New Aproaches for High-Resolution Optical Microscopy

Denis E. Trancă and George A. Stanciu

Center for Microscopy-Microanalysis and Information Processing, University Politehnica of Bucharest, Splaiul

Independentei, Bucharest, Romania

1 RESEARCH PROBLEM

The optical microscopy field has been largely

developed in the past decade and many efforts have

been made to get improvements in respect to the

optical resolution. A special attention has been paid

to the detection techniques of the evanescent waves,

which are not affected by diffraction, but

unfortunately they do not propagate into space.

Among these techniques, the apertureless Scanning

Near-field Optical Microscopy (a-SNOM) (Patane et

al., 2004) have been particularly proved efficient in

many ways. Usually it is build as an upgrade to an

Atomic Force Microscope (AFM) and it make use of

an external laser beam, which is focused on the tip

of the AFM probe (Brehm et al., 2005). This

configuration allows for simultaneous s-SNOM and

AFM imaging. The investigation is done by

maintaining the probe in a fixed position, and

moving the sample point-by-point in a raster scan

manner.

Depending on the type of the interaction between

the tip, the focused external laser beam and the

surface of the sample, three major a-SNOM

techniques exist, which return complementary

optical information: a) scattering - Scanning Near-

field Optical Microscopy (s-SNOM) (Knoll and

Keilmann, 2000, Hillenbrand, 2007); b)

Fluorescence Apertureless Scanning Near-field

Optical Microscopy (FASNOM) (Fragola et al.,

2003, Huang et al., 2006), and c) Apertureless

Scanning Near-field Optical Microscopy with

Second Harmonic Generation (ASNOM-SHG)

(Zayats and Sandoghdar, 2001).

In the first approach (s-SNOM), the illuminated

nano-tip of the investigating probe is equivalent to

an oscillating electric dipole (Brehm et al., 2005).

The electric field component of the incident beam

creates this dipole in the tip and determines the

dipole to oscillate with the same frequency as the

electric field intensity. As a result, the oscillating

electric dipole will re-emit light, behaving like an

antenna. Because in a typical AFM investigation the

tip is located in the proximity of the sample, the

oscillating dipole will be highly influenced by the

presence of the sample’s surface. In this case, the

surface is equivalent to a mirror dipole (located in

the sample, close to the surface), which will interact

with the original dipole. The interaction is reciprocal

and the light emitted by the oscillating electric

dipole will carry optical information related to the

optical characteristics of the sample’s surface.

The second approach (FASNOM) exploits the

fact that metallic and semiconductor nano-structures

have complex influence on fluorescence phenomena

(Huang et al., 2006). More precisely, it relies on the

fact that placing a metallic structure in the proximity

of a fluorophore leads to fluorescence quenching or

enhancement and also to a modification of both the

radiative and the non-radiative rates of the probe,

inducing changes in both the fluorescence lifetime

and the in emission intensity. These changes can be

exploited to obtain sub-wavelength resolution

imaging.

The third approach (SHG-ASNOM) is based on

a nonlinear optical effect in which either the tip or

the sample is SHG active. In this configuration, the

local second harmonic field generated at the

sample’s surface is probed.

While all these approaches are capable of sub-

wavelength optical resolution (on the order of

curvature radius of the tip, usually few nanometers),

the a-SNOM techniques are widely limited by the

difficulties in understanding the collected data. This

situation is generated by the fact that the existing

mathematical models are either incomplete or

extremely complicated (Brehm et al., 2005). In the

same time, the experimental acquisition data process

is highly affected by background light (representing

noise), which appear due to multiple light reflections

between the sample’s surface and the probe’s body;

this background light arrives on the detector and has

a higher intensity than the light emitted from the

near-field of the sample, assessing thus difficulties

in the detection process.

E. Tranc

a D. and A. Stanciu G..

New Aproaches for High-Resolution Optical Microscopy.

