Waveguide Evanescent Field Microscopies for Application in

Cell- and Bacteria- Biophysics

Silvia Mittler

Department of Physics and Astronomy, The University of Western Ontario, London, ON, N6G2P6, Canada

Keywords: Waveguide, Evanescent Illumination, Microscopy, WEEF and WEFS, Cells, Bacteria, Fluorescence,

Scattering, Distance Mapping, Granularity, Application as a Sensor.

Abstract: Two evanescent field microscopy technologies based on glass slab waveguides with permanent coupling

gratings are presented: waveguide evanescent field fluorescence microscopy (WEFF) and waveguide

evanescent field scattering microscopy (WEFS). The technologies are briefly described: the experimental

setup is based on a conventional inverted microscope. A comparison to TIR and TIRF microscopy is given.

The advantages of the waveguide method are clearly addressed. Various examples from for WEFF and

WEFS microscopy are given. For WEFF: static distance mapping with a multimode waveguide, dynamic

solubilisation studies of cell plasma membranes and the kinetic response of osteoblasts to trypsin. For

WEFS: bacteria sterilization as well as cell adhesion and granularity studies. The latest development is a

mass producible all-polymer-waveguide-chip to bring the technology to the interested scientific community.

1 INTRODUCTION

With the aim of developing new medical devices

with direct tissue contact, drug delivery vehicles,

and tissue engineering scaffolds, there has been

increasing interest in recent years in the interactions

of cells with both synthetic and natural biomaterials

(Niu et al., 2005; Storrie et al., 2007). In particular,

the study of the contact regions between a cell and

its substratum is of considerable interest as its

investigation delivers inter alia information about

the cytocompatibility of the substratum - the affinity

of cells towards that particular surface. Promotion or

inhibition of cell adhesion to synthetic and natural

biomaterials is often crucial to the proper function of

a particular device. Some information concerning

these interactions, e.g. the lateral location and the

density of the adhesion sites, as well as their

relationship to the actin stress fiber system, part of

the cell's cytoskeleton, can be inferred from

fluorescence microscopy of immunolabeled

molecules involved in adhesion; typically, vinculin,

a protein located within the multi-protein complex

that anchors the adhesion to the cytoskeleton inside

the cell (Burmeister et al., 1998). These methods

only deliver signals from the focus volume and no

information about adhesion distances to the

substratum. However, a direct and quantitative

method to address the distance to the substratum is

highly attractive. To address this need, different

microscopic techniques based on electron

microscopy (Chen and Singer, 1982) and optical

means such as evanescent fields and interference

techniques have been developed. Total internal

reflection fluorescence (TIRF) (Burmeister et al.,

1998; Burmeister et al., 1994), surface plasmon

resonance microscopy (SPRM) (Giebel et al., 1999),

interference fluorescence microscopy (IRM)

(Verschueren, 1985), fluorescence interference

contrast (FLIC) microscopy (Braun and Fromherz,

1997) and combinations thereof (Burmeister et al.,

1998; Atilgan and Ovryn, 2009) have been used to

visualize and quantify these contacts. The contacts

themselves had been discovered by interference

reflection microscopy (IRM) in the 1970s

(Abercrombie et al., 1971).

Bacteria, on the other hand, are the most

metabolically diverse group of organisms found in

all natural environments including air, water and

soil. Bacteria commonly occur with food sources

and are also found within and on our bodies.

However, concerns exist over contamination of

food, water, and air by pathogenic bacteria (Sapsford

Mittler, S.

Waveguide Evanescent Field Microscopies for Application in Cell- and Bacteria- Biophysics.

DOI: 10.5220/0005618302010212

In Proceedings of the 4th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2016), pages 203-214

ISBN: 978-989-758-174-8

203

and Shiver-Lake, 2008) that can enter our bodies

through ingestion, inhalation, cuts or lacerations

(Pizarro-Cerda and Cossart, 2006). Therefore, there

is an increasing interest in bacterial contamination

and the need for anti-bacterial surfaces not only for

application in the food industry but also for medical

and hygienic purposes (Oliver, 2005). Over two

million hospital-acquired cases of infection are

reported annually in the USA, which lead to

approximately 100,000 deaths annually and added

nearly $5 billion to U.S. healthcare costs (Madkour

and Tew, 2008; Madkour et al., 2009).

Contamination of medical devices (e.g., catheters

and implants) has been attributed to 45% of these

infections (Stamm, 1978).

