Supporting materials
Detailed apparatus
To generate 200fs 830nm laser beams, a high power Yb laser was modified to operate at
75MHz. The homemade Yb laser can produce pulses ranging from 100fs to 1ps, at power
levels of 6 to 11W at a wavelength of 1040nm. More than 60% conversion to the second
harmonic generation at 520nm is routinely achieved with an angel tuned LBO crystal at
room temperature (theta=90 degrees, phi=13 degrees, 4mm long, Casix). The doubled
520nm is then used to pump a homemade optical parametric oscillator (OPO), which uses
a temperature tuned 6mm long LBO crystal. By changing the temperature of the crystal,
the signal wave is tunable from 680nm to 1000nm, and the idler wave is available at
wavelengths from 1080nm to more than 2000nm.
In the current work, 300mW of 200fs 830nm beam is generated from the OPO signal
wave, and the power at the focus is reduced to less than 10mW to avoid photodamage.
For the current work, the homemade laser and OPO can also be replaced with a
commercial ultrafast Ti: Sapphire laser.
The femtosecond excitation beam and the cw probe laser beam (785nm, Sacher
Lasertechnik, TEC510-785) are spatially overlapped with a dichroic mirror. The
1
excitation beam is modulated by an acoustio-opitcal modulator (AOM) (model 3080-122,
Crystal technology) at 100kHz which is driven by a square-wave function generator.
Excitation and probe beams are coupled into a modified laser scanning inverted
microscope (IX71, FV300, Olympus). The beam size is matched to fill the back-aperture
of objective. A 60X 1.2 N.A long-working distance objective (UPlanSApo, water,
Olympus) is used for excitation for all cell images (red blood cells and cytochrome
imaging) ; A 20X 0.75 N.A objective (UPlanSApo, air, Olympus) is used for excitation
for all tissue images (mouse ear vessels and zebrafish gill). A 20X 0.95 N.A. longworking distance objective (XLUMPlanFI, water, Olympus) is used as a condenser.
Another lens is used to image the scanning beams onto a silicon amplified photodiode
(PDA36A, Thorlabs) to avoid beam movement during laser scanning. Two high OD low
pass filters (3RD800SP, Omega) are used together to block the excitation beam
completely and only transmit the probe beam. An iris diaphram with adjustable aperture
is mounted on a two dimensional translational stage and is placed in the probe beam path.
The output of the photodiode is low-pass filtered ( DC-1.9MHz, Mini-circuits) to
suppress the strong signal at the pulsing repetition rate (76MHz), and is terminated into
50Ω. A high-frequency lock-in amplifer (SR844, Stanford Rserach) is used to
demodulate the stimulated emission signal. The analog on phase componet x-output of
the lock-in amplifier is fed into the A/D converter of the microscope input. The time
2
constant is set for 100us for the imaging experiments. All images are collected with
200μs pixel dwell time, which takes about 13 seconds for a 256×256 pixels image.
Sample Preparations :
Human embryonic kidney (HEK) 293 cell line was obtained from American Type
Culture Collection (ATCC, Rockville), HEK 293 cells are maintained in DMEM (ATCC)
supplemented with 10% fetal bovin serum (ATCC) at 37 oC in a humidified 5% CO2 air
incubator. Cells are then plated on uncoated glass bottom dishes (P35G-1.0-14-C,
MatTek Cooperation) for imaging.
Mouse skin tissue from wild-type white mice is obtained from Harvard Mouse Facility. A
mouse ear is harvested for imaging soon after sacrificing the mouse. The mouse ear tissue
is then sandwiched between two No.1 coverslips with a drop of water for imaging.
Zebrafish ruby mutant is obtained from Harvard MCB Zebrafish Facility. The larval
zebrafish is euthanized 7 days after fertilization. Immediately after euthanization, the fish
is trapped in methyl cellulose and sandwiched between two No.1 coverslips with a drop
of water on top for imaging. The fish is placed with one side facing the objective.
3