusted so that the laser reection light
from the samle 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 lter and the Cain
57
4. Showing the surface role data after the measurement.
Menu,Window,Surface Prole should be selected.
Click
Menu/Window/Surface Profile
3D Surface Plot Window
Figure 3.31: The surface role data
Note}
The surface role data with other data format can be done with the Menu,File,.
Hint}
Automatic focusing is ossible after the surface roling measurement. On
the microscoe monitor, jut right-clicl and select Set Focus here so that the
the oint at the cursor is brought into the focus.
3.6 Shutting down RAMANplus
1. Closing RAMAN Imager with clicking Close button at the top
right of the window.
2. Switching o the power supplying unit with turning the key switch
left.
3. Shutting down the computer.
Attention The order of switching o should be 1. RAMAN lmager and
2. the ower suly unit.
58
Figure 3.38: The shut down of RAMANlus
59
Chapter 4
Software RAMAN Imager
RAMAN lmager is the software controlling RAMANlus
This chater exlains RAMAN lmager version 1.2 in detail.
4.1 Outline
4.1.1 lnterface
There are three arts in the interface of RAMAN lmager. Lach art with sections
in detail will be exlained as follows.
Common control anel
The common control that is mainly for otical microscoe is done in this
art.
Measurement setting anel
The measurement arameters are set. The Measurement tye is divided
with the tab anel.
Standard Raman Measurement,RAMAN
Surface roling measurement,Surface
Raman imaging in an wide-eld area, WF-Raman (Otional}
Otical microscoe observation in an wide-eld area,WF-Observation
(Otional}
Multi-well (m x n} automatic measurement, m x n(Otional}
61
Menu
Menu rovides 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 lmager
4.1.2 How to start RAMAN lmager
1. Turning on the Computer.
2. Turning on the power supply unit.
note} The sequence of turning on the comuter and the ower suly unit
can be converse.
3. Starting RAMAN Imager with double-clicking the icon of RAMAN
Imager.
RAMAN lmager starts to initialize devices in RAMANlus

While initializing,
only the rogress bar of the initialize is shown. After nishing the initialize,
62
the main window will show u. lt tales several minutes by the end of the
initialize and main window showing u.
Attention Because RAMAN lmager communicates the devices, it is nec-
essary to turn on the Power suly unit rst.
Figure 4.2: The icon of RAMAN lmager
Figure 4.3: The main window of RAMAN lmager just after showing u
4.1.3 How to quit RAMAN lmager
1. Closing RAMAN Imager with clicking Close button at the top
right of the window.
63
Attention lt is necessary to quit RAMAN lmager before turning o the
ower suly unit. Otherwise the comuter will hung u because of the
intercetion between RAMAN lmager and devices.
Figure 4.4: How to quit RAMAN lmager
4.2 Common control panel
Aellations of arts in the Common control anel are as below.
64
1. Miroscope monitor
2.Illumination contoroller for
microscope
3. Z stage
controller 4. Memorandum
to be saved
5.Display assist
Figure 4.5: Common control anel
1. Otical microscoe monitor
The otical microscoe monitor shows the microscoe image of samles
talen with a CCD camera.
2. lllumination controller for microscoe observation
Transmission lam
The controller for illumination of transmission observation. This is
controlled with 255 stes.
Reection lam
The controller for illumination of reection observation. This is con-
trolled with 255 stes.
3. Memorandum
This is the eld that an oerator write down memorandum about mea-
surement condition. The memorandum with measured data is transfered
to data of RAMAN Viewer at every measurement.
65
Oerator Name
Samle Descrition
Comments
4. Z stage controller
U and Down buttons
These buttons move the stage with the distance of Ste.
Ste box
Travel distance of one ush of U and Down buttons. The unit
is m.
Pos
This box shows the relative height of the stage.
Z Rest button
This button resets the origin of the stage height and inuts 0 m
in Pos
Focus Handle button
This button switches the seed of the z stage movement with three
stes.
5. Dislay assistant
Scale
This checl-box controls the show,hide of the scale bar on the micro-
scoe monitor.
Cursor
This checl-box controls the show,hide of the cursor indicating laser
irradiation osition on the microscoe monitor.
6. XY stage controller Otional
lf a motorized XY stage is equied, the xy osition is controlled.
4.3 Measurement setting panel
The anel of Standard Raman measurement Raman is main anel. ln addi-
tion, There are Surface roling measurement Surface, Raman imaging in an
66
wide-eld area WF Raman, microscoe observation of an wide-eld area WF
Observation and automatic multi well (m n} measurement m n (Auto
multi-well measurement}. These anels are selected by clicling the tab located
at the to of the anel.