 2015 SCITEPRESS (Science and Technology Publications, Lda.)

2 OUTLINE OF OBJECTIVES

The objectives of the presented work are focused on

addressing the major issues faced by a-SNOM

techniques. Thus, an advancement regarding the

mathematical models associated with the physical

phenomena is on the objective list. Analyses of the

mathematical models will provide clues for

improvements with respect to data interpretation

process – that is another major objective. Because of

the difficulties associated with a-SNOM techniques,

few investigation areas had been explored till now.

Thus, an increased attention is given to new research

areas where a-SNOM can be used successfully – this

being the third major objective of the presented

work.

3 STATE OF THE ART

In the frame of near-field optical investigations, two

major categories of microscopes are available: (a)

aperture-SNOM, and (b) apertureless-SNOM. The

first class uses a metal-coated tapered optical fibre

as a probe (NOVOTNY et al., 1995). Although

SNOM with aperture is more popular and already

commercially available, it suffers from a major

drawback, as a compromise is necessary between the

dimension of the aperture and the power of the

incident laser beam. For higher resolution, the

aperture needs to be smaller; in the same time, for an

efficient transmission, a smaller aperture requires a

greater optical power, which may be harmful to the

probe’s tip. This situation limits the lateral

resolution to about 100 nm.

The second class of near-field microscopes is

represented by the so-called “apertureless-SNOM”.

Its functioning principle has been already presented

in the introductive part of the paper. As this class of

near-field microscopes present several advantages

over SNOM with aperture (better resolution;

independence on the radiation wavelength), it itself

confronts with a series of difficulties. The major

drawback is the presence of a background light

(noise), which affects the detection process. Several

techniques have been developed to date in order to

discriminate between the near-field signal and the

signal generated by the background light. Higher

harmonic demodulation (HHD) (Maghelli et al.,

2001) takes advantage over the nonlinear

dependence of the intensity of the light emitted from

the near-field with the distance between the tip and

the sample’s surface. Thus, oscillating the probe in a

sinusoidal movement above the sample (as in AFM

tapping mode) and detecting the signal on a higher

harmonic of the oscillation frequency f

, the signal-

to-noise ratio (SNR) will increase. This is due to the

rather linear dependency of the background light on

the tip-sample distance.

However, HHD is not sufficient for efficient

background reduction. Along with it, interferometric

detection is also used. During the last decade, three

major interferometric methods were used: homodyne

(Knoll and Keilmann, 2000), heterodyne (Gomez et

al., 2006) and pseudoheterodyne detection (Ocelic et

al., 2006). Among these, the last one proved to be

particularly efficient and easy to implement. Is

consists in a modified Michelson interferometer, in

which one arm is used for the illumination of the tip

and for collection of the near-field emitted light

(together with the background). The other arm is

travelled by a reference beam, which is phase-

modulated by an oscillating mirror. This reference

mirror oscillates with a certain frequency M (much

smaller than the oscillating frequency of the probe)

and certain amplitude, A. The two beams finally

interfere on the detection path, on which a detector

is placed. Without the reference beam, the Fourier

spectrum of the electric signal generated by the

detector will contain signal components situated at

the probe’s oscillation frequency and at its

harmonics. When the reference beam is present, the

phase modulation determines the appearance of two

side-bands around each initial harmonic component.

These side-bands contain components situated at

±mM. It had been demonstrated that the

background light has a minimal influence on these

side-bands; thus, detection on a frequencies situated

in these side-bands assures for best SNR.

A recently reported alternative to pseudo-

heterodyne detection is represented by the

combination between synthetic optical holography

and s-SNOM (Schnell et al., 2014). In this case, the

resulting image is a hologram, which contains both

amplitude and phase data of the near-field emitted

light.

Regarding the mathematical models, two major

models are largely used: Oscillating Point Dipole

Model (OPDM) (Knoll and Keilmann, 1999) and

Finite Dipole Model (FDM) (Cvitkovic et al., 2007).

