Microscopy techniques for studyingpolymer-polymer blends

Master’s Thesis in Engineering Physics

Sandra Mattsson (saamon04@student.umu.se)

April 8, 2019

Microscopy techniques for studying polymer-polymer blendsMaster’s Thesis in Engineering Physics

Author: Sandra Mattsson, sandramattsson86@gmail.comSupervisors: Petter Lundberg and Mattias Lindh, Department of Physics, Umeå UniversityExaminer: Ludvig Edman, Department of Physics, Umeå University

© 2019 Sandra Mattsson

Abstract

Semiconductors are used in many electronic applications, for example diodes, solar cells andtransistors. Typically, semiconductors are inorganic materials, such as silicon and gallium ar-senide, but lately more research and development has been devoted to organic semiconductors,for example semiconducting polymers. One of the reasons is that polymers can be customized,to a greater extent than inorganic semiconductors, to create a material with desired properties.Often, two polymers are blended to obtain the desired function, but two polymers do not usuallyresult in an even blend. Instead they tend to separate from each other to varying degrees. Themorphology of the blend affects the material properties, for example how efficiently it can convertelectricity to light.

In this project, thin films consisting of polymer blends were examined using microscopytechniques for the purpose of increasing our understanding of the morphology of such blends.One goal was to investigate whether a technique called correlative light and electron microscopycan be useful for examining the morphology of these films. In correlative light and electronmicroscopy, a light microscope and an electron microscope are used in the same location in orderto be able to correlate the information from the two microscopes. The second goal was to learnabout the morphology of the thin films using various microscopy techniques.

The polymers used were Super Yellow and poly(ethylene oxide) with large molecular weight.Super Yellow is a semiconducting and light-emitting polymer while poly(ethylene oxide) is anisolating and non-emitting polymer that can crystallize. In the blend films, large, seeminglycrystalline structures appeared. The structures could be up to 1 mm in the lateral direction, whilethe films were only approximately 170 nm thick. These structures could grow after the films haddried and their shapes were similar to those of poly(ethylene oxide) crystals. Consequently, thereis reason to believe that it is the poly(ethylene oxide) that makes up the seemingly crystallinestructures, but the structures also emitted more light than the rest of the film, and Ramanspectroscopy showed that there was Super Yellow in the same location as the crystals.

Among the microscopy techniques used, phase contrast microscopy was particularly inter-esting. This method visualizes differences in optical path length and was useful for studyingpolymer blends when the polymers have different indices of refraction. Correlating light andelectron microscopy showed that there was a pronounced topographical difference between theseemingly crystalline regions and the rest of the thin film. Light microscopy has a limited resolu-tion due to diffraction, but as long as the resolution of the light microscope is sufficient for seeingphase separation, correlative light and electron microscopy turned out to be a good method forstudying the morphology of thin films of polymer blends.

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Sammanfattning

Halvledare är viktiga för många elektroniska ändamål eftersom de kan användas till exempelvisdioder, solceller och transistorer. Traditionellt används inorganiska halvledande material somkisel eller galliumarsenid, men på senare tid har allt mer forskning och utveckling inriktat sigmot organiska (kolbaserade) halvledare, såsom halvledande polymerer, bland annat eftersom det ihögre utsträckning går att skräddarsy de organiska materialen så att de får önskvärda egenskaper.Ofta blandas två polymerer med varandra för att skapa ett material med nya egenskaper som ärönskvärda, men två polymerer brukar inte blandas jämnt utan tenderar att separera från varandrai olika utsträckning. Hur blandningen ser ut (morfologin) påverkar materialets egenskaper, tillexempel hur effektivt det omvandlar ström till ljus.

Med syfte att öka förståelsen för hur morfologin ser ut hos en blandning av två polymerer,har detta projekt gått ut på att undersöka tunna filmer av polymer-blandningar med hjälpav mikroskopiska tekniker. Ett delmål var att ta reda på om en teknik som heter korrelativljus- och elektronmikroskopi är en bra metod för att undersöka morfologin hos dessa filmer. Vidkorrelativ ljus- och elektronmikroskopi används både ett ljusmikroskop och ett elektronmikroskoppå samma plats för att kunna korrelera informationen som de båda mikroskopen ger. Det andradelmålet var att undersöka vad de olika mikroskopi-teknikerna kan säga om morfologin hos detunna filmerna.

De polymerer som använts är Super Yellow och poly(etylenoxid) med hög molekylmassa.Super Yellow är en oordnad halvledande och ljusemitterande polymer medan poly(etylenoxid) ären isolerande och icke-emitterande polymer som kan kristallisera. I de blandade filmerna uppstodstora kristall-liknande strukturer som kunde vara upp emot 1 mm breda trots att filmerna baravar ungefär 170 nm tunna. Dessa strukturer kunde växa fram efter det att filmerna redan hadetorkat och påminde i form om kristaller som kan bildas av poly(etylenoxid). Det finns alltsåskäl att tro att det är poly(etylenoxid) som kristalliserats, men de kristall-liknande strukturernavisade sig emittera mer ljus än vad resten av filmen gjorde, och Raman-spektroskopi visade attdet även fanns Super Yellow på samma plats som kristallerna.

Bland de mikroskopitekniker som testades utmärker sig faskontrastmikroskopi, som visarskillnader i den optiska vägskillnaden (det vill säga faktisk vägskillnad multiplicerat med bryt-ningsindex). Det visade sig vara en intressant teknik för att studera polymerblandningar när debåda polymererna har olika brytningsindex. Genom att korrelera ljus- och elektronmikroskopivisade det sig att det fanns en tydlig skillnad i struktur mellan de kristall-liknande områdena ochresten av den tunna filmen. Ljusmikroskopi har begränsad upplösning på grund av ett fenomensom heter diffraktion, men så länge som ljusmikroskopets upplösning är tillräcklig för att sefasseparation visade det sig att korrelativ ljus- och elektronmikroskopi är en bra metod för attstudera morfologin hos tunna filmer av polymerblandningar.

