TRAINING MANUAL Multiphoton Microscopy LSM 510 META-NLO September 2010
Multiphoton Microscopy Training Manual Multiphoton microscopy is only available on the LSM 510 META-NLO system. This system is equipped with the Chameleon laser, which is a Ti:Sa femtosecond laser. The pre-requisite for the multiphoton training is the Introduction to Confocal Microscopy training. Many of the steps for setting up and acquiring a multiphoton image is the same as for single photon imaging. Therefore this training only covers the steps that differ from the introductory training. Multiphoton Training includes: A. Introduction to Multiphoton Microscopy B. Start-up Procedure C. Turning on the Chameleon Laser D. Setting the Configuration E. Scan Parameters F. Laser Safety G. Shut-down Procedure Please refer to the LSM 510 META Training Manual for a more complete description of all of the steps.
A. INTRODUCTION - Multiphoton Microscopy Multiphoton microscopy is another type of laser scanning microscopy that also provides a single plane image, however multiphoton microscopy achieves this without using a pinhole. Instead, only the fluorescent molecules within the plane of focus are excited eliminating the need for the pinhole since there is no excitation and subsequent emission outside the focal plane. Excitation of only the fluorescent molecules in the focal plane is achieved by a femtosecond laser that sends photons of light with lower energy (longer wavelength) in rapid succession. Multiple photons of the lower energy light must be absorbed in order for the fluorochrome to reach the excited state and the focal plane is the only place where there is sufficient number of photons (in both space and time) for this to occur. Single Photon Multiphoton 2. Transition 2. Transition 1. Excitation 3. Emission 1. Excitation 3. Emission 1 photon 2 photons 1 photon with higher energy (Shorter wavelength) is required for excitation 2 photons with lower energy (Longer wavelength) is required for excitation Single Photon Multi Multiphoton Photon Although the wavelengths used for single and multiphoton excitation are different, the emission spectrum is the same. Emission Intensity (%) 100 Single photon excitation 75 50 25 Emission Intensity (%) 100 75 50 25 2 photon excitation 450 475 500 525 550 575 600 650 700 450 475 500 525 550 575 600 650 920 wavelength (nm) wavelength (nm) wavelength (nm)
A. INTRODUCTION Continued... Advantages of Multiphoton Microscopy: Longer wavelengths (Red or Near-Infrared light) Longer wavelength light is less biologically damaging Excitation is limited to the focal plane The only place where there is sufficient number of photons (in both space and time) is the focal plane therefore very little, if any, out-of-focus fluorescence occurs ( therefore a pinhole is not required) Photon Specimen Excitation is restricted to the focal plane due to photon crowding (In both space and time) Since excitation is limited to the focal plane, photobleaching is also limited to the focal plane Single Photon Excitation Multiphoton Excitation Coverslip } FOCAL PLANE Slide Photobleached Fluorochrome Photobleaching is limited to the focal plane with multiphoton excitation
A. INTRODUCTION Continued... Advantages of Multiphoton Microscopy Continued... Image thicker specimens Light Scatters Both single and multi-photon excitation and resulting fluorescence can be scattered in the specimen The extent of scattering depends on the specimen Generally the laser power is increased to compensate for scatter } FOCAL PLANE In both single and multi-photon microscopy the excitation laser is scattered in thick specimens. The laser intensity is increased to compensate for this scatter. Single Photon Multiphoton Emission in multiple planes with single photon excitation { FOCAL Out-of-focus emission scattered and passes through the pinhole to the detector FOCAL PLANE quality (contrast) of the image by collecting out-of focus fluorescence PLANE In-focus emission scattered and blocked by the pinhole in emission collected from the focal plane In-focus emission passed through the pinhole and is collected Excitation only occurs in the focal plane because the absorption of 2 photons at the same time only happens in the focal plane. Therefore, emission only originates from the focal plane Since there is no pinhole in multiphoton microscopy scattered emission (along with nonscattered) is able to reach the detector Detector Detector
A. INTRODUCTION Continued... Some Disadvantages or Limitations of Multiphoton Microscopy Less lateral and axial resolution When light from the various points of a specimen passes through the objective and is reconstituted as an image, the various points of the specimen appear in the image as small patterns (not points) known as Airy patterns. This is caused by diffraction or scattering of the light as it passes through the specimen and the circular back aperture of the objective Airy Pattern Resolution the minimum detectable distance between two closely space specimen points the limit at which 2 airy disks can be resolved into separate entities (Rayleigh Criterion) These points can be resolved 3 critical characteristics set the resolution limit of the microscope These 2 points cannot be resolved as separate points NA at a fixed, size of airy disk, resolution Numerical Aperture: the light cone size decreases with NA (at a fixed ) and produces a corresponding decrease in the size of the Airy disks ( resolution) Refractive Index: numerical aperture can be dramatically increased by immersion medium such as oil, glycerin or water Wavelength: an increase in the illumination wavelength at a fixed NA will result in an increase in Airy pattern ( resolution) multiphoton microscopy uses longer excitation compared to single photon (therefore multiphoton microscopy has resolution) Increasing illumination Increasing NA illumination size of airy disk resolution
