Wild card macros - aao5520

advances.sciencemag.org/cgi/content/full/3/12/eaao5520/DC1

Supplementary Materials for

Deep tissue optical focusing

and optogenetic modulation with

time

reversed ultrasonically encoded light

Haowen

Ruan, Joshua

Brake, J.

Elliott

Robinson, Yan

Liu, M

ooseok

Jang, Cheng

Xiao,

Chunyi

Zhou,

Viviana

Gradinaru, Changhuei

Yang

Published 8 December 2017,

Sci. Adv.

, eaao5520 (2017)

DOI: 10.1126/sciadv.aao5520

PDF file includes:



method S1. Calculation of the focal spot size of

TRUE

and conventional f

ocusing.



method S2. Viral injection surgery.



method S3. Electrophysiological recordings.



method S4. Daily alignment procedure.



fig. S1. Setup.



fig. S2. Electrical signal flow diagram.



fig. S3. Ultrasound pulse

echo image of the tip of the glass pipette ele

ctrode.



fig. S4. Electrophysiological photocurrent traces from neurons in 500

and 300

μm

thick acute brain slices.



fig. S5. Electrophysiological photocurrent and membrane voltage traces

comparing ultrasound on and off conditions.

Supplementary Materials

method S1. Calculation of the focal spot size of

TRUE

and conventional focusing.

To calculate the focal spot size of TRUE focusing from the images captured with the observation camera

(Camera2 in

ig.

1), the image was first cropped to a region (82 × 82 pixels) surrounding the focus

profile, and was low

pass filtered by a 3 × 3 averag

ing filter to smooth the speckle. Then, the focus

profile was fit using a 2D Gaussian function













( , )

exp

(

)

fxyAxxyyB







   





, where



are the fitting parameters. The full width at half maximum (FWHM) focal spot size was

obtained by FWHM =

2 2 ln 2



. The focal spot size of the conventional focusing was calculated in a

similar way. However, since the focal spot size was m

uch larger than that of TRUE focusing, the fitting

was applied to a region composed of 1040 × 1392 pixels.

method S2. Viral injection surgery.

Mice were anesthetized with an isoflurane gas/carbogen mixture and placed to a stereotaxic frame (David

Kopf Inst

ruments, CA, USA). After sterilizing the skin with chlorohexidine, a midline incision was made

with a sterile scalpel, the skull surface was cleaned with autoclaved cotton swabs, and the bregma and

lambda were identified and leveled to be on the same z

axi

s. AAV

CaMKII

bReaChES

YFP (500

nL) was injected into the prelimbic cortex (AP + 1.65 mm, ML ± 0.2 mm, DV − 2.25 mm) using a blunt

gauge microinjection needle within a 10 μL microsyringe (NanoFil, World Precision Instruments, FL,

USA), which was c

ontrolled by a microsyringe pump (UMP3, World Precision Instruments) connected to

a controller (Micro4, World Precision Instruments). Following injection, the needle/syringe was held in

the same location for an additional 10 min to allow further diffusion.

To prevent potential backflow, the

needle/syringe was slowly withdrawn over approximately 10 min. After infusion, the scalp was sutured

and the mouse was returned to the vivarium for post

operative recovery. Animal husbandry and all

experimental procedure

s involving animal subjects were approved by the Institutional Animal Care and

Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of

Technology under IACUC protocol 1650

method S3. Electrophysiological recor

dings.

Mice expressing CaMKII

bReaChES

YFP were euthanized with carbon dioxide and transcardially

perfused with ice

cold sucrose

based cutting solution saturated with 95% O

/5% CO

(carbogen) that

contained (mM) 85 NaCl, 75 sucrose, 2.5 KCl, 1.25 NaH

, 4.0 MgCl

, 0.5 CaCl

, 24 NaHCO

and 25

glucose. The brain was removed and 300

–

800 μm thick coronal slices that contained the mPFC were

prepared in oxygenated cutting solution using a vibratome (VT

1200, Leica Biosystems, IL, USA). Slices

were recovered

at 32°C for one hour prior to recording in carbogenated ACSF containing (mM): 125

NaCl, 2.5 KCl, 1.2 NaH

, 1.2 MgCl

, 2.4 CaCl

, 26 NaHCO

, and 11 glucose. Slices were transferred

to the recording chamber and perfused (1.5

–

2.0 mL/min) with carbogen

turated ACSF at 32 ± 0.5°C.