Bacterial contamination

of any surface typically begins with the initial

adhesion of only a few cells that can then develop

into a more structurally cohesive biofilm in less than

24 hours when provided with suitable nutrient

conditions sustaining metabolism and cell division

(Hetrick and Schoenfisch, 2006). Therefore, a better

understanding of bacterial adhesion to surfaces is

important for technical surface development and in

biomedical applications. However, the precise

measurement of bacterial adhesion to surfaces are

difficult and time consuming because bacterial cells

typically occur on the micrometer-scale and their

adhesion forces are generally low‚ typically 0.1–100

nN (Christianson, 2004). Recent studies on the

detection of bacteria on surfaces have focused on

similar imaging systems as with cells such as optical

(Vasilev et al., 2009) and fluorescent microscopy

(Pires et al., 2013) to image the bacteria themselves

or luminescence measurement of the presence of

cells by ATP (adenosine triphosphate) detection

systems (Pera et al, 2010). Surface Plasmon

Resonance (SPR) sensors (Taylor et al., 2008),

Nucleic Acid Detection (Schmidt et al, 2006),

Optical Waveguide Lightmode Spectroscopy

(Cooper et al., 2009), Optical Leaky Waveguide

Sensors (Zourob et al., 2005), and Evanescent Mode

Fiber Optic Sensors (Mazhorova et al., 2012) have

also been applied in order to detect biochemical

toxins as signatures of bacteria.

In conclusion, it is important to have methods

which are able to investigate interfaces between a

technical surface and a bacterium or cell.

In recent years, Total Internal Reflection

Fluorescent (TIRF) microscopy has been

demonstrated to be an effective method for studying

cell-substrate interactions that occur at surfaces and

interfaces. Using TIRF microscopy, the behaviour of

various types of cells (Bauereiss et al., 2015: Liu,

2015) and bacteria (Smith et al., 2002, Vigeant et al.,

2001) near surfaces has been characterized. Total

Internal Reflection (TIR) Microscopy utilizes the

basic technology of TIRF without any fluorescence

dyes present in the sample by creating an optical

contrast due to scattering (Byrne et al., 2008).

Recent studies have also demonstrated the use of

TIR for imaging microbial adhesion.

The waveguide evanescent field scattering

technique was developed by Thoma et al. (Thoma et

al., 1997; Thoma et al., 1998) for ultrathin technical

structures on surfaces using ion exchanged

waveguides. Later, Waveguide Evanescent Field

Fluorescence (WEFF) microscopy was developed

(Grandin et al., 2006; Agnarsson et al., 2009;

Horvath et al. , 2005; Hassanzadeh et al., 2008) as a

straightforward alternative to TIRF microscopy for

imaging ultrathin films and cell-substrate interaction

using fluorescence dyes located in the plasma

membrane.

This paper will give an overview on biophysical

applications of WEFF and WEFS microscopy on

cells and bacteria and a short outlook on current

developments to offer the methods to a broader user

base.

2 EXPERIMENTAL

2.1 Waveguides

In this study home-fabricated, glass on fused silica,

step-index slab waveguides or ion exchanged

waveguides with holographic coupling gratings were

used (Hassanzadeh and Mittler, 2011; Halfpap et al.,

2012). The waveguides were reusable various times

after thorough cleaning. A typical cleaning

procedure consisted of a submersion in 70% ethanol

(Aldrich, Canada) with sonication (Branson 2510,

Branson, USA) for 20 min and a blow-dry with

nitrogen gas. To remove organic material, the dried

samples were cleaned with Nano-Strip (KMG

Chemicals Inc., Fremont, CA) at 80°C for 5 minutes.

After the Nano-Strip application, the substrata were

rinsed extensively in Milli-Q water and blown dry

again.

2.2 Cell Culture

The osteoblastic cell line MC3T3-E1 (subclone 4,

ATTC Catalog 3 CRL-2593) were cultured in flasks.

The cleaned waveguides were sterilized for 3 hours

by UV light. Growth medium was prepared from

17.8 ml α-minimum essential medium 1X (MEM;

Gibco), 2 ml fetal bovine serum (FBS; Gibco) and

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0.2 ml antibiotic-antimycotic solution 100X (Anti-

