Renishaw invia Raman Microscope (April 2006)

Transcription

1 Renishaw invia Raman Microscope (April 2006) I. Starting the System 1. The main system unit is ON all the time. 2. Switch on the Leica microscope and light source for reflective bright field (BF) imaging. 3. Power on the desired laser(s): a. 514 nm laser turn on main switch; switch on the key; and switch system to run from standby mode make sure that output power is set at 15 mw. b. 785 nm laser switch on the key. 4. Start the Renishaw WiRE 2.0 software. II. Running a Calibration on Si (111) 1. Load the Si(111) calibration sample to the XYZ stage. Make sure the silicon is positioned to the defined orientation for consistency. 2. Switch to BF filter by selecting A position on the filter wheel and focus with the 40x dry objective to identify Si(111) surface. 3. Switch from the A position to the L4 position on the filter wheel to activate Raman collection channel. Ensure that the side port valve, which is located between Leica Microscope and Ranishaw Raman Microscope, is open for collection. Close eyepiece shutter by sliding it out to photo position. 4. Open Measurement>New>Spectral Acquisition and use Spectral acquisition setup to define experimental parameters. 5. Use Measurement>Cycle to collect static LIVE Raman spectra of Si(111) at 520 cm -1 and use Leica focus knob to optimize signal to maximum counts. 6. Use Measurement>Run to collect static Raman spectra of Si(111) at 520 cm Zoom into the peak at 520 cm -1. Right click to bring up options to check peak position. If it is not in the cm -1 range, calibrate the system by clicking Tools>Quick Calibration. 8. Repeat steps Typical spectra of Si(111) at 514 nm and 785 nm excitation wavelengths are given in Appendix 1. 1

2 III. Collecting Data on Samples 1. Load samples to the stage and focus on the surface. 2. Move the Region of Interest (ROI) to the center of cross hair to which laser is positioned. Do NOT change the position of cross hair A video image taken using a 40x objective 3. Setup acquisition parameters for your experiment: a. Laser excitation source and corresponding optics. b. Static vs Extended scans. c. Standard vs High confocality modes d. Exposure time and number of accumulation e. Pinhole selection for 785 nm laser 4. Use Measurement>Run or the Run button to collect spectra. 2

3 IV. Switch Lasers: Please wear safety glasses!! 1. Enter the desired laser and grating combination in the Spectral Acquisition menu. 2. The light path must be adjusted for the following lens combinations: a. 514 nm laser requires lens A(1) b. 785 nm laser requires lens A(2) c lines/mm (higher resolution/shorter spectral range only valid with 514 laser) requires lens B(1) d lines/mm (lower resolution/longer spectral range) requires lens B(2) 3. Run an acquisition the software will prompt you as to which len(es) to switch out. 4. Put on the appropriate pair of laser safety glasses and use the key to open the system unit. Once the unit is opened the lasers should be interlocked, but it is best to take safety precautions. 5. CAREFULLY exchange filter(s) in control box. Place unused filters in the appropriate black storage box. 6. Run Si(111) calibration if necessary (repeat session II). 3

4 VI. Shutting down 1. Close the WiRE 2.0 Software. 2. Shut down the microscope and peripherals (lamps(s) and/or camera). 3. Turn off the 785 nm Diode laser. 4. Put the 514 nm Argon Ion laser in standby mode and switch off the laser. 5. Leave computer ON and logoff the computer. 6. Sign out in blue log book and on the website. 4

5 Appendix 1: Raman spectroscopy of Si(111) using 514 nm and 785 nm excitation nm excitation without pinhole Standard confocality 50 um slit and 7 pixel CCD area 4200 counts High confocality 20 um slit and 2 pixel CCD area 1500 counts nm excitation without pinhole Standard confocality 65 um slit and 25 pixels CCD area counts High confocality 20 um slit and 2 pixel CCD area counts nm excitation with pinhole Standard confocality 65 um slit and 25 pixels CCD area 7000 counts High confocality 20 um slit and 2 pixel CCD area 1500 counts 5

6 Appendix 3: Optimize your data by following suggestions as listed: To improve signal to noise ratio: a. Increase exposure time b. Increase number of accumulations To eliminate strong background due to autofluorescence: a. Change excitation wavelength b. Quench fluorescence by exposing sample to incident laser light for a period of time c. Decrease laser power To avoid saturated signal: a. Reduce laser power b. Reduce exposure time To avoid laser ablation on the samples: a. Use a lower magnification objective b. Defocus the laser spot c. Use a different laser d. Decrease laser power 6

7 Appendix 4: What is Raman Scattering? Raman scattering is a fundamental form of molecular spectroscopy. Together with infrared (IR) absorption, Raman scattering is used to obtained information about the structure and properties of molecules from their vibrational transitions. Infrared absorption arises from a direct resonance between the frequency of the IR radiation and the vibrational frequency of a particular normal mode of vibration. The IR photon encounters the molecule, the photon disappears, and the molecule is elevated in vibrational energy when the property of molecule involved in the resonant interaction between the dipole moment of the molecule and its vibrational motion. In contrast, Raman scattering is a two-photon event. In this case, the property involved is the change in the polarizability of the molecule with respect to its vibrational motion. The interaction of the polarizability with the incoming radiation creates an induced dipole moment in the molecule, and the radiation emitted by this induced dipole moment contains the observed Raman scattering. The light scattered by the induced dipole of the molecule consists both Rayleigh scattering and Raman scattering. Rayleigh scattering corresponds to the light scattered at the frequency of the incident radiation, whereas the Raman radiation is shifted in frequency, and hence energy, from the frequency of the incident radiation by the vibrational energy that is gained (Stokes Raman) or lost (antistokes Raman) in the molecule. Figure 1 shows the energy diagram for infrared absorption and stokes Raman scatting for a vibrational transition from g0 to g1. The scattering photo energy, hw, is shifted from the incident laser radiation energy by the infrared vibrational energy, hw, gained by the molecule. { ev ћω 0 ћω g1 g1 ћω ν INFRARED ABSORPTION g0 RAMAN SCATTERING g0 7

8 Appendix 5: Renishaw invia Confocal Raman Microscope CCD Collection optics Preslit focusing lens Slit Grating Conjugate image planes - Square pinhole (a) invia Spectrometer Includes, triple laser base plate, triple input mirror set, dual gratings on kinematic mount (1800 or 1200 l/mm), spectrometer lens set, manual slit, single Rayleigh filter mount, single laser input mirror set (b) Renishaw s RenCam deep depletion CCD assy. Includes front illuminated CCD with Peltier cooling to 70oC, USB interface. (c) Leica DM IRB microscope interface Standard Laser Excitation Kits a) Renishaw 785 nm excitation kit Includes 785 nm high power laser, 100 cm-1 edge filter, laser mounting kit and coupling mirrors, pinhole and interlock cable. b) Renishaw 514 nm excitation kit Includes 514 nm laser, holographic notch filter, laser mounting kit and coupling mirror, pinhole, interlock cable and plrf. 8