To date, a-SNOM has been applied successfully in

several areas, including: nano-imaging (Schnell et

al., 2010), characterisation of plasmonic structures

(Kim and Kim, 2012), near-field spectroscopy

(Stiegler et al., 2011), nano-chemical

characterisation (Berweger et al., 2013), or SHG and

fluorescence on representative samples (Huang et

PHOTOPTICS2015-DoctoralConsortium

al., 2006, Zayats and Sandoghdar, 2001). The

presented work extends the application areas of a-

SNOM in areas like biology, electronics and

measurement of electric permittivity in the visible

domain (using the OPDM). Detailed studies

regarding the influence of functioning parameters

upon the image contrast in a pseudo-heterodyne

scheme was also done (Tranca et al., 2014).

4 METHODOLOGY

The methodology is divided in three sections, which

follow the three major objectives described before.

Thus, advances in mathematical modelling are

represented by improvements regarding the

oscillating dipole model (OPDM), which are

obtained by advanced mathematical analysis of

signal spectra. Theoretical results are correlated with

the experimental data in the purpose of advancing

the data interpretation process. A quest for new

applications for s-SNOM investigations is worked

up by using the results obtained in the previous steps

and experimental data on samples containing

dielectrics or biological tissues.

4.1 Signal Analysis

The intensity of the near-field scattered light σ is

proportional with the amplitude of the incident light

phasor E

and a factor α

eff

named the effective

polarizability (Ocelic et al., 2006):

eff







(1)

According to the oscillating point dipole model, the

effective polarizability has the form:



eff



























(2)

In Eq. (2), α stands for the polarizability of the tip,

and β(ε) depends on the local sample electric

permittivity, ε

by the relation:















(3)

Assuming a sinusoidal oscillation of the probe above

the sample, this parameter can be written as:



1sin2

zft





  







(4)

Here, d

is the min. distance between tip-sample

during the probe’s oscillation above the sample, z

the oscillation amplitude of the probe, f

is the

oscillation frequency of the probe and t is time.

Rewriting Eq. (2), the intensity of the near-field

scattered light will have the following form:









1sin2

zft















  







  







 





(5)

From this point, one can spectrally analyse the

function given by Eq. (5), by using the exponential

Fourier transformation method. According to

Fourier analysis, a function can be written as a sum

of its harmonic components. In our case, the

harmonic components can be written as:



116

1sin

jnu

cE du





















 

















(6)

Although the integration calculus in Eq. (6) is not a

common one and its calculation requires special

mathematical algorithms, its complexity decreases if

it is calculated for certain values of the n factor.

Using numerical methods, the definite integral in Eq.

(6) can be easily calculated. Thus, for a certain

integral positive n and in the case of well-known

experimental conditions (regarding the AFM

functioning parameters and the characteristics of the

probe), the only variable in Eq. (6) remains β.

However, the value of the spectral component c

cannot be experimentally determined, and this is due

to the background light presence. By using the

pseudoheterodyne detection scheme and detecting

the signal on the side-bands created around every

spectral component of the initial signal, the

influence of the background light can be bypassed.

The detection on a certain component located at

±mM can be accomplished by means of a lock-in

amplifier. In this case, a reference beam is used and

its mathematical form will be (as a Fourier series):





exp 2

jm Mt









(7)

The coefficients ρ

can be written as:

exp

mm R

JA jjm







  



  

  

(8)

Here, ρ is the amplitude of the amplitude of the

reference wave phasor, A is the oscillation amplitude

of the reference mirror, λ is the wavelength of the

beam, M is the oscillation frequency of the reference

mirror, Ψ

is the mean phase difference between the

two interferometric pathways and t is time. In Eq.

(8), J

stands for the Bessel function of order m.

The resulting interference signal U between the

NewAproachesforHigh-ResolutionOpticalMicroscopy

near-field light σ will and the reference beam E

will

have the spectral components u

n,m



















nm n m n m

u real c real imag c imag



   

(9)

The above equation expresses the exact form of a

single spectral component of the higher harmonic

demodulated, pseudo-heterodyned near-field signal.

Together with the Eq. (6) and (8), it can be used to

develop a detailed analysis of the near-field signal.

The main purpose is to understand the way in which

the optical parameters of a sample influence the

detected signal. In the same time, it is important to

understand the way in which every functioning

parameter of the system influences the detected

signal and the overall image contrast.

