COL 11(11), 111102(2013) CHINESE OPTICS LETTERS November 10, 2013
Label-free biomolecular imaging using scanning spectral
interferometry
Tongzhou Wang (
ÓÓÓ
°°°
)
1†
, Liping Xie (
www
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)
1†
, Haley Huang
2
, Xin Li (
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)
1
,
Ruliang Wang (
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)
1
, Guang Yang (
111
)
1
, Yanan Du (
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)
1∗
, and Guoliang Huang (
III
)
1,3∗∗
1
Department of Biomedical Engineering, Tsinghua University School of Medicine, Beijing 100084, China
2
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21287, USA
3
National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China
∗
Corresponding author: duyanan@tsinghua.edu.cn;
∗∗
corresponding author: tshgl@tsinghua.edu.cn
†
These authors contributed equally to this work
Received July 16, 2013; accepted October 17, 2013; posted online November 9, 2013
This letter presents a label-free biomolecular imaging technique based on white-light interferometry
and spectral detection. The method measures thickness changes caused by specific binding between
biomolecules to detect the presence of certain analyte. A spectru m-shifting algorithm is developed to
resolve the thickness information from the spectrum. The axial resolution of th e experimental instrument
can reach ∼1 nm, thereby enabling detection of trace amounts (∼1 ng/mm
2
) of proteins or DNA. This
letter also presents two experiments to prove the feasibility of the method for detecting proteins and DNA
without fluorescent labeling.
OCIS codes: 110.0110, 120.0120, 300.0300, 330.0330.
doi: 10.3788/COL201311.111102.
Developments in optical microscope technology have
achieved remarkable progress from the 1980s to the last
decade. The detection resolution of technologies such
as s timulated emission depletion (STED)
[1−3]
, PALM
[4]
,
and STORM
[5]
, has reached scales as low as tens of
nanometers. All of the technologies developed thus far
use fluorescent labels to improve their resolution; how-
ever, these fluoresce nt dyes pre sent negative effects on
life activities
[6]
. Fluorescent labels also undergo photo-
bleaching, which further complicates the analysis of ex-
perimental results.
Important progress in label-free detection methods,
such as surface plasmo n resonance (SPR)
[7,8]
, micro-
resonators
[9]
, interferometry
[10,11]
, and electrochemist-
ry
[12]
, has been achieved. Among these methods, in-
terferometry is one of the most convenient and widely
applicable technologies fo r measuring small changes in
biochemical reactions
[13]
.
Unfortunately, beca use most la bel-free detection meth-
ods are indirect, which means the biochemical signal is
converted to another type of signal, introduction of er -
rors to the results is unavoidable. Because of this issue,
many researchers still regard molecular images as the
most reliable evidence of biochemical reactions. SPR
imaging (SPRI)
[14,15]
devices based on the traditional
SPR device, which produces an image that describes
energy absorption in a two-dimensional (2D) field, may
be used for sample imaging. Similar to SPR technology,
however, SPRI requires an expensive gold-coa ted chip,
which raises the cost of detection.
Interferometry, another promising method for label-
free molecular imaging, presents advantages of con-
venience, low cost, and high sensitivity. Previous
studies
[13,16−19]
on interferometry detection re port that
the spectrum of the reflected light beam can be used
to measure the pre sence of biomolecules. Thus, in-
terferometry is a novel method for detecting trace
amounts of biomolec ules without fluor e scent labelling.
Many devices, including spectral reflectance imaging
biosensors
[13]
and interferometric reflectance imaging
sensors
[19]
, have been designed to achieve label-free
biomolecular detection. These device s use phase shifts
(∆Φ) to evaluate thickness changes introduced by
biomolecular binding because interferometric measure-
ments are extremely sensitive to phase change (10
−10
rad)
[18]
. However, ∆Φ does not show a linear relation-
ship with the thickness increment. As such, the overall
∆Φ cannot represe nt the average thickness increment of
a detection area if the thickness increment within the
area is inconsistent. This limitation presents difficulties
in high-r e solution imaging because the thickness incre-
ment within the area of a pixel does not have a linear
relationship with its gray values in the res ulting images.
Thus, common post-pro cessing methods for reconstruct-
ing high-resolution images, such as deconvolution and
2D Fourier transformation, are inapplicable.
This letter presents a scanning-probe-style interfer-
ometric super-re solution microscope that can capture
high-resolution molecula r images without fluoresc e nt la-
belling . A novel a lgorithm is introduced to extract a
linear relationship between the thickness increment and
the imag e gray value. The algorithm used in our ex-
periment provides better linearity compared with the
phase-resolving algorithm used in the majority of other
studies
[18,19]
. Good linearity is extremely important in
scanning-probe-style microscopes because the device re-
quires deconvolution to cancel the a rtifacts introduced
by pixel overlapping.
The setup of the microscope is demonstrated in Fig .
1(a). The structure is similar to a Michelson interferom-
eter, exce pt for the coaxial measurement and reference
arms. The sample is attached to a SiO
2
-coated silicon
chip and illuminated by a wide-band light source, such
as a halogen tungsten or deuter ium lamp. Fig ure 1(b)
1671-7694/2013/111102(4) 111102-1
c
2013 Chinese Optics Letters