Whole

cell patch clamp recordings were obtained using a Heka EPC 10 USB amplifier/digitizer (Heka

Electronik, Germany) with data sampled at 10 kHz and filtered at 2 kHz. Patch electrodes had resistance

of 4

–

8 MΩ and were fille

d with a potassium gluconate intrapipette solution (mM): 135 K gluconate, 5

KCl, 5 EGTA, 0.5 CaCl2, 10 HEPES, 2 Mg

ATP, and 0.1 GTP.

method S4. Daily alignment procedure.

Before operation each day, the system was tuned to maintain optimal performance of

the digital optical

phase conjugation (DOPC) system by ensuring fine pixel

pixel alignment between the camera and the

SLM. This procedure was conducted using the quality assurance (QA) arm of the system by opening

shutters SH1 and SH2 as shown in

ig.

1. The following steps were performed for the daily alignment.

Playback beam normal to SLM.

To ensure the SLM was perpendicular to the sample and

playback beam (S/PB)

, we enabled the flip mirror FM

and tilted the SLM to maximize the

intensity measured by t

he QA photodiode (PD2).

Record and playback a scattering field from QA path for DOPC loop B.

We disabled flip

mirror FM

and optimized the alignment of DOPC loop B, which was enabled by setting the

shutters BSS2 and BSS3 (

ig.

). This allows the QA ligh

t beam to interfere with the reference

beam (R), so the phase map of the scattered light could be measured using four

step phase

shifting holography. After measuring the phase map, the conjugate phase map was displayed on

the SLM, so the playback beam woul

d propagate through the scattering medium (SM) and the

single mode fiber (SF2) to reach PD2. The single mode fiber enables higher quality correction

than previous quality assurance systems which use a camera to monitor the phase conjugation

quality.

Digitally search for tilt and shift misalignment (loop B).

To assess the respective contributions

of the shift and tilt misalignment between the camera and the SLM, the phase conjugate solution

was digitally shifted and tilted while monitoring the intensit

y of PD2. This allows the

misalignment to be mapped out before physically correcting the misalignment. Although we can

digitally correct for the shift and tilt, we prefer to correct these parameters physically to reduce

the computational time and improve t

he system performance during TRUE focusing operation.

Physically correct for the tilt and shift misalignment (loop B).

The tilt misalignment was

corrected by tilting mirror M10 to maximize the signal captured by PD2. Similarly, the shift

misalignment was c

orrected by translating the SLM in plane to maximize the detected signal on

PD2.

Aberration correction (loop B).

The aberration of the SLM, the playback (PB) beam, and the

associated optics in the light

path were digitally corrected by adding a compensati

on phase map

determined using a series of Walsh

Hadamard patterns

(

)

. The compensation map was saved

for later use during the TRUE focusing procedure.

Aberration correction for the CW laser.

Since loop B w

as also used for the continuous

wave

playback beam, a compensation phase map for this beam was measured as well. In this case, we

switched the playback beam from

the

PW laser to

the

CW laser by switching the shutter BSS1.

Alignment for DOPC loop A.

The PW

laser was enabled again by flipping shutter BSS1. DOPC

loop A was also enabled by flipping shutters BSS2 and BSS3. Steps 2 to 5 were repeated to align

DOPC loop A. Since loop A travels a different optical path, the physical tilt and shift correction is

dis

tinct from that of loop B. For loop A we corrected the tilt misalignment by tilting beamsplitter

BS4 and the shift misalignment by simultaneously tilting beamsplitters BS8 and BS4.

fig. S1. Setup.

(

and

)

chematic of the setup for DOPC Loop A

Loop

(

)

The physiological

setup

(the A

A section in

and

)

running in the neuron observation and patching mode.