Figure 4.6: Switch of the measurement anel with the tab
4.3.1 Raman
Measurement arameters for Raman measurement are set on the Raman anel.
1. Measure button
2. Scan mode
3. Obj.lens
4. Measurement time
5. Laser
6. Spectral setting
7. Cursor position
8. Scan setting
Figure 4.1: The Raman anel
67
1. Measure button
This button starts the measurement according the measurement arame-
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 function
is used. (Please refer to the section of Trigger measurement}
The text is Measure Ne Sectrum, Measure Si Sectrum and so on
when the Reference samle function is used. (Please refer to the section
of Reference samle
2. Scan mode
Please select the Scan mode.
4.3.2 Scan mode
There are eight scan modes as below (including two otional scan modes.}
(a} Point A sectrum at a oint is measured.
(b} x-Line Plural sectra at lural oints along a line are measured.
(c} xy-imaging Two dimensional xy sectrum imaging.
(d} xz-imaging Two dimensional xz sectrum cross-sectional imag-
ing.
(e} Point-z Plural sectra at a oint with dierent height are mea-
sured.
(f} xy-maing Two dimensional xy sectrum maing.
(g} xy-z-imaging(Otional} Plural xy-sectra imaging are measured
with z scanning.
(h} xy-z-maing(Otional} Plural xy-sectra maing are measured
with z scanning.
3. Objective lens selection box Obj. lens
The objective lens which are used currently should be selected. The correct
68
setting of the objective lens give correct information about the las
4. Measurement time
Lxosure time Lxosure s,shot
The exosure time that is equivalent to laser irradiation time at every
scan should be inut.
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 equal
to the exosure time.
For XY-imaging mode, the total measurement time is equal to the
exosure time multilied with the scanning number.
5. Laser
Laser ower monitor
Laser ower irradiated to the samle is shown with mW unit.
The laser ower is measured with the ower meter equied inside
the instrument and is shown in real time. The dislayed laser ower is
the value after calibration with the transmission of the otical system
and the objective lens.
The laser ower when the laser line beam is used is the value at
each oint along the line. The calibration coecient is derived from
the ratio of the sectral eal intensity of Silicon crystal measured
with the line and oint focus of the laser.
Laser wavelength dislay
The wavelength of the laser used to excite Raman scattering currently
is dislayed.
Laser injection current Current
The lasing ower can be controlled with the laser injection current.
The unit of the current is %.
ND lter controller ND lter
The laser irradiation ower to the samle can be controlled with
69
variable ND lter. The ower is determined with the transmission of
the NF lter. The transmission is controlled wit the ND lter slider
with 255 stes.
ND lter setting monitor
The ND lter setting is shown as the Transmission and the osition
index of 255 stes.
Checl box Checl Laser Sot
Checling this box oens the mechanical shutter stoing the laser
beam and irradiates laser beam on to the samle. This function may
be used to observe and conrm how the laser irradiation is made on
the samle surface.
The checling laser beam is convenient when the oerator brings the
samle in the focus.
6. Sectroscoy setting
Sectral Range
The range of measured Raman sectrum is shown.
Center Wavenumber and Set button
To set the sectral range, the center wavenumber of the sec-
tral range should be inut in the Center wavenumber box and
afterward the Set button should be ushed.
Crating Selection box
A grating among three gratings can be selected. By choosing the
grating, the motorized grating turret will rotate and the grating chose
is set.
1. Cursor osition Position (x: ix, y : ix}
x osition x, y ositiony
The coordinate of the cursor osition indicating laser focus sot is
shown. The unit of the coordinate is ixel of the microscoe monitor.
The width and height of the monitor are 640 ixel and 480 ixel
resectively.
The coordinate of the to and left is shown in the case that the cursor
is rectangle area or line shae for the XY-imaging, X-line mode and
so on.
70
Scanning area box
The width and height of scanning area is shown in the coordinate of
the microscoe monitor.
8. Scan Setting
The scanning setting arameter anel changes according to the scanning
mode.
point
No setting arameters.
No setting arameters.
xy-image
Y ste
The scanning number in y direction is automatically inut when the
scanning area is changed by the clicl-and-drag on the microscoe
monitor. The number is calculated so that the ixel size in x direction
is equal to the size in y direction.
The oerator can also inut any integer number manually.
The scanning interval in y direction nm
The unit is nm.
xz-image
Z ste
71
The scanning interval in z direction nm
The unit is nm.
Z Start and Read button
The beginning osition of the z scanning should be inut in the unit
of m. Pushing button inuts the current osition to the Z Start.
