CMi SOP003 Operating Procedures Manual

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CMI STANDARD OPERATING

PROCEDURE

Confocal Raman Microscope

Physiology Ground floor research lab

CONTACT

Peter Owens: 091 494036 (office) [e-mail]

Jennifer Connolly: [e-mail]

REQUIREMENTS FOR EQUIPMENT USAGE:

1. CMI user

2. Completion and signing of Microscope Safety Checklist

3. Certification by Peter Owens or Jennifer Connolly

REVISION LOG

INFORMATION:

CMI document ID: SOP003

Revision

1.0

Date

12/5/14

1. Purpose

1.1. This document specifies the work instructions for the CMI Witec Confocal Raman microscope located in
the ground floor Physiology lab, Quadrangle building. If you see an area where more clarification is
needed, if additional information is needed, or if you have suggestions on how to make this guide more

1.2. Note that this document is not a detailed instrument manual and does not intend to be one. For

useful in the lab, please contact the CMI.

detailed questions or if anything unusual happens with the system, please refer to the manuals
present in the lab, or ask CMI personnel for help.

Author

P. Owens

Changes

Initial draft

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2. Scope

These work instructions are applicable to all work that is carried out using the Witec raman microscope.

3. Important notes

3.1 Laser safety guidelines

PLEASE LEAVE ALL HARDCOPIES IN THE LAB

The WITec microscopes uses class 4 633 and 785 nm laser sources during operation. The
safe use of lasers requires that all laser users, and everyone near the laser systems, are aware of the
dangers involved.
CAUTION: Direct eye contact with the output beam from a laser will cause serious damage and possibly
blindness. The 785 nm laser has a high power output, extreme care is needed here when operating the

system.

3.1.1 Make sure the enclosure remains closed during laser scanning and measurement

3.1.2 Always close the laser shutter before opening the door of enclosure

3.1.3 Always wear the safety laser goggle when you want to open the door with laser on for
alignment procedure.

3.1.4 Participation in a Laser safety course is strongly recommended for operating this system.

3.2. Witec Alpha 500 manual (hardcopy in the lab, soft copy on the instrument PC), and training notes

3.3 The CMI access policy, available online at
http://imaging.nuigalway.ie/access%20policy/cmi_access_policy_1.6.pdf

3.4 This manual was developed to assist in the training process of users. Be aware that only the basic
operation details will be presented. Please contact the CMI staff for more assistance if required.

3.5 Changes may occur when a new software version or patch is installed. Please contact the CMI
staff if you are not sure about new features and functions.

3.6 Do not forget to sign in to the log book before you start your measurement.

3.7 Turn on the laser power when you begin, use laser shutter in-between measurements.

Responsibilities

4.

Operators of this equipment are responsible for the following:

4.1 Complying with all safety regulations.

4.2 Compliance with procedures and specifications contained in this document.

4.3 Reporting misuse of the instruments, or in a manner inconsistent with this specification, by any

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personnel, to the supervising CMI staff.

4.4 Maintaining a clean workspace. Food and drink are NOT allowed!

4.5 Reporting any and all maintenance issues/concerns to the supervising CMI staff member

5 Raman overview

Raman spectroscopy is a characterization technique that is widely used in scientific field in recent years. It
actually utilizes the unique Raman spectra for different components as a spectral “finger print” to identify
unknown samples or even further analysis based on the information from the spectra. Raman spectra can be
obtained from various kinds of materials from bulk solids such as paper and cellphone, to nanomaterials such
as thin films and nanoconstructs.

Raman Spectroscopy has many advantages among many characterization techniques, and therefore it is quite
welcome in scientists and laboratory workers. Typically, the samples need little preparation before
characterization and analysis can be carried out through many containers. Raman Spectroscopy is usually not
destructive to samples unless you use too much laser power and focus high-energy laser on a point of the
sample, leading great amount of heat in that small area and thus burning your sample.

In addition, Raman Spectroscopy is typically a fast characterization technique that can perform real-time scan.
It can acquire a Raman spectrum of most substances in seconds via Charge Coupled Device detectors (CCDs)
that have a wide dynamic range for users to select the appropriate exposure time for their sample. Depending
on the raman cross section of the sample of interest, a high resolution scan can be finished in minutes using
the system.

Raman Spectroscopy can do both qualitative and quantitative analysis. Quantitative analysis of the sample is
typically performed by measuring the relative intensities of each peak in the Raman spectrum that are directly
proportional to the relative concentrations of the compounds. Alternatively, chemometric methods can be
used for detailed information and accurate calculation. These quantitative analyses are very sensitive that can
be performed on samples with high concentrations ranging from 90-100% material of interest down to
concentration determination at parts per billion (PPB) levels.

This instruction will guide you to run a Raman spectroscopy scanning step by step and help you obtain your
first Raman spectrum/ image from the WITec Raman Spectroscopy Instrument. It can also be used as a manual
book for laboratory users in case of operational problems and troubleshooting. This instruction is separated
into sections for instrument overview, step by step alignment and calibration check, acquisition, imaging, and
troubleshooting. Readers should follow the procedures in this instruction in order to operate the Raman
instrument properly.

DANGER: Class 4 laser radiation involved during the procedure!

immediately.

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Fibre to detection

Adjustment

alignment

Push/pull rod for
Laser coupler

and filter wheel

Eye piece
camera

785nm laser

Brightfield/DIC/Ra

6 System Overview

man imaging
selection

system. Use 50 micron
fibre for 633 nm, 100
micron fibre for 785nm
excitation, fibre
diameter is part of the
serial number taped on
fibre. Extreme care

needed when handling

screws for

eyepiece/detector

The witec confocal raman microscope consists of the following devices:

6.1 Instrument Control PC, 64 bit running witec control and project software (version 1.6)

6.2 Controller unit.

6.3 Upright microscope with led light source for brightfield work.

6.4 Objectives including a 40x water dipping lens and 100 x air

6.5 Andor CCD cameras

6.6 Witec diffraction gratings for either vis or NIR operation.

6.7 Motorised stage for xyz control

6.8 Fibre optic cable for 633nm use: 50micron diameter

6.9 Fibre optic cable for 785 nm use : 100 micron diameter

Familiarise yourself with the system folders, located on the shelf above the equipment.

7 Start Up Procedure

7.1 Book equipment on CMI web booking system.

7.2 Turn on lasers or individual laser, if only one is to be used. For the 633 laser, turn on the key. For
the 785 nm laser, turn on power first and wait until the green led stops flashing. Then switch the
laser key to ‘on’ and again wait until the green led stops flashing and both yellow and green leds