A femtosecond Raman generator for long wavelength twophoton and third harmonic generation imaging.
Johanna Trägårdh,1 Jan Schniete,1 Maddy Parsons,2 and Gail McConnell1.
1 Centre for Biophotonics, SIPBS, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK
2 Randall Division of Cell and Molecular Biophysics, King’s College London, Guy’s Campus, London, SE11 UL,UK
Supplementary Material
Fig. S1: Schematic of the multi-photon microscope. The imaging experiments were performed using a home-built
laser scanning multi-photon microscope, based around an upright microscope base (Nikon Eclipse E600FN, Nikon, UK). The collimated beams of the SPRG and pump laser were coupled into the microscope using a periscope (not shown). The scan head contains the scanning galvo mirrors (Cambridge Technology H6215H, with
5mm mirrors, and MicroMax 671XX servo driver), SL=scan lens (f=80 mm) and TL=tube lens (f=160mm). The
condenser collected the signal from the sample. The band pass filter removed the excitation light. A 50 mm focal
length lens was focusing the light to the detector (PMT). To control the scanning galvo mirrors and for image capture, we used home written Labview-based software. The PMT signals were digitized and the mirror voltages
were supplied using a PCI-6110 (National Instruments) multifunction DAQ card with a 12 bit, 5 MHz, simultaneous sampling A/D converter and 16 bit, 4 MHz, D/A converter. The DAQ board also controlled a shutter (UniBlitz 510A-S7 with VCM-D1 controller) to block the excitation beam when not scanning. The images were processed using ImageJ. Bright field images were acquired by using an LED-based white light source (WL) and
placing a camera (Cam) in the back focal plane of the TL where the sample is reimaged. The microscope is constructed as a transmission microscope since THG is generated primarily in the forward direction. This means that
unless the sample is quite thick, or highly scattering, thereby redirecting part of the generated light to the objective, transmission detection is more efficient (D. Débarre, et al., Nat. Methods 3, 47–53 (2006)).
Figure S2. (a) The spectra from fig. 1(e) displayed on a log-scale, to visualize the long wavelength end of the
spectrum. The spectra are vertically offset for clarity. (b) The output power dependence on the pump power for
the Raman generator.
Figure S3 shows a spectrum from the Raman Generator, filtered using an 1100 nm long pass (LP) filter (Thorlabs
FEL1100). In addition to a Raman shifted emission, there is a significant contribution of light at shorter wavelengths (from the spectrally broadened pump), as expected for a non-linear crystal pumped by a J pulse. Since
THG from much of this light will be at wavelengths below what the microscope optics readily transmits, we filter
this away to minimize the light exposure and thus potential damage to the specimen. For all imaging experiments, we used an 1150 nm LP filter.
Figure S3. Spectrum from the Raman generator (black solid line) filtered using an 1100 nm LP filter. The pump
power is 1 W. The transmission curves of the 1100 nm LP filter, and the 1150nm LP filter used for imaging are
indicated by dashed lines.
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Figure S4. Left: Spirogyra imaged with SPRG. Center: zoomed in image of Spirogyra imaged in THG with SPRG
(1.34 mW at sample plane). Right: the same area imaged with OPO (14.4 mW at sample plane).
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Figure S5. Still from a bright-field video (Multimedia View) showing the cytoplasmic streaming in a Primrose
leaf hair after imaging for 22 minutes (Excitation power 3mW). The inset shows the corresponding THG image.
The dependence of the THG signal on the laser repetition rate
The fact that the signal in a multi-photon process depends on the repetition rate of the excitation source follows
from that it depends non-linearly on the peak power of the laser. Fewer pulses per unit time means higher peak
power per pulse for a given time-averaged power.
For a THG process the signal per pulse depends on the peak power Ppeak
𝑃𝑎𝑣 3
3
𝑆𝑖𝑔𝑛𝑎𝑙 𝑝𝑒𝑟 𝑝𝑢𝑙𝑠𝑒 ∝ (𝑃𝑝𝑒𝑎𝑘 ) ∗ 𝑡 = ( ) ∗ 𝑡
𝑓𝑡
where t is the pulse length, f the repetition rate and Pav the time-averaged power.
The signal per unit time is then
𝑃𝑎𝑣 3
𝑃𝑎𝑣 3
𝑆𝑖𝑔𝑛𝑎𝑙 𝑝𝑒𝑟 𝑢𝑛𝑖𝑡 𝑡𝑖𝑚𝑒 ∝ ( ) ∗ 𝑡 ∗ 𝑓 = 2 2
𝑓𝑡
𝑓 𝑡
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Description of Multimedia view files for fig 2, 3, and S5
Multimedia 1
(Associated with fig 2) Z-stack through a Spirogyra sp. imaged by THG. The excitation power was 1 mW at the
sample plane. The label indicates the z position i microns. The stack starts at the top of the spirogyra. The Z plane
spacing is 3.5 um.
Multimedia 2
(Associated with fig 3) THG images of cytoplasmic streaming in an Elodea crispa leaf at the end of a 30 min
imaging sequence. The excitation power was 1.2 mW at the sample plane. The label indicates the time in seconds.
Multimedia 3
(Associated with fig S5) Bright-field video showing the cytoplasmic streaming in a Primrose leaf hair after imaging for 22 minutes (Excitation power 3mW). The inset shows the corresponding THG image.
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