Z Lnd and Read button
The end osition of the z scanning should be inut in the unit of m.
Pushing button inuts the current osition to the Z Lnd.
point-z
Z ste
The scanning interval in z direction nm
The unit is nm.
Z Start and Read button
The beginning osition of the z scanning should be inut in the unit
of m. Pushing button inuts the current osition to the Z Start.
Z Lnd and Read button
The end osition of the z scanning should be inut in the unit of m.
Pushing button inuts the current osition to the Z Lnd.
72
XY-mapping
delta
The scanning interval of XY-maing is inut. The unit is nm.
x,y
The scanning numbers in x and y direction resectively are shown.
With the inut of the interval, the scanning numbers are calculated
from the interval and the area the width and height automatically.
4.3.3 Surface(Otional}
4.3.4 Wide-eld Raman measurement WF Raman(Otional}
The measurement arameter of wide-eld Raman measurement is set in the
WF-Raman measurement anel. This function is rovided with the motorized
xy-stage otion.
The measurement rocedure is exlained in the chater of the measurement.
73
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 anel
1. Measure WF Raman button
This button starts the wide-eld Raman measurement according to the
settings.
2. Measurement Area
The measurement area of the wide-eld is determined by setting the start
osition and the end osition. The start osition should be to left and
the end osition is the right bottom of the rectangle area.
Read To Start button
This button sets the current osition as the start osition.
Read To Lnd button
This button sets the current osition as the end osition.
74
Start box
This box shows the start osition in the coordinate. The unit is
mm.
Lnd box
This box shows the end osition in the coordinate. The unit is mm.
3. Obj. lens
The objective lens used currently should be selected. By selecting the
objective lens, the scale bar on the monitor is udated. The laser ower
monitor is also udated described later..
4. Measurement time
Lxosure s,shot
The signal recording time, equivalent to the laser irradiation time, at
every scanning is inut.
For the oint mode, the exosure time is equal to the measurement
time.
Total measurement time
The total measurement time is calculated by the exosure time and
the scanning number. h,m and s are the units of hour, minute
and second resectively.
5. lmage Quality of the wide-eld measurement (the resolution}
The image quality is determined automatically not to exceed the memory
of the comuter. When the are is large, the ixel of the wide-eld image is
binned in x direction. The scanning number in Y direction is automatically
determined so that the ixel size in x and y direction are the same.
Fine
The total ixel number is 160,000.
Standard
Coarse
75
Custom
The oerator can set the total ixel number.
6. The information of the wide-eld image.
The information of the wide-eld image is shown.
Pixel size
The Width and the Height are shown in the unit of m.
Pixel Number
The ixel number of the Width and the Height are shown.
1. Measurement area conrmation button
This button is used to conrm the measurement area.
Move to Start button
Move to LND button
8. The setting of the motorized xy stage.
Move Seed
The seed of the wide-eld scanning is set.
1 mm,s
0.01 mm,s
0.05 mm,s
0.005 mm,s
Move Sto
This button stos the motorized stage moving.
4.3.5 Wideeld observation WF Observation (Otional}
WF observation is used to observe and record the wide-eld microscoe image.
The microscoe images in the wideled area recorded while the xy stage scanning
and are stitched to one large image.
On the stitched wide-eld image, measurement area of WF-Raman can be de-
teremined.
76
1. Measurement buttons
2. Obj.lens
3. Measurement Area
4.WF-Raman
Measurement Area
5.Button for showing
WF-microscope image
monitor
6.Setting for
motorized XY stage
WF Observation anel
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-eld microscoe image are generated
from m by n microscoe images. m is the number of images in x
axis. n is the number os images in y axis. The orgins of these m by
n images are dened as the software Origin (SO}.
Reset SO
This button sets current osition as the software origin.
Origin setting
77
Center
The software origin is located at the centerof the wide-eld mi-
croscoe image.
TL (To Left}
The software origin is located at the to and left of the wide-eld
microscoe image.
TR (To Right}
The software origin is located at the to and right of the wide-
eld microscoe image.
BL (Bottom Left}
The software origin is located at the bottom and left of the wide-
eld microscoe image.
BR (Bottom Right}
The software origin is located at the bottom and right of the
wide-eld microscoe image.
Setting unit
Frame
The unit is the number of microscoe images.
mm
The unit is the distance. The distance will be tranlated to the
number of microscoe images automatically.
4. Setting measurement area of WF-Raman
The measurement area of WF-Raman can be set by clicl-and-drag on the
wide-eld microscoe iamge. The measurement area of WF-Raman will be
shown as the coodinates of the Start osition and the Lnd osition.
