13
Review Challenges in Applying Photoemission Electron Microscopy to Biological Systems Dana N. Peles and John D. Simon* Department of Chemistry, Duke University, Durham, NC Received 8 September 2008, accepted 22 September 2008, DOI: 10.1111 j.1751-1097.2008.00484.x ABSTRACT Photoemission electron microscopy (PEEM) is a unique surface- sensitive instrument capable of providing real-time images with high spatial resolution. While similar to the more common electron microscopies, scanning electron microscopy and trans- mission electron microscopy, the imaging technology relies on the photogeneration of electrons emitted from a sample through light excitation. This imaging technique has found prominence in surface and materials sciences, being well suited for imaging flat surfaces, and changes that occur to that surface as various parameters are changed (e.g. temperature, exposure to reactive gases). Biologically focused PEEM received significant attention in the 1970s, but was not aggressively advanced since that pioneering work. PEEM is capable of providing important insights into biological systems that extend beyond simple imaging. In this article, we identify and establish important issues that affect the acquisition and analysis of biological samples with PEEM. We will briefly review the biological impact and importance of PEEM with respect to our work. The article also concludes with a discussion of some of the current challenges that must be addressed to enable PEEM to achieve its maximum potential with biological samples. INTRODUCTION Photoemission electron microscopy (PEEM) is a unique, surface-sensitive technique capable of providing real-time images of the surface of a sample under high spatial resolution. The physical principle underlying this technique is the photo- electric effect. Simply, electrons are emitted from the surface of the sample when the associated photon energy of the incident light is above the photoionization threshold value character- istic of the sample of interest. The generated photoelectrons are accelerated through a series of electron optics and the surface of the sample is imaged at a high magnification. The first PEEM images were published in 1933 (1). Following these preliminary images tremendous development in the under- standing and instrumentation of PEEM has been achieved (2,3). PEEM and related imaging techniques are used exten- sively in the field of materials science, and there is a dedicated biannual meeting and associated conference proceedings that address scientific and instrumental advances in the field. Information about the latest meeting can be found at http:// www.leem-user.com/. From a historical perspective, O. Hayes Griffith (4) was one of the pioneers of PEEM technology and applied the technique to biological samples in 1972 (4), reporting first PEEM images of rat epididymis. Griffith and his group realized the capabil- ities of PEEM as the electron-optics analog of fluorescence microscopy and used colloidal gold and silver enhanced colloidal gold particles to selectively label portions of a biologic with a higher spatial resolution (5–7). PEEM images for cells (8–10), viruses (11,12) and DNA (11,13–15), among others (16–18), were reported using both labeling and nonla- beling techniques. In addition to the experimental evidence, reviews were written establishing PEEM as a powerful tool for biological imaging (19–21). Examples of the sensitivity, topographic contrast and spatial resolution achieved by Griffith’s group using biologi- cally focused PEEM are reproduced in Fig. 1. The sensitivity of the instrument is shown in Fig. 1a with an image of a mixture of T-4 bacteriophages and tobacco mosaic viruses (TMV) (12). In this image, the head of the bacteriophages is significantly brighter than TMV. A single helical strand of RNA characterizes TMV while highly condensed DNA characterizes the head of a bacteriophage. PEEM is sensitive to the density of the nucleic acid because it has been suggested that there are not large differences in photoemission between DNA and RNA and the photoemission of nucleic acids is greater than the protein components of the virus (12). The topographic contrast is illustrated in Fig. 1b with a PEEM image of MCF-7 human breast carcinoma cells (8). Visible among the textured surface of these cells are the protrusions of the large nuclei and nucleoli with the sides of these cellular protrusions appearing darker than the top portions. An example of the spatial resolution achieved by Griffith’s group is displayed by his PEEM image of recA-DNA (11) repro- duced in Fig. 1c. These three images, along with the multitude of others that Griffith and coworkers have produced, exem- plify the strides his group accomplished in pioneering biolog- ically focused PEEM. †This invited paper is part of the Series: Applications of Imaging to Biological and Photobiological Systems. *Corresponding author email: [email protected] (John D. Simon) Ó 2008 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/09 Photochemistry and Photobiology, 2009, 85: 8–20 8

Challenges in Applying Photoemission Electron Microscopy to Biological Systems

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Review

Challenges in Applying Photoemission Electron Microscopyto Biological Systems†

Dana N. Peles and John D. Simon*

Department of Chemistry, Duke University, Durham, NC

Received 8 September 2008, accepted 22 September 2008, DOI: 10.1111 ⁄ j.1751-1097.2008.00484.x

ABSTRACT

Photoemission electron microscopy (PEEM) is a unique surface-

sensitive instrument capable of providing real-time images with

high spatial resolution. While similar to the more common

electron microscopies, scanning electron microscopy and trans-

mission electron microscopy, the imaging technology relies on

the photogeneration of electrons emitted from a sample through

light excitation. This imaging technique has found prominence in

surface and materials sciences, being well suited for imaging flat

surfaces, and changes that occur to that surface as various

parameters are changed (e.g. temperature, exposure to reactive

gases). Biologically focused PEEM received significant attention

in the 1970s, but was not aggressively advanced since that

pioneering work. PEEM is capable of providing important

insights into biological systems that extend beyond simple

imaging. In this article, we identify and establish important

issues that affect the acquisition and analysis of biological

samples with PEEM. We will briefly review the biological

impact and importance of PEEM with respect to our work. The

article also concludes with a discussion of some of the current

challenges that must be addressed to enable PEEM to achieve its

maximum potential with biological samples.