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Acknowledgements

Thank you, Ludvig Edman, for giving me an intriguing puzzle to solve and for supporting meduring this process. Thank you, co-workers at OPEG, for helping me navigate the unknown oceanthat is experimental physics, whether it be in the form of a nice fika break or some much-neededinstructions in the lab.

The first steps towards learning how to handle microscopes were taken at Nanolab with thetechnical help of Roushdey Salh and with samples provided by Jenny Enevold and ChristianLarsen to practice on. Thank you.

All scanning electron microscope images were taken at Umeå Core Facility for ElectronMicroscopy (UCEM) and all light microscope images were taken at the Biochemical ImagingCentre Umeå (BICU). My sincerest thanks to those facilities and especially to Naga VenkataGayathri Vegesna and Cheng Choo Lee (Nikki) for technical assistance, encouragement andadvice.

The AFM measurements were done with the gracious help of Igor Iashchishyn from Med-Chem at Umeå University and the Raman spectroscopy was performed at the Vibrational Spec-troscopy Core Facility (ViSp) at Umeå University with kind assistance from Hamid Reza BarzegarGoltapehei. Thank you for lending me your expertise.

Thank you, friends, who have graciously allowed me to vent my frustrations and celebratemy successes with you, and sometimes to forget about the thesis and just enjoy the day. MaxBäckman deserves a special shoutout for teaching me how to correct the baseline of my Ramanspectra and for pointing out the obvious, twice.

Last, but not least, my deepest gratitude goes to my amazing supervisors, Petter Lundbergand Mattias Lindh, without whom I would have floundered like a fish on dry land.

Thank you.

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Contents

1 Introduction 1

2 Theory 22.1 Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Characterization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.2.1 Light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.2 Scanning electron microscopy . . . . . . . . . . . . . . . . . . . . . 32.2.3 Correlative microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 42.2.4 Stylus profilometry . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2.5 Atomic force microscopy . . . . . . . . . . . . . . . . . . . . . . . . 42.2.6 Raman spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3 Experimental details 63.1 General approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2 Ink and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3 Using light microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.4 Using scanning electron microscope . . . . . . . . . . . . . . . . . . . . . . 103.5 Achieving correlated images . . . . . . . . . . . . . . . . . . . . . . . . . . 113.6 Other characterization methods . . . . . . . . . . . . . . . . . . . . . . . . 123.7 Data post-processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4 Results and discussion 134.1 Varying the chemical composition . . . . . . . . . . . . . . . . . . . . . . . 13

4.1.1 Random compact crystals . . . . . . . . . . . . . . . . . . . . . . . 134.1.2 Seaweed crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.1.3 What makes a crystal? . . . . . . . . . . . . . . . . . . . . . . . . . 20

4.2 Varying the storage environment . . . . . . . . . . . . . . . . . . . . . . . 23

5 Conclusions and outlook 25

References 27

A1Microscope resolution

A2Atomic force microscopy

Chapter 1

Introduction

Semiconductors are important for many technological applications, for example diodesand transistors. Lately, semiconducting polymers have become an interesting topic ofresearch, since they are more versatile than traditional semiconductors and can allowfor tuning the bandgap and other material properties.1 Polymer electronics can alsobe produced at low cost, due to the ease with which devices can be produced and theabundance of organic material available.2 Polymers are often mixed with each other orwith a non-polymeric material to obtain a material with new properties. The morphologyof the blend, e.g. the homogeneity of the mixture and the size of the interfacial areabetween the constituent materials, has a large effect on the resulting properties.2

One application that frequently uses polymer-polymer blends is the light-emittingelectrochemical cell (LEC). It is a light-emitting device where mixing an electrolumines-cent semiconductor (often a polymer) with an electrolyte (often a salt dissolved in anion-transporting polymer) enables electrochemical reactions and the creation of a p-i-njunction in situ.3 This allows LECs to be made from only one thin film of active materialand suggests the possibility of large-scale, low cost production. However, polymer LECsare often quite inefficient and have a long turn-on time.4 One of the likely contributors tothese drawbacks is a phase separation that tends to appear between the two polymers.5

This separation needs to be better understood, in order to learn how it occurs and affectsthe performance of the devices.

Since LECs partially consist of a light-emitting polymer, optical microscopy can beused to observe the optical properties of a thin film. This would need to be combinedwith other techniques to learn more about the morphology of the film. Correlative lightand electron microscopy (CLEM) uses images from a light microscope and an electronmicroscope, both taken at the same location, to create an image with more informationthan either technique can manage on their own.6 This could be used to connect mor-phology to properties and thereby investigate how thin polymer-polymer films behaveunder different conditions.

The goal of this thesis is first to investigate whether CLEM can be used to characterizethin films of a polymer-polymer blend, and second to see what can be learned about themorphology of such films, using various microscopy techniques.