5. Dislaying wide-eld microscoe image
Dislay Wide Field checl box
After measuring the wide-eld microscoe image, the image is dis-
layed on the microscoe monitor. Uncheling this box changes the
miroscoe monitor from the measured wide-eld microscoe image
to real-time microscoe image.
78
Dislay realtime monitor button
This button shows the another microscoe monitor for real-time mi-
croscoe image while the measured wide-eld microscoe image is
dislayed on the microscoe monitor.
Dislay lmage Zoom slider
This slider adjusts the zoom of the wide-eld microscoe image.
Dislay osition
The current eld view osition of real-time microscoe is shown as
the red rectangle while the wide-eld microscoe image is shown.
The clicl-and-drag of the red rectangle area moves the zoomed area
shown in the microscoe image.
Dislay osition zoom slider
This slider adjusts the zoom of Dislay osition.
6. The control of motorized stage
Co To SO button
This button moves to the software origin.
Find HO button
This button nds and moves to the hardware origin.
Move Sto button
This button stos the movement of the motorized stage.
Move Seed box
The seed of the stage movement of WF-Raman and WF-observation
can be selected.
1 mm,s
0.01 mm,s
0.05 mm,s
0.005 mm,s
4.3.6 Auto well-late measurement(m n } Otional}
Auto well-late measurement if m n wells is done in this anel. This function
is rovided otionally. The number of m and n are set according to the stage
and the well tye.
79
The rocedure are exlained later,
1.Button for
starting measurement
2. Setting status
of wells
3.Obj.lens
4.Scan mode
5. Measurement time
6. Sequence
7. Measurement area
8. Sample name
9. Button
for applying the
setting to all
10. Button for stopping the
stage movement
The anel of Auto well-late measurement(m n }.
1. Start mxn measurements button
2. Overviews of wells.
The overview of well late and the status of each well setting are shown.
The status of the wells The status of the wells are shown by the
dierent color. The well sell is selected by the clicl and turns to
be ready for setting the arameter. The double-clicl moves current
osition to the well.
White
The measurement arameter setting is not done.
Pinl
The selected well for the arameter setting.
80
Blue
The measurement arameter setting is done.
Select
Aly button
Clear button
3. Objective lens
4. Scan mode
The scan mode should be selected for the current selected well.
5. Measurement time
Lxosure (s,shot} for the selected well.
Time , a samle for the selected well.
Total time for measurement of all wells.
6. Sequence of the well
The Sequence of the well should be selected. The white well will be slied.
1. Measurement area
The measurement area for the selected well is shown.
8. Samle Name box
The name of the samle in the selected well can be inut and is shown.
The name will be saved with the data.
9. Setting at once
Aly to all samles button
This button alies the arameter in the selected button to all other
samles.
Clear all samles button
This button clear the setting of all wells.
10. Sto button
This button stos the movement of the motorized stage.
81
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 Microscoe lmage
This saves the microscoe image on the microscoe monitor to an
image le.
Save Real-Time Frame
This saves the real-time confocal reection image (Plus} to an image
le.
Save Surface Prole
This saves the surface role image measured with the Plus function
to an image le.
Save Lxtended deth image This saves the extended deth image
image measured with the Plus function to an image le.
Save,Load Settings
This shows the Save,Load Settings windows. The measurement
arameters are saved or loaded with this function.
2. Function
Laser
This shows the laser setting window. From the window, the wave-
length of the laser can be selected. The laser outut ower can also
be adjusted by the injection current.
lmage Quality & Sectral ROl
lmage binning and sectral ROl are set for the urose of the com-
ressing the data size, the imrovement of the data quality and the
shortening of measurement time.
Auto Save Settings
This functions determines the auto-saving setting after measurement.
82
Trigger Setting
This function enables time-course measurement (lnternal trigger
mode}. This function also rovides the measurement with combina-
tion with external devices (Lxternal trigger mode}.
Data Calibration and Comensation
This function rovide the setting of data calibration and comensa-
tion.
Assist
This includes the sulementary functions.
Reference Samles
The reference smales equied inside RAMANlus are utilized through
this function.
Device,Sectrograh
Sectrograh setting is rovided.
Device,CCD Detector
Thermo-electrically cooled CCD detecotor setting is rovided.
Device,lllumination for Otical Microscoe
The on,o controll setting of microscoe transmission,reection set-
ting.
Device,Microscoe Z stage
The motorized z stage settinn is rovided.
3. Window
Detector Temerature
This shows the Detector Temerture widnow.
Stage controller
This shows the Motorized stage control widnow (otinal}.
4.4.2 File,Save Load Settings
Menu,File,Save Load Settings
Save Load Settings will be shown.