4.2 Data Interpretation

As the background light has a minor influence on the

components situated at frequencies nf

±mM, with

m≠0, a proportional relation can be assumed

between the value of an image pixel I

n,m

and the

amplitude of the harmonic component u

n,m

on which

the detection is employed. This can be expressed as

a linear mathematical relation. Based on this

proportionality, the spectral component c

from Eq.

(9) can be determined. Introducing c

in Eq. (6), the

value for β is obtained and with Eq. (3), the electric

permittivity of the investigated material is then

determined. Thus, the data obtained by s-SNOM

investigation can be interpreted on the basis of the

local electric permittivity of the sample. Figure 1

shows two images of a sample containing Si and

SiO

domains. The images were detected at,

respectively, 2f

+M and 2f

+2M. Both images are

necessary for electric permittivity measurement. The

figure also shows how the areas of different

materials (Si and SiO

) were selected for local

permittivity determination.

Figure 1: 2x2 μm s-SNOM images of the Si/SiO

sample;

a) detection on 2f

+M; b) detection on 2f

+2M. The

central Si region is marked with blue and the surrounding

SiO

region is marked with red.

A crucial role in data interpretation process is

played by the functioning parameters of the system,

which can easily influence the image intensity and

contrast. An example is the oscillation amplitude, A,

of the reference mirror. It has been demonstrated

that the image contrast is strongly dependent on A

and depending on the value of A, the image contrast

can be inverted and even reduced to zero. However,

the contrast variation is due to variation of the

amplitude of the spectral components of the signal

with the amplitude A. In Figure 2 is presented the

variation of the magnitudes of the spectral

components situated at 2f

+M and 2f

+2M with the

amplitude A of the reference mirror oscillation.

Figure 2: Normalized magnitudes of the spectral

components situated at 2f

+M (blue) and at 2f

+2M (red);

their variation with the amplitude A of the reference

mirror. Continuous curves: Jacobi–Anger expansion

coefficients; circled curves: Fast Fourier Transform; Black

dots: experimental determinations.

Another example is the mean phase difference Ψ

between the two interferometric pathways. Varying

the mean phase difference, the amplitude of the

detected signal will also vary, and every spectral

component will be affected. In Figure 3 is presented

the variation of the magnitudes of the spectral

components situated at 2f

+M and 2f

+2M when the

mean phase difference Ψ

is varied between 0 and

2π.

Figure 3: Normalized magnitudes of the spectral

components situated at 2f

+M (blue) and at 2f

+2M (red);

their variation with the mean phase difference, Ψ

PHOTOPTICS2015-DoctoralConsortium

4.3 New Applications

Due to the fact that such a microscopy system can

provide local optical information about the

investigated sample with sub-wavelength resolution,

it became highly attractive for many applications. In

biology and medicine, the refractive index of a live

cell has gained much attention as an attractive

indicator of cell abnormality. In cancer biology, it is

well known that the refractive indexes of cancer

cells are relatively higher than normal cells, which is

believed as a good criterion for quantitative

diagnosis of cell malignancy. The possibility to map

the electric permittivity of a biologic sample with

nano-resolution precision can provide the means for

early stage cancer diagnosis.

In materials science, the detailed knowledge of

the changes in the refractive index can be used to

pinpoint the locations of local defects.

The a-SNOM system can also be used in the

nano-electronics industry, where in the last years the

optical constants of the materials became important,

as they are related to the thickness of the films:

errors in the refractive index value translate into

errors in the film thickness, determining less

accurate results.

4.3.1 S-SNOM Investigations

Two different samples were used for s-SNOM

investigations. The first one was a calibration

sample containing Si and SiO

features and it is

relevant for nano-electronics industry. It was

prepared from a Si substrate on which a 26 nm thick

SiO

film was deposed. Holes with 500 nm in

diameter have been made in the SiO

film until they

reached the Si substrate. s-SNOM image containing

a single hole, together with the AFM image is

presented in Figure 4. The two images were taken

simultaneous.

