SUPPLEMENTARY INFORMATION
Supplementary Figure 1 (SF.1): Layer by layer film deposition
Polycation
Wash
Wash
Polyanion
Scheme of layer by layer film deposition process using glass slides and beakers. A charged solid
substrate is immersed in a oppositely charged poly-ion solution. Electrostatic attraction occurs
between the charged surface and the poly-ion solution. This reverses the charge on the solid
support. This is followed by rinsing in water or a buffer solution. The substrate is exposed to a
solution containing the oppositely charged ions. Due to electrostatic attraction between the
charged poly-ion on the substrate and the poly-ion in the solution a thin layer of poly-ion is
coated. The substrate is rinsed again in water. This process is done repeatedly till the desired
number of layers is obtained.
Supplementary Figure 2 (SF.2): Molecular picture of layer by layer deposition
Simplified molecular picture of the adsorption depicting film deposition starting with a
negatively charged substrate. A polycation layer is formed in the first step and rinsing. A
polyanion layer is formed in the second step and rinsing.
Supplementary Figure 3 (SF.3): Absorption spectra and transient absorption of WT-bR
Absorption spectrum of bR. (Inset) Transient absorption of bR solution fitted with an
exponential curve with decay constant of 10 ms.
Supplementary Figure 4 (SF.4): High resolution AFM image of bR
(b)
(a)
(a) High resolution AFM image of D96N mutant on mica substrate showing hexagonal ordering.
(b) 2D Fourier transform of the image in (a).
Supplementary Figure 5 (SF.5): Simultaneous AFM and NSOM imaging of bR
(a) AFM image of sample containing bR patches. (b) Height profile of the bR patches selected in
(a). (c) Corresponding NSOM image of the AFM image in (a). (d) Absorption profile of the bR
patch selected in (c).
Supplementary Figure 6 (SF.6): Photograph of experimental setup
NSOM tip
Piezo stage
Photograph of NSOM setup showing the piezo stage and tip assembly
Supplementary Figure 7 (SF.7): Experimental setup
NSOM Tip
The probe laser is coupled to the NSOM tip using a fiber coupler. The pump laser is directed
through a 50x objective from the bottom. Transmitted intensity is detected using a PMT and the
output is fed to an oscilloscope.
Supplementary Figure 8 (SF.8): CCD image of NSOM tip
NSOM tip
Probe laser spot 
100 nm diameter
CCD image of a NSOM tip showing the probe laser spot
Supplementary Figure 9 (SF.9): Probe and pump spots
Probe laser (532 nm)
spot from NSOM tip
Pump laser (532 nm)
spot from objective
Overlapping spots from the probe and pump lasers for pump-probe analysis
Supplementary Figure 10 (SF.10): Noise histograms for quartz
Noise histogram of bare quartz substrate (blue bars, 532 nm illumination, red bars, 532 nm along
with pump illumination). (Inset) log frequency plot of the histograms (blue dots, 532 nm
illumination, red dots, 532 nm along with pump illumination.
Supplementary Figure 11 (SF.11): Setup for humidity variation
(a)
(b)
Chamber
Scan head inside
the chamber
(a) Photograph of experimental setup for humidity measurement. (b) Close up photograph of
scan head inside the chamber.
Supplementary Figure 12 (SF.12): Noise histogram for D96N mutant under influence of
humidity
Noise frequency histogram for D96N mutant under varying humidity (blue and brown bars,
ambient humidity, black bars, high humidity).
Supplementary Figure 13 (SF.13): Noise histogram of D96N mutant sampled at 250 KHz
Noise frequency histogram for D96N sampled at 4s (blue bars, 532 nm illumination, red bars,
532 nm along with pump illumination) showing no shifts in the profile.
Supplementary Figure 14 (SF.14): Noise histogram of WT-bR sampled at 500 Hz
Low frequency noise histogram for WT-bR (green bars, 532 nm illumination, blue bars, 532 nm
along with pump illumination) showing no distinct shifts in the profiles.
Supplementary Table 1 (ST.1): Kolmogorov-Smirnov tests: 40 nm WT-bR under probe
illumination
Lognormal 40nm 532 nm
Kolmogorov-Smirnov
Sample Size
Statistic
P-Value
Rank
93
0.04128
0.99552
1
0.2
0.1
0.05
0.02
0.01
Critical Value 0.10947 0.12506 0.13891 0.15533 0.16666
Reject?
No
No
No
No
No
Supplementary Table 2 (ST.2): Kolmogorov-Smirnov tests: 40 nm WT-bR under probe
and pump illumination
Lognormal 40 nm 405 nm
Kolmogorov-Smirnov
Sample Size
Statistic
P-Value
Rank