83
Figure 4.9:
1. Name list of Measurement aramters
For saving the current measurement arameter, the name of ther ara-
menter into the list. For loading the measurement aramter to the current
measurement arameter, select the arameter name from the list.
2. Load button
This button loads the arameter on the list to the current measurement
arameter.
3. Save button
This button saves the current measurement arameter to the list with the
arameter name.
4. Delete button
This button deletes the selected arameter on the list.
4.4.3 The wavelength and the outut ower 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.Laser
4.Injection current control
84
Figure 4.10:
1. Selected wavelength box
The laser wavelegths that are quied in RAMANlus are listed. After
selecteing the wavelength, laser waveleght will start to be chaged. lt talse
a few ten seconds to nish changing the wavelength.
2. Laser ower monitor
Laser ower on the samle is shown when the laser is irradiated.
3. Lasing laser
The checl box of the laser under lasering shows the checled status. Checl-
ing the box of laser under no lasing males the laser lasing.
Some of the lasers need a few minutes to lase after checled.(e.g.185nm
laser}
4. lnjection current control
The injection current can be contolled to adjust the laser outout ower.
After moving the slider, the Set button should be ushed to set the
current.
Some of the lasers have no injection current control.(e.g. 185nm laser}
4.4.4 lmage Quality & Sectral ROl Function,lmage Quality &
Sectral ROl
Menu,Function,lmage Quality & Sectral ROl
lmage Quality & Sectral ROl window is shown.
This function rovides the setting about the data size, sectral and image
quality.
85
Figure 4.11:
Image Quality
The image quality in xy-imaging, xz-imaing mode and so on, can be
adjusted to decrease the data size and imrove the sectral quality. 400 sectra
along a line illumination are measured simulataneously by default (Fine}. ln
the case of Standard, 200 sectra are measured by combining two neighbor
sectra to one sectrum.
lt is called binning that lural ixel are combined to one ixel.Combining
lural ixel increas the sigal at a ixel. Because the number of read-out (AD
conversion} is decreased, the read-out noise is also decreased.
Fine
400 sectra er a line. No binning is done.
Standard
200 sectra er a line. 2 ixel binning is done.
Coarse
100 sectra er a line. 4 ixel binning is done.
Custom
The number of the binning is set arbitrarily.
Spectral ROI
The sectral range is trimmed to decrease the data size.
86
ROl Region of lnterest
Range inut box, From to
Trimmed sectral range shown in From to.
Add button
This button shows range inut window.
Deletebutton
This button deletes the selected range.
4.4.5 Auto Save Settings Function,Auto Save Settings
This function sets auto-save arameters.
Menu,Function,Auto Save Settings
Auto Save Settings is shown.
Figure 4.12:
Auto-save function works dierently according to the scan mode.
x-line, xy-imaging, xz-imaging, xy-maing, oint-z
All aramters for the auto-save function are available.
oint mode
The name setting is used for the measured sectral name. The Multi-
Sectral View data is not saved automatically.
xy-z-imaging, xy-z-maing
For these measurements, the folder setting for auto-save is necessary. The
measured data are always save and closed automatically.
87
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 checl box
Auto-save fuction is enbale,disable.
Auto Close checl box
Auto-close fuction is enbale,disable. Auto-close is available only when the
auto-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. The
name is determiend with the combination of 4 aramters.
4 paramters for the naming
Scan mode
Samle Name
Oerator Name
Date Time
4.4.6 Trigger function Function,Trigger
This function enables time-course measurement (lnternal trigger mode}. This
function also rovides the measurement with combination with external devices
(Lxternal trigger mode}.
Menu,Function,Trigger
Trigger settin window is shown
88
Figure 4.13:
External trigger mode starts measurements with the trigger signal from
the external device.
The trigger signal is neglected while measurement. The next measurement starts
with the trigger while the idling time.
The TTL singal is accetable for the trigger through the BNC connector
(Trigger ort} behind the ower suly unit.
Internal trigger mode starts to reeat measurements with the constant
interval Cycle(ms} using the clocl inside the comuter.
Trigger mode selection button
Trigger o
Lxternal trigger
lnternal Trirgger
Measurement interval Cycle time
This determines the interval of the internal trigger mode.
Samling count
The number of measurement of the trigger mode should be set. With the
number of 0, the measurement is reeated endlessly till Sto measure-
ment is ushed.
Measurement procedure with trigger mode.
1. Setting
Setting of Lxternal trigger or internal trigger on the trigger setting window.