Anti; Gibco). First the medium was aspirated from

the cell culture flask. Dulbecco’s phosphate-buffered

saline 1X (PBS; Gibco) was added to wash the cell

layer and aspirated subsequently. To detach the

osteoblasts from the vessel wall, 5 ml trypsin-EDTA

(0.05%, Gibco) was added and incubated at 37°C for

5 minutes. The culture was checked by phase-

contrast microscopy to confirm that cells were

released into the suspension. The trypsin was

neutralized by adding 9 ml growth medium to the

flask. The resulting cell suspension was diluted in

growth medium to 10,000 cells per ml. Waveguides

were placed in a Petri dish and 1 ml cell suspension

per substrate was applied to the surfaces. Samples

were then incubated for 24 h at 37°C, 100%

humidity and 5% CO

The waveguides were removed from the growth

medium and excess medium was aspirated. Next,

each waveguide was rinsed three times in PBS. For

fixation, the waveguides with the cells on top were

placed in a solution of 4% paraformaldehyde in PBS

for 10 minutes at room temperature. Subsequently,

samples were rinsed three times with PBS. To

prevent desiccation, samples were kept in PBS until

further treatment. A solution was prepared from 1.5

mg DiO in 1 ml dimethyl sulfoxide (DMSO) and

heated to 37°C within 5 minutes. This mixture was

sedimented for 5 minutes at 2000 rpm to separate

solid residues. Ten μl of this stock solution was

dissolved in 1 ml growth medium to form the

staining solution. The staining solution (200 μl) was

pipetted onto the corner of each waveguide and the

waveguide gently agitated until all cells were

covered with staining solution. The samples were

left in the solution for 20 minutes to incorporate the

dye. Afterwards, the staining solution was drained

and the waveguides were washed in PBS. For the

removal of all unbound dye, the samples were

immersed in PBS for 10 minutes and drained again.

The entire wash cycle was repeated two more times.

The waveguides were stored in PBS until

performing WEFF microscopy. This procedure

delivers fixed cells, cells that are “frozen” in their

habitus (Lanier, 1981; Smit, 1974; Su, 2014) with

the dye situated in the plasma membrane of the cells.

2.3 Bacteria Culture

Nitrobacter sp. 263 was cultured on R2A (Difco™)

plates at room temperature (approximately 23°C) for

two weeks. For each colonization experiment,

bacteria from one R2A plate were removed and

suspended in 1 ml of filter-sterilized (0.45 μm pore-

size) distilled deionized water to produce an aqueous

bacterial suspension (with 10

bacteria/ml). A

separate stock solution of R2B (i.e., broth/liquid

culture medium) was made by dissolving R2A in

sterile, distilled, deionized water and filtering this

solution to remove the agar constituent leaving the

dissolved nutrients for bacterial growth.

Bacterial attachment to the waveguide surface

was achieved by placing a 50 μl aliquot of the

bacterial suspension on top of the waveguide for one

hour at 37°C. After bacteria attached to the surface,

the waveguide was rinsed with sterile, distilled water

and placed in a sterile Petri dish containing 20 ml of

R2A and incubated for 24 hours at 37°C to allow the

attached bacteria to grow. The samples were not

agitated. After 24 hours incubation, the waveguides

were examined using bright field microscopy to

determine whether microcolonies had formed. Note

that all images were taken of live cells in growth

medium. Samples were then analysed using WEFS

microscopy.

Sterilization experiments were performed.

Separate bacteria suspensions of 10 ml (with 10

bacteria/ml) were placed in a sterile, open glass dish

and exposed in a low pressure collimate beam

apparatus (LPCB) to induce sterilization (Hedrick et

al., 2007; Kuo et al., 2003) at doses of 2, 4, 8, 14, 20

and 30 mJ/cm

increasing time to produce

different doses (Kuo et al., 2003). This mode of UV

photon sterilization was chosen for its common use

in industrial applications and its ability to disrupt

and dimerize neighboring DNA bases (thymine

dimerization) that hinders bacterial growth but not

viability (Berney et al., 2007; Durbeej and Eriksson,

2002). Each ‘sterilized’ bacterial suspension,

produced via the different dose exposures, was used

in an identical colonization experiments as described

above.

It is important to note that prior to the first and

second colonization experiment, separate aliquots of

all bacterial suspensions (1ml) were stained using

BacLight

(Invitrogen) Live-Dead stain and

examined using fluorescence microscopy to confirm

that the cells were viable.

2.4 WEFF and WEFS Microscope

The WEFF and WEFS microscope (Hassanzadeh et

al., 2008) consisted of an inverted microscope

(Zeiss, Oberkochen, Germany) with the waveguide

being located on the sample stage (Fig.1). The

specimen was positioned on top of the waveguide.

An argon ion laser (35 LAP 341-200, CVI Melles

Griot) operated at λ = 488 nm with a variable output

Waveguide Evanescent Field Microscopies for Application in Cell- and Bacteria- Biophysics

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power of 7- 126 mW or a HeNe laser with a

wavelength of 543.8 nm (Research Electro-Optics,

0.5 mW) were used as light sources in WEFF and

WEFS microscopy, respectively. A neutral density

filter was placed directly behind the laser for power

reduction, avoiding bleaching and overexposure. An

iris aperture controlled the beam diameter.

Figure 1: Schematic of WEFF microscope. Ap: aperture,

F1: neutral density filter, M: mirror, WG: waveguide, PD:

photo diode. For WEFS microscopy a HeNE laser was

used and the LP filter omitted.