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Chapter 2

Theory

2.1 Polymers

A polymer is a large molecule consisting of smaller repeat units that are bound to eachother with covalent bonds. Synthetic polymers can be produced at fairly low cost whiletheir properties can be tuned to better suit different purposes, but they are normallythought of as insulators. In 1977, Shirakawa, MacDiarmid and Heeger showed thatproper treatment can make polymers conductive. This inspired a considerable amountof research into, and development of, polymers for electronic applications.7

Due to their repeating units, some polymers can easily form semicrystalline solids.When a solid has its atoms arranged in a repeating fashion, it is said to be crystalline. If ithas some degree of crystallinity but is not entirely crystalline, it is called semicrystalline.The crystal structure minimizes the intermolecular energy, resulting in a more stableconfiguration. Regular polymers therefore tend to crystallize,8 for example poly(ethyleneoxide) (PEO). Zhang et. al investigated thin films of PEO and found how differentcrystal shapes appeared depending on a number of parameters, including the processingconditions.9

To make materials with new and customized properties, polymers can be mixed toget a blend, but mixing two different polymers is commonly not thermodynamicallypreferred. Such a blend will phase separate if given enough time to reach equilibrium.1

If one polymer is hydrophilic while the other is hydrophobic, this further contributes tothe phase separation.10 Moreover, if both polymers are dissolved in the same solvent,these two polymers may have different solubilities, which has been shown to cause phaseseparation during processing.1,5

2.2 Characterization techniques

2.2.1 Light microscopy

A light microscope is a category of microscopes that uses visible light to magnify andimage small samples. Several different specialized microscopy techniques fit into this

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category, such as fluorescence microscopy or phase contrast microscopy. I will refer tothese techniques collectively as “light microscopy”.

The wavelength of visible light sets the limit for what the smallest possible resolutionis in a light microscope. Typically the resolution in the lateral plane is of the order ofhalf the wavelength of the light, depending on the numerical aperture of the objectiveused. There are techniques that can be used to improve the resolution, e.g., stimulatedemission depletion microscopy, but in this project such techniques were not used.11

Fluorescence microscopy

The principle behind a fluorescence microscope is that a fluorescent sample is excited by ashort-wavelength laser. The sample then sends out fluorescence in the form of light witha longer wavelength than the laser, and the microscope separates out the fluorescencefrom the laser beam using filters.12 This results in an image with good contrast betweenregions that fluoresce and regions that do not.6

Phase contrast microscopy

Phase contrast microscopy visualizes differences in optical path length. The illuminatinglight passes through the sample, where some of the light is diffracted by the sample andthe diffracted light undergoes a phase shift that is proportional both to the difference inrefractive index and to the thickness variation.

The illuminating and diffracted light end up in slightly different locations in thediffraction plane and can be manipulated separately. The illuminating light goes througha phase plate (usually a quarter wavelength plate) to produce a phase shift similar to thatof the diffracted light, so that constructive interference is possible between the differentlight waves. Since the illumination is larger in amplitude than the diffracted light, agrey filter is used to lower the amplitude of the illumination light, greatly increasing thecontrast between the diffracted and the illuminating light.13

In most phase contrast microscopes, a higher refractive index generally leads to adarker image. However, this is not always the case. A darker image can also be causedby a thickness variation that is large enough to compensate for a lower refractive indexor by the microscope being set up in a way that produces contrast inversion.13

2.2.2 Scanning electron microscopy

In a scanning electron microscope (SEM), electrons are emitted from an electron gunand accelerated towards a sample by a series of apertures and electromagnetic lensesthat control the position, shape and size of the beam. The focused electron beam thenhits the sample surface and interacts with it in various ways that result in different signals(secondary electrons, backscattered electrons, x-rays etc).14 These signals are collectedby different detectors and the signal is converted into an image.

When an electron from the beam (a so-called primary electron) interacts with thesample, electrons from the sample (called secondary electrons) can be dislodged and

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escape. The secondary electrons have fairly low energy, less than 50 eV, and they canonly be detected near the sample surface. Because of this, the secondary electrons giveinformation about the topography of the surface.14

One of the main benefits of SEM is the large magnification and excellent resolutionpossible. An SEM has a resolution on the order of the beam size, which can be as small as1 nm. However, using SEM requires that the sample first undergoes careful preparationin order for it to be able to withstand the beam of highly energetic electrons and the highvacuum that is needed in the SEM. The preparations and the electron beam exposurecan damage the sample or render it unsuitable for future experiments. To avoid chargebeing built up in the sample surface, non-conducting samples are usually coated with athin layer of metal or carbon and the sample is mounted on a special sample holder. Thesamples are also grounded, using for example silver glue to provide a connection to the(conductive) sample holder. Samples that were conductive from the beginning also needto be glued to the sample holder to keep them in place.

2.2.3 Correlative microscopy

Correlative imaging is simply using more than one imaging technique on the same placeon the sample, in order for the different techniques to complement each other. ForCLEM, the techniques that are combined are light microscopy and electron microscopy.The purpose of doing this can be to gather more information about the sample thaneither microscope can manage on its own, or it can be to use the light microscope toeasily find a region of interest and then use the electron microscope to take a closerlook. This correlation can be done manually by keeping track of where you are on yoursample, it can be done by imaging the sample simultaneously with both microscopes inan integrated CLEM, or it can be done by using special sample holders and software thathelps keep track of positions.6

2.2.4 Stylus profilometry

Stylus profilometry is a mechanical way of mapping the height profile of a sample. Astylus probe is brought down to make contact with the sample. The force with which thestylus pushes down on the sample is kept constant as the stylus moves along the surface.To keep the force constant, the stylus needs to change its position as the surface of thesample varies in height and from this, a height profile can be calculated.15

2.2.5 Atomic force microscopy

Similar to stylus profilometry, the atomic force microscope (AFM) uses a probe to mea-sure properties of a sample surface. The probe is attached to a cantilever which deflectsas a result of the probe-sample interactions and this deflection can be converted into aforce.16 There are a number of imaging modes which are developed for different purposesand circumstances. The tapping mode is suitable for soft samples because of its rela-tively small interaction forces. In this mode, the cantilever vibrates near its resonance

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frequency and when the probe touches the surface each period, the amplitude of thevibration is reduced.17 The force needed to restore the amplitude is recorded and usedto calculate the height profile and other characteristics of the sample.