89
2. Stopped state
Closing the trigger setting window. The descrition on the Measurement
button changes to Start Waiting Trigger. With the status of Start
Waiting Trigger, the measurement will not start when internal,external
singal is detected.
3. Idling state
Changing to idling state by ushing Start Waiting Trigger button. The
measurement will not start while the trigger signal does not come. After
ushing the button, the descrition will change to Sto Waiting Trigger.
4. Measurement state
At the moment when the trigger signal comes, the measurement starts
automatically. While the measurement, the trigger signal is ignored.
5. Idling state
After the measurement, the idling state restarts. At the same time, the
measured data will be saved and closed automatically.
6. Termination of the trigger mode To terminate the trigger mode, The Sto
Waiting Trigger button should be ushed. Or the trigger mode is nished
when the the number of Samling count measurements are done.
The descrition on the button will be Start Waiting Trigger button.
h
gK
gK or
gK
Figure 4.14:
4.4.1 Function,Data Calibration and Comensation
This function sets calibration data and comensation data.
Menu,Function,Data Calibration and Comensation
90
Figure 4.15:
The following three calibration and comensation data are set. These data
should be reared in advance. The data are utilized as the data transfered to
the RAMAN Viewer with measured data.
Wavelength calibration data
This data is used as the wavelength data of Raman sectrum. Without the
setting, the wavelength data calculated from the data of the grain and its
angle automatically is used. With this setting, the wavelength data, that
the oerator reare, is used.
Sectral intensity comensation data
The sectral intensity attern is distorted with QL curves of detector and
grating, and transmittance curve of otics. No comensation is roceeded
without this setting.
Flat-eld comensation data.
This data comensates the sensitivity dierence between ixels of the de-
tector. No comensation is roceeded without this setting.
4.4.8 Function,Assist
Menu,Function,Assist
91
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 ray
noise. This function males measurement time longer.
Bee sound
This function set the bee sound informing the end of measurement. No
bee sound is set as default. Two linds of sounds are equied.
Otical Line Shaer
As default, an otical element otical line shaer is used to form the
line shaed laser illumination for xy-imaging measurement so on. Without
92
the otical line shaer, the quasi-line illumination, so-called ying spot
illumination is utilized for xy-imaging.
Line illumination method Line length ower at a oint at a moment
Otical line shaer Fixed Low
Flying sot Arbitrary High
Wobbling for the line illumination
Wobbling is used with otical line shaer. Cenerally the intensity attern
of line illumination by the otical line shaer is not uniform along the
line. The high intensity at the center and the lower intensity at the edges.
To obtain uniform intensity attern with the otical line shaer, the non-
uniform attern is wobbled in the line direction (atented}. The length of
the wobbling is selected.
Long
Medium
Short
Automatic Shutter OPLN,CLOSL
The mechanical shutter stos laser irradiated to the samle while no mea-
surement (CLOSL state}. To irradiate the laser to the samle, the shutter
should be removed from the laser ath (OPLN state}. While the measure-
ment, the shutter reeats OPLN and CLOSL state synchronizing with the
exosure time as default.
With this function disabled, the shutter lees CLOSL while measurement.
This function is uses to measure the baclground signal of this instrument.
Auto ND Filter Adjustment at Shutter OPLN,Close
This function is auto-adjustment of ND lter when the laser is irradiated
to the samle by Checl Laser Sot button. As default, this function is
enable and the ND lter is set at the osition of the minimum laser ower.
Set button inuts current ND lter osition as the osition when the
shutter is oened.
93
4.4.9 Standard samle Function,Reference Samles
Silicon crystal is equied inside the instrument as a standard samle. The
sectrum of the Si can be measured through this function. The neon lam is
also quied and its sectrum can be measured.
Other standard samles may be rovided otionally. ln the case the otional
standard samle is transarent such as quartz, the sectrum of the samle on
the stage can be measured with the sectrum of the standard samle. The
standard sectrum of the standard samle gives the exact information of the
unlnown samle.
Menu,Function,Reference Samles
Figure 4.11:
Silicon crystal
Neon lam for the wavelength calibration.
Quartz (otional}
4.4.10 Function,Device,Sectrograh
Menu,Function,Device,Sectrograh
94
The following three arameters can be set. The unit of sectrum is nm.
Crating
Range of wavelength
Slit width
60 m width is set as default. Narrower slit width, higher sectral and
satial 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
95
Figure 4.18:
The following three arameters can be set.
ADC Seed
AD conversion seed of the ixel of the CCD is selected between 2MHz and
100lHz. Higher ADC seed, the bigger readout noise. 2 MHz is selected
as default.
2MHz
lt tales about 0.25 second to transfer the signal of 400 1340 format.