The laser beam was coupled into a chosen

waveguide mode by a coupling grating located on

the waveguide. In the case of WEFF microscopy, the

undesired excitation wavelength was blocked with a

long pass filter with a cut-off wavelength of λ

cut-off

490 nm (3RD490LP, Omega Optics, Brattleboro,

VT) which was fitted between the objective and the

camera. The out-coupled intensity at the end of the

waveguide was captured with a large active area

photodiode (FDS1010, Thorlabs, Newton, NY) for

determining the coupling efficiency when needed. A

cooled CCD-camera (Persuit - XS 1.4 Diagnostic

Instruments Inc., Sterling Heights, MI), controlled

with SPOT 5 Basic (Spot Image Solutions, Sterling

Heights, MI) was connected to a computer. Image

data were exported for processing. Additionally,

bright field microscopy images of the samples were

captured with the same field of view/objective lens

as the WEFF/WEFS microscopy images and

processed with Image Pro Express (Media

Cybernetics, Rockville, MD).

3 TIRF/TIR AND WEFF/WEFS

COMPARISON

Both microscopy suits, TIRF/TIR and

WEFF/WEFS, employ evanescent fields for sample

illumination at the surface of a substrate which are

produced by total internal reflection. In modern

TIRF/TIR microscopes a laser beam is guided opto-

mechanically within the microscope and the

objective lens to allow a laser beam to undergo total

internal reflection at a high refractive index substrate

which carries the specimen and is located above the

objective lens. Costly state-of-the art equipment and

objective lenses with specially designed high

magnification and high numerical aperture

objectives and built-in optical path control are

necessary. Theoretically, all angles above the critical

angle of TIR can be achieved in this way, giving the

possibility to achieve different penetrations depth

with different angles. This can be used to measure

distances from the substrate surface (Truskey et al.,

1992). Practically, the microscopes are set to

particular angles, typically to receive a high quality

TIRF or TIR image and a high quality epi-

fluorescence or bright field image, respectively,

taken with a transmitting beam.

On the other hand, operating a TIRF/TIR

microscope manually can easily lead to a loss of the

evanescent mode and to a full specimen exposure to

the laser beam resulting in a damaged sample.

Reviewing the literature and in particular the use

of TIRF microscopy for distance measurements

shows that besides Burmeister’s excellent work in

the middle of the 1990s (Burmeister et al., 1994)

during the development phase of TIRF microscopy

only little has been published on exploiting different

penetration depths.

TIR microscopy is performed identically but

excluding the dye from the samples and the

necessary filter sets. Scattered photons instead of

fluorescence photons are collected. Bright field

images are taken for comparison since epi-

fluorescence is not possible. Little distance work has

been published involving TIR microscopy (Smith et

al., 2002). This is not surprising since the scattering

intensities are hard to analyse because all refractive

index fluctuations present in the evanescent field

contribute to the signal and these are not necessarily

controllable, in particular with living cultures

producing extracellular matrix in the case of cells or

extracellular polymeric substance (EPS) when

imaging live bacteria.

In WEFF/WEFS microscopy the resonances of

the waveguide modes dictate the available

evanescent fields and penetration depths. So the

number of choices is limited by the number of

modes propagating in the waveguide. In TIRF and

TIR microscopy, the penetration depth of the

evanescent field is limited to a maximum of ~ 200

nm, whereas a waveguides can produce penetrations

depths from below 100 nm to over a μm by tuning

the refractive index and thickness architecture of the

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core and cladding layers (Agnarsson et al., 2009).

Planar waveguides also offer an extended

illumination area over macroscopic dimensions only

limited by the attenuation of the propagating

waveguide mode.

In addition, the beam in WEFF/WEFS can never

escape the waveguide; therefore WEFF and WEFS

microscopies carry the intrinsic safety mechanism of

avoidance of sample overexposure and damage.

In well characterized waveguides the evanescent

fields and penetration depth are well known

quantities and can be used for quantitative

measurements (Hassanzadeh et al., 2009).

WEFF and WEFS microscopy do not desire

state-of-the-art microscopes or objective lenses.

WEFF and WEFS technologies are based on a few

simple accessories and attachments to a standard

inverted microscope. It is therefore straightforward

to image the specimen in any magnification and

field of view available due to standard long distance

objective lenses by just turning the objective lens

revolver without the necessity of beam stirring. Due

to the evanescent field formation being taken care of

by the substrate and completely independent from

the entire microscope, different field of views or

magnifications still deal with the same illumination

conditions allowing direct comparison of images or

measurements after changing magnification.

Epi-fluorescence images can be achieved by

simply enhancing the integration time of image

acquisition. This is due to non-perfect waveguides:

every waveguide scatters slightly and therefore

supplies the 3D volume of the specimen with

excitation or scattering photons.

Comparing TIRF and WEFF images of the same

samples has shown identical image information

(Hassanzadeh et al., 2010). Both microscopy

technology suits are diffraction limited, therefore the

lateral resolution depends on the chosen laser

wavelength and the highest possible magnification

lens supported by the microscope used. The

resolution in z-direction (perpendicular to the

substrate) lies in both types of microscopes in the

order of ~7 nm.

To achieve a wide use of WEFF and WEFS

microscopy in the interested research communities it

is necessary to have simple access to and supply of

inexpensive waveguide substrates. Therefore it is

necessary to develop a mass producible waveguide-

chip.