2.2.6 Raman spectroscopy

The physical process behind Raman spectroscopy is the inelastic scattering of light whenit interacts with molecular vibrational modes. This scattering transfers some of the en-ergy of the probing light into molecular vibration, or conversely, transfers some molecularvibration energy into light, resulting in a different wavenumber of the light after it hasinteracted with the sample. A sample is illuminated by monochromatic light in the formof a laser source. By measuring the spectrum of the light after it has interacted withthe sample and comparing it to the original laser beam, the Raman shift - or change inwavenumber - can be calculated and this corresponds to vibrational energy modes in themolecules present in the sample, enabling identification of the molecules.18

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Chapter 3

Experimental details

3.1 General approach

While this project has not been focused on making functioning LEC devices, I haveworked with materials that are often used in polymer LECs.

Super Yellow (SY) is the common name of a light-emitting polyphenylene vinylene co-polymer. It has a broad emission spectrum19 and is often used in organic light-emittingdiodes and LECs as an electroluminescent semiconductor. SY is hydrophobic20 and hasits peak absorption at a wavelength of around 440 nm and the electroluminescence peakis around 550-600 nm.19 Poly(ethylene oxide), or PEO for short, is a more hydrophilic21

polymer. PEO is produced in a large variety of molecular weights. High-molecular weightPEO is often used as the ion transporter in LECs10 because of its good ion-dissolvingproperties and electrochemical stability.22 PEO is known to crystallize and can formnumerous crystal shapes, depending on molecular weight and crystallization conditions.9

Potassium trifluoromethanesulfonate (KTF for short) is often used as the ion sourcein polymer LECs,23 because this salt can provide a fast turn-on time and long-termstability.10 Both PEO and KTF absorb moisture from the air and need to be handledin an inert, low-moisture atmosphere. Figure 3.1 shows the chemical structure of SuperYellow, PEO and KTF.

Figure 3.1 – Chemical structure of Super Yellow, PEO and KTF. The value of n (numberof repeat units in PEO) varies with the molecular weight chosen.

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I have looked at thin films of a mixture of SY, PEO and KTF. First, the ratiobetween the two polymers was varied. Then one ratio was selected and new samples wereproduced while the storage environment was varied. When varying the ratios betweenthe constituent materials, the films were produced in ambient conditions and the ratioof SY to PEO varied while the amount of KTF was held as a constant fraction of thetotal mass. The selected ratios were SY:PEO:KTF = 1:1:0.2, 1:2:0.3 and 1:3:0.4. Whenvarying the storage environment, three sets of films were prepared with a fresh batch ofSY:PEO:KTF = 1:3:0.4 solution in nitrogen atmosphere. After drying, the films werestored for one month. One set was stored in nitrogen atmosphere, one set in ambientconditions and one set in a climate chamber with increased temperature and humidity.

The films were produced to facilitate large phase separation between the polymers.Large amounts of PEO were used since it was hypothesized to result in a PEO-enrichedcrystalline phase.

3.2 Ink and sample preparation

Magnetic stirrers and vials were cleaned with 2-Propanol (VWR Chemicals) and dried.The empty jars with stirrers were weighed on a scale (Sartorius TE64) with a precisionof 0.1 mg.

PEO (5·106 g/mol, Sigma-Aldrich), KTF (Potassium trifluoromethanesulfonate, Sigma-Aldrich) and SY (Livilux®PDY-132, Merck, Germany) were separately dissolved in cy-clohexanone (Sigma-Aldrich) in a concentration of 10 mg/ml to produce “master inks”.Table 3.1 summarizes the production details of these master inks in two separate sets,termed 1 and 2.

Table 3.1 – Production details on master inks of PEO, KTF and SY in cyclohexanone.Concentrations of all is 10 mg/ml.

Set Chemical Preparation environment Stirring1 PEO Nitrogen1 50 °C overnight1 KTF Nitrogen1 Shaken at room temperature1 SY Ambient 50 °C overnight2 PEO Nitrogen1 50 °C overnight2 KTF Nitrogen1 Shaken at room temperature2 SY Nitrogen1 50 °C overnight

1Nitrogen-filled glovebox with low oxygen (<1 ppm) and water (<0.5 ppm) content to provide aninert environment.

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“Composite inks” were then mixed by blending the master inks. The composite inkswere stirred on a hotplate at 50 °C for varying amounts of time. Table 3.2 summarizesthe production details.

Table 3.2 – Production details of composite inks made from master inks.

Ink From set Ratio SY:PEO:KTF Atmosphere Stirring timeInk-12 1 1:1:0.2 Ambient 4 hInk-2 1 1:2:0.3 Ambient 3 hInk-3 1 1:3:0.4 Ambient 3 hInk-4 2 1:3:0.4 Nitrogen1 6 h

For the film deposition, thin glass microscope coverslips with varying sizes were usedas substrates. The substrates were cleaned using subsequent baths in Acetone (VWRChemicals) and 2-Propanol (VWR Chemicals) in an ultrasonic cleaner (VWR Ultrasoniccleaner) for 15 minutes each. The spincoater Spin150 (SPS Europe) was used for de-positing the films, using 2000 RPM for 60 s at an acceleration of 1000 RPM/s. All filmswere dried on hotplates at 70 °C for different time durations. Table 3.3 shows the varyingproduction parameters.

Table 3.3 – Production details of films

Film Ink Age ofink

Substrate(mm2)

Dryingtime

Productionenviron-ment

Storageenviron-ment

SY1 SY fromset 1

2 months Cut3 4 h Ambient Ambient

Mix1 Ink-1 5 days 20x20 4 h Ambient AmbientMix2 Ink-2 1 day Cut3 Overnight Ambient AmbientMix3 Ink-3 3 weeks Cut3 Overnight Ambient AmbientCLEM1 Ink-3 1 month 22x22 4 h Ambient AmbientCLEM2 Ink-4 1 day 22x22 2.5 h Nitrogen1 Nitrogen1

CLEM3 Ink-4 1 day 22x22 2.5 h Nitrogen1 AmbientCLEM4 Ink-4 1 day 22x22 2.5 h Nitrogen1 CC4

1Nitrogen-filled glovebox with low oxygen (<1 ppm) and water (<0.5 ppm) content to provide aninert environment.