A. INTRODUCTION Continued... Some Disadvantages or Limitations of Multiphoton Microscopy Continued... Excitation spectra difficult to predict and measure few sources of multiphoton excitation information available twice the wavelength of the single photon excitation may not necessarily be the most efficient for multiphoton excitation Single Photon Excitation Multiphoton Excitation FITC Rhodamine Absorption 400 500 600 700 800 900 1000 1100 Wavelength Thermal damage to specimen Water molecules absorb more energy from longer wavelengths of light (NIR and IR) compared to shorter wavelengths (VIS and UV) Heat is generated as water molecules absorb the energy from NIR and IR light. Since the excitation wavelength in multiphoton microscopy is in the NIR and IR range, thermal damage can be a problem as water molecules (both intracellular and Extracellular) in the sample absorb the excitation light generating heat
B. START-UP PROCEDURE - Multiphoton Microscopy 1. If the chiller is in Standby Mode, press the Standby/Run button to switch the chiller from o Standby Mode to Run Mode. (The chiller is set to ~25 C and the current temperature will be displayed.) NOTE: DO NOT OPERATE THE CHAMELEON LASER WITH THE CHILLER IN STANDBY MODE!!!! THE CHILLER MUST ALWAYS BE TURNED ON WHEN USING THE CHAMELEON LASER!!!! 2. Turn on the mercury burner 3. Turn both the PC and Components buttons on the REMOTE CONTROL switch to the ON position 4. Turn on the computer 5. Log into user profile - press Ctrl, Alt and Delete simultaneously - enter Userid and password 6. Double click on the LSM 510 icon start the LSM software 7. In the LSM 510 Switchboard window select Scan New Images and then Start Expert Mode The system will go through an instrument initialization and then the main LSM Toolbar will appear
C. TURNING ON CHAMELEON LASER - Multiphoton Microscopy Before turning on the Chameleon laser: make sure the chiller is in Run Mode (not in Standby Mode) THE CHILLER MUST BE ON AT ALL TIMES WHEN THE CHAMELEON LASER IS TURNED ON!!!!! make sure the key on the Chameleon power supply (under the table) is in the On position If the chiller and Chameleon is in Standby Mode, go to step #1 in the Multiphoton Microscopy Start-up Procedure (Section A) 1. click on Chameleon to select the Chameleon laser 2. Click the On button to turn the laser on Mode Locked 3. Click on Modify to open the Laser Modify Control window 4. Enter the excitation wavelength (720-930 nm only) for the Chameleon laser and then click on Store. Wait a few seconds until Mode Lock is re-established Close the laser control window if all of the lasers needed are turned on DO NOT adjust the fine tuning AO-Frequency!!!
D. CONFIGURATIONS - Multiphoton Microscopy All of the steps for setting the configurations are the same for both single photon and multiphoton microscopy. There are only a few things to keep in mind. multiphoton can be used in combination with single photon (ie. Argon & HeNe lasers) use as a single or multi-track use Ch S, Ch2 or Ch3 always use a KP (short pass) main dichroic beam splitter always use either a BP or KP emission filter (never use a LP filter) Refer to the General Confocal Microscopy Training Manual for a more detailed description and instruction on the configurations 1. Select Channel Mode 2. Select Single Track or Multi Track 3. Click on Excitation to open the Laser Control Window 4. Activate the excitation wavelengths (check box) and setting of excitation intensities (slider) NEVER SET THE CHAMELEON LASER >10%!!!! * * 5. Click on main dichroic beam splitter (HFT) to open the list of beam splitters and select the appropriate dichroic for the excitation laser line(s) you have activated. The main dichroic beam splitter separates excitation from emission NT 80/20 = no filter * HFT = main dichroic beam splitter KP = short pass (LSM 510 META - NLO only) - use with the Chameleon laser Always use a KP beam splitter with the Chameleon laser
D. CONFIGURATIONS Continued... 6. Click on the first secondary beam splitter (NFT) to open the list of options for this position. This beam splitter directs the appropriate wavelengths of emission to Channel S and/or Channel 2 and 3. Select the appropriate mirror or filter. None = all emission will pass through to Channel S Mirror = all emission will be directed to Channel 2 and 3 Plate = all emission will pass through to Channel S NFT = secondary dichroic beam splitter KP = short pass filter 7. Click on the second secondary beam splitter to open the list or options for this position. This beam splitter directs the appropriate wavelengths of emission to Channel 2 and/or Channel 3. Select the appropriate mirror or filter. Mirror = all emission will be directed to Channel 2 NFT 490 = all emission with wavelengths less than 490 nm will be directed to Channel 2 and emission with wavelengths greater than 490 nm will pass through to Channel 3 NFT 545 = all emission with wavelengths less than 545 nm will be directed to Channel 2 and emission with wavelengths greater than 545 nm will pass through to Channel 3 BG 39 = all emission will pass through to Channel 3
D. CONFIGURATIONS Continued... 8. Click on Ch S and select the adjust the sliders or click on the Emission Filter for Channel 2 and Channel 3 to open the list of emission filters available. 9. Select the appropriate emission filter and then check the box to turn on the corresponding channel. Use either a KP or BP filter for multiphoton microscopy. Do not use a LP filter. Band Pass (BP) - permit only wavelengths of emission within the range indicated to pass through to the PMT - Band pass filters can be used with all of the lasers Short Pass (KP) - permit emission with wavelengths below the threshold indicated to pass through to the PMT Channel 2 Emission Filters Channel 3 Emission Filters X X - Short pass filters are used only with the Chameleon laser and not the Argon or HeNe lasers. Long Pass (LP) - Long pass filters permit emission with wavelengths above the threshold indicated to pass through to the PMT - Long pass filters can be used with the Argon and HeNe lasers, but should not be used with the Chameleon laser 10. Click on the colour box to o select the appropriate pseudo-colour for that particular channel. 11. Add another track and repeat steps #1-11 if necessary.