Figure 4: a) AFM and b) s-SNOM investigation on a

Si/SiO

sample.

The second sample for s-SNOM investigations

was a biological tissue from a rat retina. In Figure 5

there can be observed retinal ganglion cells. Again,

simultaneous acquired AFM and s-SNOM are

presented.

Figure 5: a) AFM and b) s-SNOM investigation on a rat

retina tissue.

The results presented here demonstrate not only the

capability of the s-SNOM system to image samples

relevant for micro- and nano-electronics industry

and for biology, but they also demonstrate the

nanometric-scale optical resolution capability.

4.3.2 FASNOM Investigations

A sample containing collagen fibres deposed on a Si

substrate was used to investigate the optical near-

field with the modulation of the fluorescence

(FASNOM). This sample is also relevant to biology

and medicine areas, as the collagen is an important

compound of many biological tissues. In Figure 6

are presented the AFM and FASNOM images.

Figure 6: a) AFM and b) FASNOM investigation on a

sample containing collagen fibres.

4.3.3 SHG-SNOM Investigations

The collagen sample used for FASNOM imaging is

also relevant for SHG-SNOM investigation. In

Figure 7 are presented the AFM and SHG-SNOM

images on this sample.

A second sample on which SHG-SNOM imaging

was performed is a rabbit cornea tissue. A good

SHG signal was detected, as investigated tissue is

NewAproachesforHigh-ResolutionOpticalMicroscopy

rich in collagen, which is known to generate second

harmonic optical frequency light.

Figure 7: a) AFM and b) SHG-SNOM investigation on a

sample containing collagen fibres.

Figure 8: a) AFM and b) SHG-SNOM investigation on a

sample containing a rabbit cornea tissue.

5 EXPECTED OUTCOME

Regarding the s-SNOM imaging, the expected

outcome is related to the possibility to map at

nanoscale precision the local electric permittivity of

complex samples, such as biological samples. Such a

map can provide useful information regarding the

cell abnormality or details of the sub-cellular

features. Such investigations are of high importance,

as the s-SNOM system does not require special

preparations or treatments of the samples. To

achieve this, firstly the influence on the detected

signal of all involved functioning parameters of the

system need to be evaluated. Secondly,

investigations in well-known functioning conditions

need to be performed on simple samples containing

known materials, in order to calibrate the system and

to confirm the method for electric permittivity

determination. The third major step comes with the

complex samples containing biological cells, on

which the mapping of the local electric permittivity

is performed.

For FASNOM and SHG-SNOM systems, the

expected outcome is related to the correlation with

their corresponding far-field optical investigations.

If their only gain (against the fluorescence

microscopy and SHG microscopy, respectively) is

the improved resolution, then this is sufficient for

new important scientific findings, as fluorescence

optical investigations or SHG investigations with

nanometric resolution were impossible before. For

this, large areas on relevant samples can be

investigated both with far-field and near-field

methods, in order to find correspondences at micro-

scale. If correspondences are possible, then

fluorescence and SHG near-field investigations can

be performed at nano-scale, which will provide

detailed optical data.

6 STAGE OF THE RESEARCH

The stage of the presented research is sufficiently

advanced for s-SNOM imaging system. As shown in

the Methodology section, the possibility to measure

the local electric permittivity with nano-resolution

was demonstrated on a calibration sample. New

applications for s-SNOM in the biology area are

expected, as biological tissues has already been

investigated by s-SNOM (see section 4.3.1). The

next step is the mapping of the electric permittivity

of complex samples containing biological cells.

Regarding the FASNOM and SHG-SNOM, the

detection capabilities was demonstrated on relevant

samples; however, far-field investigations need to be

performed on the same samples, in the next steps, in

order to compare them with the near-field images.

ACKNOWLEDGEMENTS

This work is supported by the Sectoral Operational

Programme Human Resources Development (SOP

HRD), financed from the European Social Fund and

the Romanian Government under the contract

number POSDRU/159/1.5/S/137390/.

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NewAproachesforHigh-ResolutionOpticalMicroscopy