2Additional cyclohexanone was added to this composite ink to make it more dilute. The resultingink had a concentration of 8 mg/ml.

320x20 mm2 coverslip cut to approximately one fourth of the original size with a diamond knife tobetter fit in the SEM.

4Climate chamber (Dongguan Haida Equipment Co, Ltd.) with a temperature of 39 °C and 40 %relative humidity

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3.3 Using light microscopes

For the light microscope images, the microscope Zeiss Axio Imager Z1 with the softwareZEN was used. For the phase contrast microscopy the channel TL Phase was used.For the fluorescence microscopy, the channel Alexa Fluor 488 with the bandpass filterFITC BP 450-490/BS 495/BP 500-550 was used. This channel excites at 488 nm andselects emission in the wavelength interval 500-550 nm, which partially overlaps with theabsorption and emission spectra of SY.

The objectives used were 10X EC-plan-Neofluar 10x/0.30 Ph1, 20X Plan Apochromat20x/0.60 Ph2, 20X Plan Apochromat 20x/0.8 M27 and 100X EC-plan Neofluar 100x/1.30Oil Ph3 M2. Together with the bandpass filter, this results in a lateral resolution ofapproximately 875 nm for the 10X objective, 438 nm for the first 20X objective, 328 nmfor the second 20X objective and 210 nm for the 100X objective. Appendix A1 showsa demonstration of the resolution of the fluorescence microscope when using the 100Xobjective.

I did not want to use fluorescence microscopy when searching for good places on myfilms to acquire images from, because the fluorescence can “bleach” when a fluorescentmaterial is exposed to the laser for too long, reducing the amount of fluorescence andresulting in bad pictures. At first, differential interference contrast was used, but verylittle detail could be seen. It turned out that phase contrast microscopy was a good choice,because not only could it be used to find interesting features to image in the fluorescencemicroscope, but it also provided information about the films that fluorescence microscopycould not. This is because there is a large difference in refractive index between SY andPEO. PEO has a refractive index of around 1.45,24 while the refractive index of SY ishigher across the entire visible range with the highest value being 2.10 at a wavelengthof 485 nm.25 Figure 3.2 shows one of the thin films in differential interference contrast,fluorescence microscopy and phase contrast microscopy.

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Figure 3.2 – Micrographs of a photoluminescent film, taken at the same place and at thesame magnification using different microscope modes in order to demonstrate the differencebetween them. The micrographs are taken near a corner of the substrate, showing somefilm damage. (a) was taken using the differential interference contrast mode, (b) usingfluorescence microscopy and (c) using phase contrast microscopy.

3.4 Using scanning electron microscope

A Carl Zeiss Merlin Field-Emission Scanning Electron Microscope with the softwareZEISS SmartSEM was used to obtain the SEM images. Samples were coated with a 2 nmlayer of iridium, using the Quorum Q150T-ES Sputter Coater (Quorum Technologies Ltd,UK). Iridium was selected because it results in an even layer that does not change thesample topography much, even if the features are very small. An accelerating voltage of5 kV and a beam current of 150 pA was used in the SEM. The detectors used were thesecondary electron detectors InLens and HE-SE2.

Soft materials can be damaged by the high-energy electron beam. One example isheating due to prolonged exposure during electron beam alignment. Because of this,focus and stigmator settings were adjusted on one spot at high magnification, and onceI was happy with that, I reduced the magnification and moved to a nearby spot (thatdid not contain the damaged region) for image acquisition. When taking pictures withdifferent magnifications, I started at the lowest magnification that I wanted and workedmy way up to the highest magnification.

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3.5 Achieving correlated images

The light microscope can take simultaneous images with several filters, which means thatcorrelation of images with different light microscopy modes (for example fluorescencemicroscopy and phase contrast microscopy) is very easy. In order to correlate lightmicroscopy with SEM, a bit more work is needed. It is possible to do this using differentmethods, but I chose to use the Zeiss Shuttle & Find system. The sample was placed ina special sample holder with three calibration markers. The position of each marker wasrecorded in the light microscope for calibration purposes and when taking pictures theposition of each image relative to the markers was also recorded.

The sample was then removed from the holder to be coated with a conductive iridiumlayer before being placed in the holder again and transferred to the electron microscope.Inside the electron microscope, the markers were again used to calibrate the positionof the sample holder. Images from the light microscope were loaded and when double-clicking on a region of interest from those images, the holder was automatically movedto the position of that region. The imaging software could be used to correlate theimages and automatically superimpose them in the same image26 if three points werefirst manually identified in both the light and SEM micrographs.

Due to technical limitations, the 100X objective could not be used with Shuttle &Find. Figure 3.3 shows why this is the case. The sample is placed in a holder whichpartly covers the sample near the edges. Using high magnification requires oil immersionobjectives and a coverslip between the sample and the objective would be needed toprotect the sample from being contaminated with the oil. Due to the holder extendingover the sample, the coverslip could not be placed directly on top of the sample, so thefocal plane of the objective would be far above the sample. With biological samples,this is normally solved by using a smaller coverslip, but this was not an option for mycomparatively dry samples because the coverslip would then adhere to the microscopeoil instead of the sample.

Figure 3.3 – Cross-sectional sketch of Shuttle & Find holder, demonstrating that a cov-erslip can not be placed directly on the sample.