4 STATIC DISTANCE MAPPING

WITH A MULTIMODE

WAVEGUIDE

A waveguide with a thickness of 651 ± 2 nm and a

refractive index of n = 1.840 ± 0.001 was used for

mapping the distances of the dye located in the

plasma membrane of fixed osteoblasts. The volume

above the waveguide was assumed to be water with

a refractive index of 1.33 for simulating the

evanescent fields. Two images taken with the TM

and TM

mode were used to calculate the dye

distance map (Fleissner et al., 2015).

The WEFF image in Fig.2 depicts four

osteoblasts well spread and indicating the nuclei and

some cell extensions. A false colour representation

of Fig.2 can be found in (Fleissner et al., 2015). The

dye distance map depicts lower distance grey values

(dark) in the area of the cells from close to 0 to ~

130 nm. In the unoccupied area, the unstained

medium, where the raw data do not show

fluorescence intensities, only noise is present. This is

depicted as distances in the order of the penetration

depth of the evanescent field: ~160 ± 40 nm (noisy,

dotty area). In addition, isolated spots in the no-

sample area (outside the cells) are visible in black.

These spots are correlated to un-physical distance

values below zero caused by microscopic damages

of the waveguide. These un-physical distances

should always be omitted in image interpretation.

Figure 2: B/w representation of a dye distance map with

four osteoblasts. The inset represents a WEFF image with

increased integration time of the same field of view. The

scale bar represents 50 μm.

All four osteoblasts can be found in the distance

map and show cell outlines similar to the cells

depicted in the “epi-fluorescence” image. However,

the lamellipodia and the thinly spread cell body are

even clearer in the distance map. In Fig.2, the

distance map does not depict any information about

Waveguide Evanescent Field Microscopies for Application in Cell- and Bacteria- Biophysics

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the nuclei. Not the entire cell body reached down

very close to the surface, as expected. At some of

the cells’ outer lines and at some extreme tips of the

spread cells, small regions – only a few pixels in

diameter – were found with distances of ~10 - 25

nm, typical of a focal adhesion (Chen and Singer,

1982; Tawil et al., 1993).

Twice line like accumulations of dense focal

adhesions are found (very dark lines with distances

around 10 - 25 nm). Between the focal adhesions,

there are regions in lighter dark grey depicting

distances around 40 -50 nm as well as grey areas

depicting distances around 70 - 80 nm. Lamellipodia

of the cells, which are very faintly seen in the epi-

fluorescence images, are clearly visible in the

distance map as thin spikes with a dark grey

(possible focal adhesions or point contacts) or lighter

dark grey (possible extracellular matrix contacts)

center and bright grey to white surroundings (Chen

and Singer, 1982).

Fig.3 (b in false colour representation in

(Fleissner et al., 2015)) depicts one well spread

osteoblast in epi-fluorescence WEFF and grey scale

distance map imaging. Fig.4 shows the two z-cuts

through the distance map: one randomly through the

cell, (Fig.4a) and one through an area including the

smallest distances of the cell (Fig.4b).

Figure 3: Single osteoblast. a) Epi-fluorescence WEFF

image and b) b/w representation of the dye distance map

with z–cuts. The scale bars represent 25μm.

The area outside the cell is nearly

homogeneously dark grey. The existing noise level

in the no-sample regions is clearly depicted in the z-

cut data; it is the noisy data at an average distance of

~ 90 nm on both sides of the cell. The cell itself is

shown by the depressions in the z-cuts with the dips

indicating adhesions. The spreading of the cell is

excellently depicted by the distance map. The cell is

attached at all extreme spreading points, however

not necessarily as focal adhesions since, distances

above 40 nm and up to 50 nm, possible close

contacts, are found. In the centre of the cell, focal

adhesions are present.

The z-cuts show the position of the plasma

membrane/dye location along the cut line in nm. For

the random cut ‘a’, three “small” distances in the

order of ~ 55 nm are found, as well as a couple of

more bends towards the substratum with distances of

~ 62 - 67 nm. The maximum heights of the plasma

membrane from the waveguide surfaces between the

bends towards the substratum are found to be

between 62 and 75 nm.

Figure 4: z-cut through cell at random position ‘a’ through

cell at smallest distance locations at position ‘b’ in Fig.3.

The cuts in Fig.3 from bottom to top are represented in

Fig.4 from left to right. The scale bars represent 25μm.

Arrows depict cell position.

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In the z-cut ‘b’ through the small distance

adhesions one focal adhesion at 18 nm is found as

well as contacts with distances of 25 – 35 nm. The

maximum heights of the plasma membrane from the

waveguide surfaces in this case are 37 and 45 nm.