( 400 1340 2000000 = 0.25}
100lHz lt tales about 5 second to transfer the signal of CCD of 400
1340 format. ( 400 1340 100000 = 5}
Readout ort
There are two readout orts.
Low Noise(High Sensitivity}
300,000e- (ty.}, 250,000e- (Min}
High Caacity
1,000,000e- (ty.}, 150,000e- (Min}
Cain
lt is the gain of CCD. lt determines the number of electron generation for
a single hoton. The high gain is set as default.
High
16e- with High Sensitivity ort, 4e- with High Caacity ort
96
Medium
Low
4.4.12 Automatic on,o of microscoe illumination. Function,lllumination
for Otical Microscoe
The microscoe illumination both for reection and transmission is turned o
automatically while the measurement. This setting can disable this automatic
on,o. This is enable as default.
Menu,Function,Device,lllumination for Otical Microscoe
Figure 4.19:
LPl LLD
LLD for reection.
Transmission LLD
LLD for transmission.
4.4.13 Motorized z stage Function,Device,Microscoe Z stage
Menu,Function,Device,Microscoe Z stage
97
Figure 4.20: The window for the setting of motorized z stage
Ste width
The ste width when U,Down button is ushed.
Tolerance
Motorized Z stage equied with linear encoder and moves with closed
feedbacl system. The tolerance can be set.
98
Chapter 5
To understand Raman scanttering
lt is imorant to understand of basis of Raman scattering to utilize the many
linds of functions of RAMANluseectivellly. ln this chater, lt is exlained
about Raman scattering including the relation between molecular vibration and
Raman sectrum, a concet of a sectral image, and eal shift imaging.
5.1 Raman spectrum
The acquirable data of RAMANlusis the Raman sectrum. Figure below de-
icts a Raman sectrum of articles observed with an otical microscoe. The
measured area is given by the yellow crossed-cursor in the microscoic image.
The sectrum accords well with that of olystyrene, the material of which the
articles are in fact art of. The Raman sectrum of any oint on the samle
can be measured by simly clicling the mouse while observing a microscoic
eld-of-view on a monitor.
99

Polystyrene
Microscope image
Figure 5.1: The sectrum of olystyrene
5.2 Raman scattering
lncidental light gets scattered by materials (Figure below}. lf the incidental
light is of a single wavelength the scattered light is also of a single wavelength
(Lx. Creen incidental light leads to green scattered light}. However, recisely
analyzing the color of scattered light revealed extremely weal light whose color
slightly diers from that of the incidental light. This henomenon is called Raman
scattering and the scattered light is Raman light, both of which are named after
the discoverer of them, C. V. Raman.
Raman scattering is caused by the interaction between the incidental light and
molecular vibrations of a material. When the incidental light gets scattered by
the molecular vibrations the light gains (or loses} amounts of energy corresond
to the molecular vibrations and the wavelength of the scattered light shifts from
incidental. The relationshi between the energy and wavelength of light can be
exressed with the following equation:
E = h = hc/
(L: energy of light, : wavelength, : frequency of light, c: light seed, h:
Plancls constant}
To reresent the change in Raman light color a grah called the Raman sec-
trum (Figure 3.3} is used in which the horizontal axis lots the energy (color,
100
wavelength} shift amounts of Raman light while the vertical axis lots the Ra-
man light intensity with each shift in energy. Because the energy of molecular
vibrations is discrete the amount of energy shifts of Raman light also becomes
discrete, thus maling the Raman sectrum discrete along the horizontal axis.
Or that is to say, the Raman sectrum generically has a number of shar eals
(called Raman eals} within it. The energy of the molecular vibrations cause
disrutions in a secic range of the sectrum through uctuations caused by
thermal vibrations, and the band width of the Raman eals is nite.
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 sectrum
Abscissa of Raman sectrum is the dimension of envergy dierence that is
inverse of wavelength and is called wavenumber or Raman shift idiomatically.
The unit is cm
1
101
Wavenumber(cm
1
) = 1/
laser
1/
Raman scanttering light
The Raman scattering light which has higher energy (shorter wavelength} is
called anti-Stoles Raman and the Raman scattering light which has lower energy
(longer wavelength} is Stoles Raman. The eal ositions of stoles,anti-stoles
Raman scattering light from the Rayleigh light are the same while the intensity
of Raman light with Stoles raman is bigger than that of anti-stoles.
Raman sectrum is identical for each molecule. The sectra below are of
ribose and glucose. These sectra has many eals at the dierent osition. lt
is ossible to identify the material from Raman sectrum.

Different molecule has
different set of peaks.