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3.6 Other characterization methods

The thickness of the films was measured using the DektakXT Stylus profiler along withthe software Vision64 (Bruker). A scratch was made in the films and several measure-ments were made across the scratch to determine the depth of the scratch and therebythe thickness of the film. The stylus force was set to 1 mg and a 500 µm stretch wasmeasured at a speed of 10 seconds per measurement.

The BioScope CatalystTM BioAFM (Bruker) was used for AFM measurements. Thebuilt-in microscope was used to locate a good place for measurement and the AFM wasthen used in tapping mode (Peak Force QNM).

For the Raman spectroscopy, a Renishaw inVia Raman spectrometer with a laserwavelength of 633 nm and a power of 50 W was used. The Raman shift of SY wasacquired by taking a measurement on the film SY1 which consisted of SY on a glasssubstrate. The Raman shift of a clean glass substrate was also measured so that thissignal could be corrected for. The Raman shifts from three different spots of a mixedSY/PEO film were then acquired.

3.7 Data post-processing

Images from the light microscope were processed in ImageJ while images from the electronmicroscope were processed in Gimp. Brightness and contrast were altered with the goalto have as much contrast as possible without losing any information in very dark orbright regions. Scalebars were also added to the images. After acquiring the images fromboth microscopes, correlated images were obtained in Gimp. The image files from bothmicroscopes were opened and because the microscopes image the sample with differentorientations, one of the images was rotated approximately 180°. The larger of the twoimages was then resized until both images were of the same size.

The AFM data was processed using the software Gwyddion. The data was levelledby subtracting the mean plane and horizontal scars in the data were removed with thesoftware. A few manually selected defects were removed, using Pseudo-Laplace interpo-lation to calculate a new value for those pixels. Lastly, the minimum data value wasshifted to zero.

The data acquired from the Raman spectroscopy studies was processed in Matlab.A baseline correction was performed using the Asymmetric Least Squares method withλ = 108 and p = 0.1. For the SY spectra, the Raman shift contribution from the substratewas subtracted.

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Chapter 4

Results and discussion

The films turned out to have large structures with seemingly crystalline characteristicsbut it is not determined whether they are actually crystals. For the sake of simplicity, Iwill nonetheless call them ’crystals’ from here on.

4.1 Varying the chemical composition

For the films produced and stored in ambient conditions while varying the ratio ofSY:PEO:KTF, two main crystal shapes could be seen: the random compact crystaland the seaweed crystal, to follow the same naming convention as Guoliang Zhang et. alin their study of thin PEO films.9

4.1.1 Random compact crystals

In all films produced in ambient conditions, regardless of ratio of SY to PEO, randomcompact crystals could be seen. Some examples are shown in figure 4.1, where the largerstructures are the random compact crystals. Smaller bright and dark spots are mostlikely a feature of the PEO, because they also appear in films with only PEO but notin films with only SY, and might be undissolved PEO. The random compact crystalsappear to be more rare when the PEO amount increases, and there is also a tendencyfor them to be smaller in average size with a higher amount of PEO.

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Figure 4.1 – Phase contrast micrographs of random compact crystals found in films madeof SY, PEO and KTF. (a) is from the film Mix1 with SY:PEO = 1:1. (b) is from the filmMix2 with SY:PEO = 1:2, and (c) is from the film Mix3 with SY:PEO = 1:3. Note that(c) is taken at a higher magnification than the others.

When using fluorescence microscopy to look at the random compact crystals, theyturned out to be more fluorescent than the surrounding film as shown in figure 4.2.

Figure 4.2 – Micrographs of random compact crystals found in the film Mix2. (a) isa fluorescence micrograph showing that the crystals have a larger degree of fluorescencethan the rest of the film, (b) is a phase contrast micrograph for comparison.

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The random compact crystals could also be seen in SEM. Figure 4.3 shows one suchcrystal at two different magnifications. Note that in the image with higher magnification,the morphology is slightly different in the crystalline region compared to the regionoutside.

Figure 4.3 – SEM micrograph of the film Mix1, consisting of equal amounts of SY andPEO. In (a) is an entire random compact crystal at a relatively small magnification. Theboxed in region is magnified in (b), giving a closer look at the interface between thecrystal and the surrounding region. The approximate location of this interface is markedwith white dots. Note that there is a difference in the morphology between crystal andnon-crystal regions.

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4.1.2 Seaweed crystals

Seaweed crystals could be seen in films with the ratios SY:PEO = 1:1 and 1:3, but not1:2. In some cases they could be seen by the naked eye, as shown in figure 4.4.

Figure 4.4 – Photograph of the film CLEM1 consisting of a mixture of SY, PEO andKTF. A large number of seaweed crystals are visible.

These were easy to see in both phase contrast and fluorescence. In figure 4.5 two suchcrystals are shown in phase contrast, where one is from a film with ratio SY:PEO = 1:1and the other has SY:PEO = 1:3. With increasing amount of PEO, there was a tendencyfor the seaweeds to be easier to distinguish from the background and have narrowerfeatures in the lateral plane.

Figure 4.5 – Phase contrast micrographs of two thin films with seaweed crystals visible.The scale is the same for both images. (a) shows the film Mix1 with SY:PEO = 1:1 and(b) shows the film Mix3 with SY:PEO = 1:3.

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The seaweed could be very large in the lateral plane, despite the restriction in thethird dimension. Figure 4.6 shows several such crystals, one of which is more than 1 mmlong from end to end. While the crystals are large in the lateral dimension, profilometricstudies show that the film is approximately 170 nm thick which would make the crystalsvirtually two-dimensional. However, that is not the full story. Measuring across a seaweedcrystal shows that there are significant height differences across the seaweed crystals,typically in steps of the order 50-100 nm. One such measurement is shown in the figure.An AFM height measurement was also performed on a seaweed crystal and can be foundin appendix A2.