The bending of the membrane towards the

cytoplasm between these adhesions points is clearly

depicted. The relative straight lines between the

“maxima and minima” in the distance curve bear a

resemblance to a stretched rubber band. One needs

to keep in mind that the surface tension of the

plasma membrane tries to minimize the surface area,

trying to force the cell into a spherical shape. The

adhesions are obvious biological disruptions of the

physical effect of surface minimization.

With the current set-up it is not possible yet to do

time laps distance mapping. An automatic motorized

mirror adjustment for M4 (Fig.1) needs to be

implemented.

5 DYNAMIC SOLUBILISATION

STUDIES OF CELL PLASMA

MEMBRANES

Detergent-membrane interactions have been the

subject of many studies (Ngassam et al., 2012).

Functional membranes typically exist in the fluid

state also called the liquid-disordered state. Due to

difficulties of working with authentic cell

membranes, simplified membrane models - such as

supported lipid bilayers or liposome mimicking

biological systems - have often been used to

investigate detergent-membrane interactions

(Ngassam et al., 2012). Model membranes were

helpful in exploring the basic membrane functions.

However, in comparison to a living cell, with

integral and peripheral proteins, cholesterol

molecules and oligosaccharides in and on their

plasma membrane, artificial membrane models

cannot mimic all aspects of plasma membrane

function. In addition, studying the interaction

between lipids and detergents in the form of vesicles

(liposomes) or supported lipid bilayers has several

other disadvantages. For example, in supported lipid

bilayers, the quality of the deposited film plays a

major role. The direct contact with the underlying

substrate affects the bilayer’s structure and fluidity,

and blocks access of solutions to both sides of the

membrane.

The results of lipid-detergent interaction studies

using bio-membrane models have been related to a

Figure 5: Normalized, integrated intensities of three cells

versus time. Triton X-100 (0.013 w/w%) was added where

indicated by the arrow.

three-stage model, which was described by

Lichtenberg et al. (Lichtenberg et al., 1985). In stage

I, with increasing detergent concentration, detergent

incorporates into the bilayer. At this stage,

solubilization does not occur, but the bilayer

becomes saturated with detergent. At stage II, with

further increase in detergent concentration, the

bilayer starts to solubilise. Lipid vesicles saturated

with detergent form and coexist with mixed micelles

of lipid and detergent. At stage III, the entire

membrane solubilises, and only mixed micelles exist

(Csucs and Ramsden, 1998; Helenius and Simons,

1975).

Osteoblast were cultured on the waveguides and

imaged alive with time laps WEFF microscopy. At a

certain time Triton X-100 was added to the medium

to start solubilisation. Fig.5 shows the normalized

integrated intensity of the WEFF fluorescence signal

of three example cells imaged with time.

In the absence of detergent, the integrated

intensities are constant indicating negligible photo

bleaching. In the presence of the detergent, three

reproducible kinetic stages were found: i) an

increase in fluorescence intensity, ii) a plateau, and

iii) a decrease in intensity. Therefore, a comparison

to or an adaption of the established three-stage

model is possible. In stage I, the membrane takes up

detergent and the concentration of detergent rises in

the plasma membrane. The integrated fluorescence

intensity increases due to suppression of fluorophore

quenching by dilution of the dye with detergent

(Silvius, 1992) in the cell membrane. In this stage,

solubilisation does not occur. According to the

model, stage I ends when the membrane becomes

saturated with detergent. The end of stage I is seen

in Fig.5 when the intensity increase ends and the

plateau starts.

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In stage II of artificial membrane solubilisation,

the detergent-saturated lipid bilayer undergoes a

structural transition and converts partially into lipid-

detergent mixed micelles; however, these micelles

are not yet mobile, but still incorporated in the

membrane. Therefore, stage II is seen in our data as

the plateau in which intensity remains constant as

the dye is not leaving the evanescent field. At this

time, the dye is still located either in the membrane

or in formed micelles in unquenched conditions

mixed with detergent.

During stage III, the micelles become mobile and

leave the evanescent field, leading to a decrease in

integrated intensity. Individual micelles are too

small to be seen with the WEFF microscope.

By changing the Triton X-100 concentration the

duration of all three phases changed: the higher the

detergent concentration the quicker the solubilisation

stages (Hassanzadeh et al., 2012).

WEFF microscopy confirmed that living

osteoblasts are solubilized in the same way as model

membranes.

6 KINETIC RESPONSE OF

OSTEOBLASTS TO TRYPSIN

Trypsin is a serine protease and cleaves peptide

chains. Therefore, trypsin is used in laboratories to

cleave proteins bonding the cultured cells to the

dish, so that the cells can be suspended in fresh

solution and transferred to fresh dishes.

Figure 6: The impact of a 0.05% trypsin containing

medium on an individual focal adhesion: intensity and size

decrease with time. The lines are guides to the eye.