Figure 5.4: Raman sectra of dierent molecules

Figure 5.5: Dierence of structure also leads to dierent Raman eals
102
Not only the interaction with the energy of molecular vibrations but also the
interaction with the energy of the lattice vibrations induces Raman scattering.
Figure 3.5 gives the Raman sectra of grahite, coal and diamonds. All of them
consist of carbon but each has its own individual Raman sectrum because the
crystal structure and energy of the lattice vibrations dier from each other. The
width of a Raman eal reects the urity of the crystals (singularity of the lattice
vibration energy} while the osition of a Raman eal reects the energy of the
crystal vibrations, resectively. ln addition, the stress within the crystals distorts
the crystal lattice, thereby resulting in a shift in the osition of a Raman eal.
This therefore allows ositional shift analysis of Raman eals to be utilized in
stress analysis.
5.3 Raman imaging (distribution of materials)
An image of the material distribution of a samle can be created by measuring
the Raman sectra from every oint (ixel} in the eld-of-view of an otical
microscoe and identifying the tye of material that exists at each oint. Fig-
ure below gives a Raman image of the material distribution within the yellow
rectangular area selected in the eld-of-view of an otical microscoe. Otical
microscoic images do not reect the material of an object but a Raman im-
age reveals that it contains two linds of material there. ln this Raman image,
the Raman eal at 1610cm-1 is colored green while at 1138cm
1 it is red, the
former being inherent with olystyrene while the latter PMMA from database
information. The sectrum of the green area and the red area in this Raman
image reveals the tyical forms of Polystyrene and PMMA, resectively.
103

Raman image
Raman spectrum
Optical microscope image
Figure 5.6: Otical microscoic image and Raman image
This Raman image is 400 ixels (in the x direction} x 400 ixels (in the y
direction}, for a total of 160,000 sectra, with one sectrum dislaying 1,340
oints along the horizontal axis (wave-number}. The data structure of a Raman
image, therefore, can be assumed to rovide a data set of 400(x} x 400 (y} x
1340(}. As described above an image that contains sectrum information at
each ixel is called a sectrum image. To enable a material to be more intuitively
understood and analyzed 3 x-y sections were selected at 1440cm-1, 2840cm-1
and 2930cm-1, colored blue, green and red, resectively, and then overlaid in the
color image given in Figure 3.1. ln addition, RAMANlus includes the function
of Peal-shift imaging through use of which the amount a articular Raman eal
shifts at each ixel can be visualized (Figure}. lt rovides a articularly a good
method for analyzing the stress distribution of a crystal.
104

Color image
W
a
v
e
n
u
m
b
e
r (c
m
-1
)
Figure 5.1: Data structure of Raman image
Distortion in a crystal induces a frequency shift in Raman light when comared
to a crystal without any distortion. The comressive stress in silicon shifts the
Raman eal (520cm-1, 2g otical mode} to a higher frequency while the
tensile stress shifts it to a lower frequency. RAMANlusincludes other methods
of roviding sectrum images, which are exlained in the data dislay and analysis
manual.
105

Stress distribution of silicon substrate
(Peak shift imaging)
160,000 spectra (400 x 400 pix)
Raman shift [cm
-1
]
R
a
m
a
n

i
n
t
e
n
s
i
t
y

[
a
.
u
.
]
Figure 5.8: Peal-shift image
5.4 Sectional Raman imaging (xy Raman image)
RAMANlusincororates confocal otics, thus maling internal sectional Raman
images of transarent samles ossible. As described above, otically gaining
internal sectional images of a samle, without actually having to slice a samle
into sections, is called otically slicing or otically sectioning a samle. Figure
below gives internal sectional Raman images of a cell. Four Raman images are
obtained by dislacing the focal lane in increments along the z axis.

Optical sectioning image
with confocal optics
Raman images
of living HeLa Cells
at different hieght.
Figure 5.9: Sectional Raman imaging of living cell
106
5.5 Cross-sectional Raman imaging (xz Raman image)
The confocal otics mounted in RAMANlus enables cross-sectional Raman
imaging of transarent samles (Figure below}, thus eliminating the need for
samles to be cut and their cross-sections exosed.

Multi-layer analysis
Green: Polyethylene (PE)
Orange: Nylon (PA)
Blue: Polypropylene (PP)
Figure 5.10: 400 120 = 480,000 sectra (480,000 ixels}
107
Chapter 6
Support
Please contact with Nanohoton cororation when the roblems about this rod-
uct that is not solved within this oerational manual haens.
Nanohoton cororation
Tel 81-6-6818-9911
Fax 81-6-6818-9912
Lmail: infonanohoton.j
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