(

)

The

physiological setup running in the neuron stimulation mode

Abbreviations: ACSF, artificial

cerebrospinal fluid; AOM, acousto

opti

c modulator; BB, beam block; BS, beamsplitter;

BSS, beam

selecting shutter;

CL, camera lens; CP, chopper; CW laser, continuous

wave laser; DL, delay line; FM,

flip mirror; HWP, half

wave plate; L, lens; LS, light source; M, mirror;

MF, multi

mode fiber;

neutral

density filter; NP, Nomarski prism; OI, optical isolator; P, polarizer;

PB, playback beam;

PBS,

polarizing beamsplitter; PD, photodiode; PH, pinhole; PN, plane; PP, pipette; QA, quality assurance; R,

reference beam; S, sample beam; SC, sample cha

mber; SF, single

mode fiber;

SH, optical shutter;

SLM,

spatial light modulator; SM, scattering medium; TS, tissue slice; UST, ultrasonic transducer; ZB, zero

order block (a black 100

diameter disk printed on a transparency).

fig. S2. Electrical

signal flow diagram.

The experiment can be divided into two phases. In the first phase,

the TRUE focusing system searches for the wavefront solution using

a pulsed

wave (PW) laser.

After that,

switch to

a continuous

wave (CW) laser by controlling the sh

utters

using software trigger

To obtain

a correct wavefront solution, we implement

nine

iteration

the

TRUE

focusing

process

by switching

the shutters that select between

DOPC loop A and B. In each DOPC loop,

four

step phase

shifting

holography approach

used to measure the

optical

field of

the

ultrasound modulated light. In this case,

an arbitrary

function generator (AFG1)

used to synchronize the camera and

the

phase

of the reference

beam

he frequency of

the reference beam

shifted

by 50.020 MHz by AFG2. The reference beam

clock with

the

ultrasound pulses and lasers pulses, both of which are triggered at 40 kHz. A 7.46

s delay

used to compensate for the propagation time of the ultrasound

wave

traveling to the ult

rasound

focus.

Abbreviations: AFG

, a

rbitrary

function

generator; AMP

, a

mplifier

; AOM

, a

cousto

ptic

odulator

;

, d

elay

enerator;

UST

, u

ltrasound

ransducer.

fig. S3. Ultrasound pulse

echo image of the tip of the glass pipette electrode.

focus ultrasound to

the targeted neuron that is patch

clamped by the glass pipette, the ultrasound transducer is scanned to

image the pipette

tip

pulse

echo mode. The echo is recorded at each

scanning position

to form an

image of the tip where the neuro

locate

. The transducer is then moved to

location

so that

the

ultrasound focus overlap

with the

neuron. Scale bar

100

fig. S4. Electrophysiological photocurrent traces from neurons in 500

and 300

μm

thick acute

brain slices.

TRUE focusing enables

photocurrent enhancement at depths of 500

μm

(

)

and 300 μm

(

)

in addition to the 800 μm thickness shown in the main text

fig. S5. Electrophysiological photocurrent and membrane voltage traces comparing ultrasound on

and off

conditions.

(

) Photocurrent traces when performing the TRUE focusing procedure with the

ultrasound

on and off. When the ultrasound is

a phase

map

measured which enables light to be

focused

to the location of the ultrasound focus

and the

neuron

cell

body

In contrast

, when the ultrasound

disabled

by turning off

its driving

amplifier,

an incorrect

phase map

is measured so that the playback

beam

is not

focus

onto the

neuron

As a result, we see a decrease in the amplitude of the photocurrent.

(

)

Membrane voltage traces when performing the TRUE focusing procedure with the ultrasound on and

off.

hen the ultrasound is on

the

playback beam

focused onto the neuron and elicit action potentials.

In comparison, when the ultrasound is off, light is no

t focused onto the neuron and therefore no action

potentials are elicited. It should be noted that the photocurrent and membrane voltage are lower with

ultrasound off than those with ultrasound on followed by a shifted TRUE phase map displayed on the

SLM (

Fig. 3) which leads to an artificially increased photocurrent enhancement since the overall energy

in the “US OFF” condition is lower than in the “No Shaping” condition.

This is because the random phase

map measured with ultrasound off has a higher spatial

frequency (1/pixel size) than that of a normal phase

map, and these high frequency components are filtered out by the limited numerical aperture of the

optical system. In contrast, the shifted phase pattern in the “No Shaping” condition does not change th

spatial frequency distribution of the phase map measured with ultrasound on and therefore maintains the

same background intensity level as the background of the TRUE focus