Healthy osteoblast cells were grown directly on

the waveguide and monitored with time laps WEFF

microscopy. Trypsin was used at 0.05% and 0.02%

concentration. Upon addition of 0.05% trypsin, the

cells were lifted very fast and only individual focal

adhesions could be imaged. However, with the lower

concentration changes in cell morphology could be

observed, such as cell retraction.

The quick disappearance of an individual

adhesion point at the high trypsin concentration was

examined. The focal adhesion point had the

appearance of a bright circular dot. A series of

images were taken with time and analyzed. Fig.6

depicts the kinetic behaviour of the adhesion point’s

disappearance, with respect of its integral intensity

and size. Clearly both the size and the integral

intensity of this individual focal adhesion point

decreased in an S-shaped curve and provided

basically identical kinetic information about the

detachment of the cell.

A sample was treated with 0.02% trypsin. The

cells have shown cell retraction, and partly detached

from the surface, leaving a black featureless

evanescent image. After the trypsin treatment the

medium was exchanged carefully to a trypsin-free

environment. The imaging was continued. The

osteoblasts, still alive, re-synthesise new adhesion

proteins for the formation of new adhesion points.

The kinetics of the adhesion process, unit the cell

population died and lost adhesion again, is depicted

in Fig.7.

120 140 160 180 200 220

-2

intensity [a.u.]

time [minute]

Figure 7: Integrated, intensity of 5 individual re-appearing

adhesion points after exchanging a trypsin-containing

medium at t = 0 to a trypsin-free medium.

7 BACTERIA STERILIZATION

Studies on the attachment of bacteria onto surfaces

using WEFS microscopy detection is a quick method

for investigations regarding bacterial sterilization

treatment (Nahar et al., 2014). We hypothesized that

non-potent, sterilized cells do not attach to surfaces

and do not form microcolonies. Therefore, we have

treated identical bacteria sample batches with

different UV doses (2, 4, 8, 14, 20 and 30 mJ/cm

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After the UV illumination the viability was

measured. The UV illumination did not result in

bacterial death. As a control, one sample was left

without UV treatment. All bacteria illuminated with

different UV doses and the control were cultured

identically and examined using WEFS microscopy

after 24 h. Fig.7 shows a series of WEFS and bright

field images of the control and UV treated bacteria.

The relative signal attributed to attached colonies

and individual bacteria on the waveguide surface

decreased as exposure to UV illumination was

increased (Fig.8). It is significant to note that the

highest dose of 30 mJ/cm

was not sufficient to

completely prevent bacterial attachment.

Figure 8: WEFS and bright field microscopy images of

UV illuminated, sterilized bacteria after 24 h of culturing:

a) and e) control: 0 mJ/cm

), b) and f) 8 mJ/cm

, c) and g)

20 mJ/cm

, d) and h) 30 mJ/cm

. The scale bars are 50 μm.

Both WEFS and bright field microscopy

demonstrated that the highest dose resulted in the

attachment of primarily individual bacteria,

demonstrating that while attachment still occurred

with increasing UV-dose, microcolony formation

was prevented.

Figure 9: Percentage of occupied area of bacteria versus

applied UV dose. The line is a guide to the eye only.

In order to yield quantitative data, a Matlab

program was written to investigate the intensity

distribution of each WEFS image and to calculate

the percentage of area (i.e., pixels with signals above

the defined threshold) occupied by bacteria (i.e.,

individual cells and cells comprising distinct

colonies). Fig.9 shows the percentage of area on a

sample occupied by bacteria versus the applied UV

dose. Although the percentage of surface area with

attached bacteria was decreasing exponentially, it

did not reach zero. Bacteria were still attached to the

waveguide surface despite the UV treatment.

A rough extrapolation of the exponential curve

suggests that a dose of >100 mJ/cm

would be

required to completely hinder all bacterial

attachment to the surface. If this had been a water

sterilization experiment, at least a dose of 200

mJ/cm

(double safety) should be applied before

release to the user.

8 CELL GRANULARITY AND

ADHESIONS

Fixed osteoblasts were imaged with WEFS

microscopy. Fig.10 shows a bright field image of a

single osteoblast and the corresponding WEFS

image. In the WEFS image the nucleus can be

located: it is the dark area in the cell centre. In

addition, the granular structures in the cell body and

the adhesion sites at the cell outline are visible.

Fig.10 indicates with the arrow the propagation

direction of the waveguide mode. The cell’s

boundary first hit by the propagating light is shown

very clear and with many adhesions points. The

other three outer lines depict the adhesion points but

not the complete cell boundary. At this chosen

integration time the WEFS image depicts adhesions

due to the evanescent illumination and the cell

granularity due to 3D scattering of the waveguide.

Cell-substrate adhesions could be distinguished

from scattering centres located further away from

the substrate, the granularity of the cell, by varying

the integration time. This is shown in Fig. 11.

Figure 10: a) Bright field image and b) WEFS image of an

osteoblast taken with an exposure time 3000 ms. The

green arrow indicates the direction of light propagation.