139
0.04775
0.89394
1
0.2
0.1
0.05
0.02
0.01
Critical Value 0.09101 0.10373 0.11518 0.12876 0.13817
Reject?
No
No
No
No
No
Supplementary Table 3 (ST.3): Lognormal fits for WT-bR films with 40 nm and 100 nm
heights
S.No.
1.
2.
Variable
Sample
max
Height (nm)
(Hz)
Lognormal distribution fit parameters
f ( x;  ,  ) 
1
x 2

e
(ln( x )  )2
2 2
σ

532 nm
40
505
0.36
6.29
With pump
40
630
0.45
6.52
532 nm
100
630
0.72
6.41
With pump
100
720
0.48
6.54
Supplementary Note 1 (SN.1): Estimation of number of bR molecules illuminated by a 100
nm NSOM tip.
bR occurs as trimers arranged in a hexagonal lattice. The lattice constant is typically 6-10 nm
depending upon the intermediate
1-3
. Area of a circular beam of radius 50 nm is 7.9 x 103 nm2 .
The area of a trimer is typically 36 nm2 as reported by Muller et. al4 . In a 100 nm2 area (taking a
10 nm lattice constant) there will be 3 trimers. Hence an illumination area of 7.9 x 103 nm2
would excite  220 trimers in a monolayer.
Supplementary Note 2 (SN.2): Matlab code for data analysis
%%This program evaluates the noise frequency distribution of a
bacteriorhodopsin thin film
function freqdis
N=input('how many files '); % Number of traces to take
Fs=250000; %Sampling rate for WT-bR data, 500 Hz for D96N
m=0;
f=(0:8192)*Fs/16384; %construct the frequency axis
p=input('enter name of file ','s');
e=zeros(249897,1);
k=1;
for i=1:N
%loop to read in individual files
fid = fopen(p, 'r');
c = dlmread(p,'\t',5);
d=c(:,2);
wordcount=size(p,2);
wordcount=wordcount-4;
for j=1:15
%split the file into sub-windows
e=d(1+m:16384+m);
pxx=fft(e,16384);%%fft of time data
pxxz=log(abs(pxx(2:size(pxx))).^2);%leave out DC contribution
freqm=find(pxxz>=max(pxxz)); %find the peaks in fft
freqmax(k)=freqm(1,1)
k=k+1;
m=m+16384;
end
m=0;
if i<10
p(wordcount-1)=num2str(0);
p(wordcount)=num2str(i);
else
p(wordcount-1:wordcount)=num2str(i);
end
end
counts = hist(freqmax*15.26,f); %histogram of frequencies
plot(f,counts,'o');
fclose('all');
References:
1. Takeda, K. et al. Crystal structure of the M intermediate of bacteriorhodopsin: allosteric
structural changes mediated by sliding movement of a transmembrane helix. J.Mol.Biol.
341, 1023-1037(2004).
2. Schobert, B., Brown, L.S. & Lanyi, J.K. Crystallographic structures of the M and N
intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water
molecules between Asp-96 and the retinal Schiff base. J.Mol.Biol. 330, 553-570 (2003).
3. Sato, H. et al. Specific lipid-protein interactions in a novel honeycomb lattice structure
of bacteriorhodopsin. Acta Crystallogr.,Sect.D 55. 1251-1256 (1999).
4. Muller, D. J., Schabert, F. A., Buldt, G. & Engel, A. Imaging purple membranes in
aqueous solutions at sub-nanometer resolution by atomic force microscopy Biophy. J. 68.
1681-1686 (1995).