Multiphoton Imaging
Safety
IR laser is not visible but can still blind you or burn you. Do not put your hand underneath the laser
when scanning, do not put any mirrors under the laser (glass slides are OK) these could reflect the
laser into you eyes.
Do not move any of the equipment behind the microscopes, the lasers are very accurately aligned
and any movement will misalign the system causing it to fail and may make the system unsafe.
Useful Points
The multiphoton laser can be used by both microscopes at the same time if using the same
wavelength. When doing this each microscope will only get half the power.
For 100% power to the upright confocal set the black dial on the first tube coming out of the laser to
100. For 100% power to the inverted confocal set the black dial on the first tube coming out of the
laser to 140. For 50% power to the upright confocal and 50% to the inverted set the black dial to 120
The laser power varies for different wavelengths. When set to 780nm the power output is 3.6
Watts. This is the highest power.
At lower and higher wavelengths the power decreases as described below, and so it is necessary to
increase the % transmission to compensate for this.
Power
600 mWatts
2.5 Watts
3.6 Watts
2.5 Watts
1.6 Watts
300 mWatts
Wavelength
690nm
720 nm
780nm
850 nm
930nm
1000nm
The multiphoton laser
The multiphoton laser produces a femtosecond pulsed laser beam, of a longer wavelength than the
standard confocal laser. The laser causes less damage to the sample and is also able to penetrate
deeper into the sample. Multiphoton excitation requires the near-simultaneous absorption of two
photons and so does not occur outside the focal plane. Consequently the pinhole should be set to
maximally open. This is done using the “Scan” window and selecting the “channel” tab.
In single photon microscopy the excitation wavelength is shorter than the emission wavelength. In
multiphoton microscopy the excitation wavelength is longer than the emission wavelength. The
excitation peak is also less specific than for single photon excitation e.g. DAPI can be excited
between 720 and 800nm
Multiphoton Walkthrough
Before entering the LSM software turn the key on the box under the table from STANDBY to ON.
Only 1 PC can control the multiphoton wavelength at a time and depends on where the multiphoton
is connected. This is generally the PC for the upright confocal, but it can easily be changed; ask for
advice if you wish to do this.
To switch on the laser, do as for the other lasers using the LSM software and selecting the “laser”
window and the Chameleon laser from the list. The software will indicate MODE LOCKED when
ready to use.
To adjust the wavelength click MODIFY and type in the wavelength needed.
If you change the wavelength during the session the laser will become unticked in the scan box. This
is to avoid you changing the wavelength but not changing the power. If unsure always start at 2%
laser power and work up.
N.B The wavelength will be whatever was last set so it is necessary to check that it is the one you
expect.
For two-photon work always set the pinhole fully open in SCAN box.
To decide the best laser power, the detector gain should ideally be between 500 and 700 when the
image is optimised. If this is not the case increase or decrease laser power until the detector gain is
in this range.
Samples with one fluorophore
As with imaging a single label using the single photon laser, it is possible to select the appropriate
configuration from the list of saved options.
N.B. The primary dichroic used should always be a short pass version i.e. there is the prefix KP
If you have only one fluorophore in the sample KP 660 will allow you to see all fluorescence.
Samples with multiple fluorophores
All Alexa dyes have a peak in the UV range. E.g. for Alexa 488, Alexa 543 and DAPI you can excite
them all at 780 and then use specific filters to separate the emission fluorescence. Alternatively it is
possible to image the Alexa 488 and Alexa 543 as before using single photon lasers and add
multiphoton as an additional track in the config box for the DAPI component.
It is also possible to use the spectral imaging to separate the fluorescent components within a multilabelled sample. If you require help using spectral imaging please ask for advice.
Second Harmonic Generation (SHG)
This can be used to detect proteins such as collagen and elastin. Emission from SHG is exactly half
the excitation wavelength. E.g. exciting a collagen sample at 900nm requires detecting emission at
450nm.
Excitation Fingerprinting
This can be used to determine the best multiphoton excitation wavelength for a given fluorophore. It
may be that there are useful peaks for excitation that don’t cause cross talk with another
fluorophore, or remove problems with autofluorescence.
Due to the difference in laser power across the wavelengths a calibration curve for laser power
should first be made. This calibration curve is specific to the objective and primary dichroic used and
so it may be that you need to calibrate your particular set up if one has not yet been created. To do
this follow the procedure below
Calibrating the objective and filters
Place the power meter sensor under the objective and lower down until touching.
Use the correct dichroic setting that you are going to require for the actual experiment. Unless you
do this you will not be able to load the calibration data later so it is important to set these properly.
In scan control select “frame” and bidirectional. Set zoom to max (x 40) by moving the slider to the
far right. Set the scan speed to max
First select Macro – Exc. Fingerprint and select AOM-calibration
Click “start” on the scan continuous option
Set the laser transmission at 780 nm and change the % attenuation to give a reading around 5 mW.
Record this exact value. Change the wavelength to 690 nm. The power will fall, so change the %
attenuation to get back to same power. You can hold control and click left mouse button on the
arrow to increase by 0.1%. When the power is at the same value as it was at 780 nm, click “add”. Use
the manual option and increase wavelength in 10 nm steps. At each point alter the % attenuation to
get the same power and click “Add” for each result.
N.B Do not attempt to tune to a new wavelength while laser status is shown as busy. If laser does
not mode lock after several minutes press “recovery” and wait for mode locking (3 to 5 mins).
When all wavelengths have been checked store the calibration file as raw data first, and then select
normalise (to 100%) and store again as a separate normalised file. Store in D: drive AOM Calibration
and include the details of dichroic and objective used.
Characterising a fluorophore
Place your sample on microscope, use the single channel and one of the appropriate multiphoton
configurations and scan as normal. Try using an excitation wavelength which is twice the normal
single photon wavelength, or if this does not work try using 800 nm. Don’t worry about optimising
the image at this stage – you only need to be sure that your sample is in focus.
Select excitation fingerprint and lambda stack
To be able to load the calibration file you must have the SAME objective and HFT filter selected as
used for the calibration step. It also seems to help if you select these first in the channel mode
before going to lambda mode.
Load the correct calibration curve, select the laser excitation range that is required and click “start”
(you can actually use a smaller range and/or step size if wished. It does NOT have to correspond to
the range/step size in the calibration curve)
The macro will perform an excitation scan for each emission wavelength, varying the attenuation to
keep the same power.
Once the lambda stack has been produced for each excitation wavelength you can see the image for
each excitation wavelength, by selecting the gallery option
If you then select “mean” it all gets combined into 1 image. You can then select a specific ROI to get
the excitation spectrum for that area and the optimum excitation wavelength can be determined.
Useful references [1, 2]
1.
2.
Georgakoudi, I., et al., Optical Spectroscopy and Imaging for the Noninvasive Evaluation of
Engineered Tissues. Tissue Engineering, 2008. 14(4): p. 1-20.
Schenke-Layland, K., et al., Two-photon microscopes and in vivo multiphoton tomographs -Powerful diagnostic tools for tissue engineering and drug delivery. Advanced Drug Delivery
Reviews, 2006. 58(7): p. 878-896.