E. INITIAL SCAN PARAMETERS Set the initial Scan Parameters the same as for single photon with the exception of setting the pinhole: When using the Chameleon laser click on Max to open the pinhole multiphoton microscopy does not require a pinhole emission only occurs when 2 or more photons hit the fluorophore at the same time this only occurs within the focal spot F. LASER SAFETY Before you put your sample on the stage there are some very important laser safety precautions that need to be taken. But first, a little about the hazards and classification of lasers: Lasers are classified into 4 groups according to the maximum power or energy of the beam, wavelength output, and pulse repetition Generally, the classification indicates the capability of the laser or laser system to produce injury with higher class numbers indicating a greater potential hazard. Class 1 - lasers or laser systems that do not pose a hazard under normal operating conditions Class 2 - low power visible lasers that are incapable of causing eye injury unless viewed directly for extended periods of time Class 3A - lasers or laser systems that have a low risk of injury but normally would not injure the eye unless directly viewed through binoculars or similar optical devices Class 3B - lasers or laser systems that can produce accidental injuries to the eye from viewing the direct beam or a specular reflection, but normally is not a hazard from diffuse reflection (unless viewed through an optical instrument) Class 4 - lasers that have eye and skin hazards from the direct beam, specular reflections and diffuse reflections. This is a very brief description of the lasers classes. Please refer to the UWO Laser Safety Manual for a more detailed description.
F. LASER SAFETY Continued... Specifically for our systems, the Argon, HeNe and 405nm Laser Diode are all Class 3 lasers whereas the CHAMELEON LASER IS A CLASS 4 LASER!!!! When enclosed within the microscope system, all of the lasers pose very little risk and are therefore considered Class 1. Regardless, there still very important safety steps that must be followed when using the Chameleon laser. Since the Argon, HeNe and 405nm Laser Diode lasers emit in the visible range, the laser beam can be seen when running. The Chameleon Laser however emits in the near infrared range, THEREFORE YOU CANNOT SEE THE LASER BEAM!! Class 4 lasers can damage your eyes and skin from the direct beam and REFLECTED BEAM!!! NEVER MANIPULATE ADJUST YOUR SLIDE WITH THE LASER ON to avoid potentially reflecting the laser beam into your eyes and/or exposing your skin to the laser beam. There are 3 main steps to take before you do any manipulation of your sample on the stage to ensure the laser is turned off: 1. Click Stop in the Scan Control Window 2. Click Vis in the main toolbar 3. Tilt the light carrier back to disconnect the laser circuit. There are also goggles and shields available for your protection
G. SHUT-DOWN PROCEDURE 1. Close all open windows, except the main LSM Toolbar 2. Open the Laser Control box Cool the Argon Laser on Standby highlight the argon laser click on Standby and then Off wait until cooling in the status bar switches to connected IMPORTANT: the argon laser must be cooled before turning the system off Turn the NeNe lasers off highlight the appropriate laser click Off Turn off the Chameleon laser highlight the Chameleon laser click Off turn key on the Chameleon power supply (under the table) to Standby 3. Click on File in the main LSM Toolbar and then Exit. Click Exit in the main switchboard. ( A window will open reminding you the lasers are on and do not turn the system off. Click OK) 4. Log off the computer (ctl + alt + del) and record use in the log sheet. 5. CLEAN THE OBJECTIVE!!!! if not cleaned properly. These objectives are very expensive and are damaged very easily To clean: wipe off any oil on the objective with lens paper use lens paper damp with lens cleaner to clean the objective dry the objective with a clean and dry piece of lens paper IF YOU DO NOT CLEAN THE OBJECTIVES PROPERLY, YOUR MICROSCOPE PRIVILEGES WILL BE TAKEN AWAY.