The scale bars are 25 μm.

Waveguide Evanescent Field Microscopies for Application in Cell- and Bacteria- Biophysics

211

With a very short integration time only a few

spots appeared on the image in the areas where the

cell was well spread. These spots are the cell’s

adhesions within the evanescent field. With

increasing integration time, more and more features

appeared, such as the cell nucleus area, the cells

boundary and the cell granularity.

Figure 11: a) Bright field image of a single osteoblast and

b)-d) corresponding WEFS images with integration times

of 500 ms, 1000 ms and 1500 ms, respectively. The

arrows point to the features mentioned in the text: a)

adhesions, b) granularity and cell boundary, and c) nucleus

and cell boundary.

These few experiments show that not necessarily

fluorescence staining needs to be carried out for

imaging focal adhesions and hence getting some

cell-substratum interaction measures. As in WEFF

microscopy larger integration times lead to 3D

information of the cell.

Further detailed analysis, e.g. whether WEFS

data are comparable with flow cytometry (scattering

mode), need to be done.

9 MASS PRODUCIBLE

WAVEGUIDE CHIPS

In order to allow WEFF and WEFS microscopy to

be used by the interested communities, typically

biophysics, biology, biochemistry and medical

laboratories, the waveguide chips need to be

available and at a reasonable cost. Mass production

is the only way to achieve this. An all-polymer-

waveguide chip with an imprinted coupling grating

is one way to achieve this goal.

We have designed an all-polymer-waveguide

chip on the basis of a PMMA substrate. The

imprinting was performed into the PMMA with a

home-fabricated silicon stamp and in a subsequent

step a polystyrene waveguide was spin coated on

top.

Fig.11 shows SEM images of an imprinted

grating with a periodicity of 670 nm and a depth of

200 nm.

Figure 12: SEM image of a PMMA imprinted coupling

grating. The periodicity is 670 nm.

First experiments with the all-polymer chips

have produced promising WEFF imaging results.

However, still development towards mass produced

chips is necessary. The grating of Fig.11 was

fabricated by imprinting one grating into one

PMMA substrate. The imprinting and waveguide

spinning procedure needs to be scaled up to do 16,

25, 36 or even 49 chips in parallel on one substrate

with one imprinting procedure and a subsequent spin

coating process.

10 CONCLUSIONS

We demonstrated a simple, cost effective and safe

(sample safety) approach to perform TIRF and TIR

microscopy on an existing inverted microscope by

applying a slab waveguide as a sample substrate. No

expensive, extremely low loss waveguides were

needed. In contrary, the 3D scattering which is

always present in non-sophisticated waveguides

allowed to do epi-microscopy by simply enhancing

the integration time.

We applied WEFF and WEFS to a variety of

problems: from simple imaging of adhesions to

quantitative investigations like distance maps and

kinetic phenomena.

In order to spread the technology, the availability

of mass produced, inexpensive waveguide-chips is

necessary. The engineering towards this goal is on

its way.

Both WEFF and WEFS microscopy will allow

all kinds of interface and surface related imaging

and measurements. Both methods carry the

possibility to also be used in sensor technology.

Various options exist for using WEFF microscopy in

sensing. One can think to operate a WEFF

microscope with two simultaneously propagating

modes at different wavelength for any kind of pump

probe experiment. Or one can use a sensing scheme

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involving a Förster transfer changing the

fluorescence yield upon binding of an analyte within

the evanescent field and therefore detecting an

image intensity change. WEFS microscopy is

sensitive to any changes in the size or the density

(refractive index) of the scattering entity.

Recognition reactions on a surface could easily be

detected by enhancing the scattering power by a

gold nanoparticle (Klein, 2008) or by increasing the

size of a scattering entity due to the binding.

Surface functionalization of the all-polymer-

waveguide chip is possible with silane chemistry as

–OH groups can easily be produced by oxygen

plasma or UV ozone treatment (Kandeepan et al.,

2015).

ACKNOWLEDGEMENTS

Many co-workers, students, PDFs and colleagues are

thanked for their contribution in the past, the present

and the future for developing and applying WEFF

and WEFS microscopy: Frank Thoma, John J.

Armitage, Huge Trembley, Michael Nietsche,

Abdollah Hassanzadeh, Rebbeca Stuchburry, Sabiha

Hacibekiroglu, Daniel Imruck, Christopher Halfpap,

Michael Morawitz, Qamrun Nahar, Darryl K.

Knight, Susanne Armstrong, Jeremia Shuster,

Frederik Fleisser, Mihaela Stefan, Rony Sharon,

Hong Hong Chen, Jeffrey S. Dixon, Stephen Sims,

Kibret Mequanint, Uwe Langbein, Beth Gillies,

Rene Harrison, Gordon Southam, Donglin Bai,

Doug Hamilton, Cheryle Seguin, and Elisabeth

Pruski.

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