Figure 4.6 – Low magnification SEM micrograph of the film CLEM1. Image showsseveral seaweed crystals. Inset in the picture is a graph showing height profile along thewhite line across one of the seaweed crystals.

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When looking at the seaweed crystals in the fluorescence microscope, they were morefluorescent than the rest of the film, just like the random compact crystals. Figure 4.7 is acorrelative fluorescence and scanning electron micrograph, showing how the fluorescenceand topography varies between the seaweeds and surrounding regions. Seaweeds have acreased surface with larger topographical variation than the non-crystal regions, whichis in agreement with what the profilometric measurement showed in figure 4.6.

Figure 4.7 – CLEM micrographs of the film CLEM1. (a) is an SEM micrograph of aseaweed crystal and (b) is a fluorescence micrograph of the same place and magnification.The boxed-in region in (a) is shown at higher magnification in (c), showing a distinctdifference in morphology between the seaweed crystal and the outside region. The boxed-in region in (c) is magnified again in (d).

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Most of the seaweed crystals were located at the surface of the film, as indicated bytheir topography in SEM, but some seaweeds looked indistinct in all of the microscopes.The fact that they were visible (albeit blurry) in the SEM suggests that they are justbelow the surface. This implies that the crystals can grow mainly in the plane, andmore importantly, that their formation does not require interacting with the surroundingatmosphere. Figure 4.8 shows such a crystal in SEM.

Figure 4.8 – SEM micrograph of the film CLEM1, showing a hard-to-see seaweed crystal,likely located just beneath the film surface. Top left in the image is a nearby surface crystal,for comparison. Contrast has been dramatically enhanced in order for the crystal to beclearly visible.

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4.1.3 What makes a crystal?

To try and understand the crystals better, Raman spectroscopy was done on a film withseaweed crystals. First, to get a reference spectrum for SY, Raman spectroscopy wasdone on a film with only SY, see figure 4.9. The Raman shift is consistent with the onepublished by Hassan et al.27

Figure 4.9 – Raman shift of a SY film. Numbers in the graphs show the wavenumber ofthe peaks.

After this, Raman spectroscopy was done on one of the films containing seaweedcrystals. Figure 4.10 shows the resulting Raman spectra from three different points onthe film. The inserted micrograph shows the position each spectrum was taken from.The peaks are similar to those of pure SY except for 919 cm−1, which is from the glasssubstrate.

Figure 4.10 – Raman spectra from a seaweed crystal in the film Mix3, consisting ofSY:PEO = 1:3. The inserted micrograph shows the positions the Raman spectra weretaken at. The peaks match those of SY, with the exception of 919 cm−1, which is from theglass substrate. Numbers in the graph indicate where the peaks are. Two of the spectraare shifted in the vertical direction for the purpose of making it easier to distinguish thedifferent spectra.

20

From the Raman shift, we see that there is SY in the same region as the seaweedcrystals. Despite this, I believe that we are seeing PEO crystals. PEO is known tocrystallize and the seaweed and random compact crystals look very similar to ones foundin thin films consisting of only PEO.9 To explain the Raman shift and the fact that thecrystals fluoresce, it might be the case that SY is separated from PEO in the thicknessdirection, or it may be intermixed with the crystalline PEO.

Many of the seaweed crystals appeared in the vicinity of film damage that had oc-curred after the film had dried and it looked as though the crystals had grown out fromthese damaged regions. To investigate whether the crystals kept growing after the filmhad dried, observations of the same places were made eight days apart. Figure 4.11 showsthat there was crystal growth in the time interval between observations.

Figure 4.11 – Phase contrast micrographs of the film Mix3 showing the growth of seaweedcrystals. The scale is the same for all images. Images are taken from two positions on thesample at two different times. The top images were taken when the film was one day old,the bottom images when the sample was nine days old. In the images from position onethere is film damage visible that the crystals appear to have grown out from.

The fact that the seaweeds could grow after the film had dried indicates that therelikely is no large-scale movement of molecules happening in conjunction with the crys-

21

tallization. Both the PEO and SY molecules are very large and they should not havesufficient energy to move around in the solid state. Based on this, the regions of the filmwhere crystals have grown should have approximately the same chemical composition asthey did prior to crystallization, only organized in a different manner. The two polymerslikely phase separate during spin coating, as they were both dissolved in the same solventbut the solubility of PEO in cyclohexanone is lower than that of SY. If this is the case,the resulting film would have some regions with a higher percentage of PEO than therest of the film. These PEO-enriched regions may be where the crystals start formingduring the drying process. After the film has dried, a seed crystal can likely affect thePEO in its vicinity to join in, one partial alignment at a time.

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4.2 Varying the storage environment

The higher level of fluorescence of the random compact and seaweed crystals comparedto other regions of the films was intuitively not expected. Since SY is the fluorescentmaterial and PEO is crystalline, it seemed that the crystals should have little or nofluorescence. One proposed explanation was that the SY had been oxidized in the ambientatmosphere.28 If so, this may have caused the fluorescence to decrease over the entire film,except where crystals had formed to protect the underlying SY. The crystals would thenappear to be more fluorescent in comparison with the degraded fluorescence elsewhere.If this was the case, then identically produced films would display different behavioursdepending on which environment they had been stored in. A film that had been storedin a nitrogen atmosphere, for example, is not exposed to much oxygen and its crystalsshould not be more fluorescent than the rest of the film, while a film stored in a climatechamber with elevated temperature and humidity should show a larger effect. However,it turned out that the crystal shapes in this set of films were different, and that regardlessof storage environment they were more fluorescent than the rest of the film, as shown infigure 4.12.

Figure 4.12 – Fluorescence micrographs of three different films, showing that the crystalsare more fluorescent than the surrounding region. The scale is the same for all images.(a) is the film CLEM2, which was stored in nitrogen atmosphere. (b) is the film CLEM3,stored in ambient conditions. (c) is the film CLEM4, which was stored in a climatechamber.

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While no large seaweed crystals appeared in these films, the surface morphology wasvery similar to that of a seaweed crystal and it did not seem to depend on the storageenvironment. Figure 4.13 shows films from different storage environments at the samemagnification in SEM. The creases in the surface are very similar, and there appears tobe an additional directionality of the topographical features if the image was taken neara crystal. Note that the creases are similar to those found in the seaweed crystals, asshown in figure 4.7c.

Figure 4.13 – SEM micrographs, showing the morphology of three different films. Thescale is the same for all images. (a) is the film CLEM2, which was stored in inert nitrogenatmosphere. (b) is the film CLEM3, which was stored in ambient conditions. (c) is thefilm CLEM4, which was stored in a climate chamber.

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Chapter 5

Conclusions and outlook

I have shown that crystals can grow in a thin polymer-polymer film after it has dried,that the crystals appear to be growing from damaged regions in the film and that theirformation does not require interaction with the atmosphere. The shapes of the crystalsare similar to PEO crystals, but Raman spectroscopy shows that they also contain SY.Further, I have shown that the crystals are more fluorescent than the rest of the film andthat this is not due to the crystals protecting the underlying SY from being oxidized.The exact chemical composition of the crystals, how they can grow when the film is inthe solid state, and why the crystals are more fluorescent, are questions that we do notyet know the answers to. Understanding what mechanisms are behind this could haveimplications on various devices made with the same or similar materials, and furtherexperiments should hopefully be able to tell us more.

Phase contrast turned out to be a good optical microscopy method for investigatingSY/PEO blends, due to the different refractive indices of the constituent materials. Itcould give information about the films by itself and correlating it with fluorescence andelectron microscopy proved to be helpful for learning even more about the materials. Onemicroscope can not tell everything about a material, so combining different characteri-zation techniques makes sense. I found that fluorescence microscopy was interesting tocombine with SEM, since fluorescence could give information that related to the opticalproperties of the material and SEM was able to provide insight into the surface structureat the same locations.

Originally the thought was that I would find complete phase separation, such thata region consisted only of SY or only of PEO, but I did not see any that consisted ofonly PEO. In all of my fluorescence images, the entire film fluoresces to some degree.The likely reason is that any phase consisting of only PEO occupies too small a regionto be resolved in the fluorescence microscope. This illustrates the main limitation ofCLEM: the resolution of the light microscope. For some applications, diffraction-limitedresolution is just not good enough because the structures are too small. To get bet-ter resolution while still investigating the fluorescence characteristics of the material,stimulated emission depletion microscopy might be a suitable alternative.

25

Knowing how different morphologies occur and what their effects are is important fordeveloping better devices. CLEM can be a valuable method for improving our under-standing of this. For thin films of photoluminescent polymer-polymer blends, I believethat phase contrast and fluorescence microscopy in combination with scanning electronmicroscopy can really help further our understanding of these materials.

26

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[9] Guoliang Zhang, Xuemei Zhai, Zhenpeng Ma, Liuxin Jin, Ping Zheng, Wei Wang,Stephen Z. D. Cheng, and Bernard Lotz: "Morphology Diagram of Single-LayerCrystal Patterns in Supercooled Poly(ethylene oxide) Ultrathin Films:Understanding Macromolecular Effect of Crystal Pattern Formation andSelection", ACS Macro Lett. 2012, 1, 217-221 (2011), DOI 10.1021/mz2001109

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[10] J. Mindemark, L. Edman: "Illuminating the electrolyte in light-emittingelectrochemical cells", J. Mater. Chem. C, 2016, 4, 420, DOI 10.1039/c5tc03429a

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[15] Nanoscience Instruments: "Stylus Profilometry",https://www.nanoscience.com/techniques/optical-profilometry/stylus/ (retrievedFebruary 26, 2019)

[16] Yves F. Dufrêne: "Atomic Force Microscopy, a Powerful Tool in Microbiology",Journal of Bacteriology (2002), DOI 10.1128/JB.184.19.5205-5213.2002

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Appendix A1

Microscope resolution

The films produced in this project are highly fluorescent, compared to the small numberof fluorophores that are often used in life science applications of fluorescence microscopy.It was originally hypothesized that this high level of fluorescence might make it difficultto see contrast between regions with different levels of fluorescence and that even if acontrast can be detected, perhaps we would not be able to get down to the theoreticalresolution of the microscope.

This was tested by using the highest magnification available and looking at the small-est visible contrast difference to measure its spatial dimensions. In figure A1.1a is a smallfeature that can just barely be seen as a darker vertical line. Across this darker line is awhite line along which intensity and distance has been measured in A1.1b to get an ideaof the width of the darker line and thereby an upper limit to the resolution.

The graph in A1.1b shows that the darker line is approximately 5 % darker than thesurrounding region and it is approximately 400 nm wide, which means that the resolutionis 400 nm or better.

Figure A1.1 – (a) high magnification fluorescence micrograph of the film Mix3, showinga less fluorescent (darker) line. A white line has been drawn across the dark line. (b)graph of gray value (intensity) along the white line, showing how the intensity varies.

Appendix A2

Atomic force microscopy

To try and verify the results from the profilometer in figure 4.6, an AFM study was doneon a region containing the boundary between a seaweed crystal and the surroundingfilm, see figure A2.1. It was difficult to interpret the data, because the surface was veryadhesive which caused several spikes in the data and the resulting height map showedseveral discontinuities. However, the results are also consistent with the profilometermeasurement and the height jumps may in fact be a feature of the seaweed crystals.

Figure A2.1 – AFM height image across the interface between a seaweed crystal and theoutside region, taken on the film Mix3.