INTRODUCTION

Photoemission electron microscopy (PEEM) is a unique,

surface-sensitive technique capable of providing real-timeimages of the surface of a sample under high spatial resolution.The physical principle underlying this technique is the photo-

electric effect. Simply, electrons are emitted from the surface ofthe sample when the associated photon energy of the incidentlight is above the photoionization threshold value character-istic of the sample of interest. The generated photoelectrons

are accelerated through a series of electron optics and thesurface of the sample is imaged at a high magnification. Thefirst PEEM images were published in 1933 (1). Following these

preliminary images tremendous development in the under-standing and instrumentation of PEEM has been achieved

(2,3). PEEM and related imaging techniques are used exten-

sively in the field of materials science, and there is a dedicatedbiannual meeting and associated conference proceedings thataddress scientific and instrumental advances in the field.

Information about the latest meeting can be found at http://www.leem-user.com/.

From a historical perspective, O. Hayes Griffith (4) was oneof the pioneers of PEEM technology and applied the technique

to biological samples in 1972 (4), reporting first PEEM imagesof rat epididymis. Griffith and his group realized the capabil-ities of PEEM as the electron-optics analog of fluorescence

microscopy and used colloidal gold and silver enhancedcolloidal gold particles to selectively label portions of abiologic with a higher spatial resolution (5–7). PEEM images

for cells (8–10), viruses (11,12) and DNA (11,13–15), amongothers (16–18), were reported using both labeling and nonla-beling techniques. In addition to the experimental evidence,

reviews were written establishing PEEM as a powerful tool forbiological imaging (19–21).

Examples of the sensitivity, topographic contrast andspatial resolution achieved by Griffith’s group using biologi-

cally focused PEEM are reproduced in Fig. 1. The sensitivityof the instrument is shown in Fig. 1a with an image of amixture of T-4 bacteriophages and tobacco mosaic viruses

(TMV) (12). In this image, the head of the bacteriophages issignificantly brighter than TMV. A single helical strand ofRNA characterizes TMV while highly condensed DNA

characterizes the head of a bacteriophage. PEEM is sensitiveto the density of the nucleic acid because it has been suggestedthat there are not large differences in photoemission betweenDNA and RNA and the photoemission of nucleic acids is

greater than the protein components of the virus (12). Thetopographic contrast is illustrated in Fig. 1b with a PEEMimage of MCF-7 human breast carcinoma cells (8). Visible

among the textured surface of these cells are the protrusions ofthe large nuclei and nucleoli with the sides of these cellularprotrusions appearing darker than the top portions. An

example of the spatial resolution achieved by Griffith’s groupis displayed by his PEEM image of recA-DNA (11) repro-duced in Fig. 1c. These three images, along with the multitude

of others that Griffith and coworkers have produced, exem-plify the strides his group accomplished in pioneering biolog-ically focused PEEM.

†This invited paper is part of the Series: Applications of Imaging to Biologicaland Photobiological Systems.

*Corresponding author email: [email protected] (John D. Simon)� 2008TheAuthors. JournalCompilation.TheAmericanSociety ofPhotobiology 0031-8655/09

Photochemistry and Photobiology, 2009, 85: 8–20

8

Despite the capabilities demonstrated by Griffith andothers, the technique did not flourish in the biological sci-ences and applications of PEEM to biological systems have

lagged behind complementary electron imaging techniques,such as scanning electron microscopy (SEM) and transmissionelectron microscopy (TEM). Instead, as an ultra high vacuum

(UHV) instrument suited for relatively flat samples, PEEMfound prominence in the fields of surface and materialssciences (2,22). The applications of PEEM in surface and

materials sciences go beyond imaging. For instance, surfacedynamics can be imaged during in situ experiments (e.g.heating, cooling, exposure to reactive gases, etc.) by monitor-ing the changes in the electronic properties of the surface

(22–28). If the resulting work function of the modified surfaceis below (above) the photon energy of the incident light animage will (will not) be observed. Subsequently, and more

importantly for the purposes of this review, tunable mono-chromatic light sources make it possible to scan a series ofphoton energies and obtain the work function or selectively

turn on or off a particular region of the sample (29–31). Thus,PEEM has become a critical tool in surface and materialssciences that is capable of achieving valuable and unique

information.More recently, the determination of electrochemical prop-

erties (work functions) through PEEM has emerged as asignificant application for biological systems (32–38). This

particular technique is widely applicable to those systemswhose electrochemical properties are central to understandingtheir function, but are impossible to determine using conven-

tional approaches. The electrochemical properties are obtainedby fitting the threshold data with functional fits based ontheory rigorously defined for photoelectric curves of semicon-

ductor and metallic surfaces. A series of concerns arise whenapplying techniques (analysis and instrumentation) that arerigorously established and developed for metallurgic, surfaceand materials sciences to biological sciences. The aim of this

review was to identify and establish the key issues that affectthe acquisition and analysis of photoionization thresholds forbiological samples and briefly review the biological impact and

importance of threshold-PEEM with respect to our work withhuman melanosomes. We will conclude with a discussion ofthe current issues of PEEM that need to be addressed in order

to derive a deeper molecular understanding from the dataobtained using this technique.

INSTRUMENTATION

A schematic of the photoelectron emission microscope isshown in Fig. 2. The instrument consists of a main chamber, a

lens column and a detection center, all of which are held underUHV. Emission of photoelectrons occurs when light of anappropriate wavelength is illuminated on the surface of a

Figure 1. (a) A PEEM image of a mixture of T-4 bacteriophage andtobacco mosaic virus (TMV). Reprinted from Houle and Griffith (12)with permission from Elsevier. The image displays the sensitivity of thePEEM technique illustrated by the differences in brightness. (b) PEEMimage revealing the topographic contrast on the surface of MCF-7human breast carcinoma cells. Reprinted with permission fromHabliston et al. (8). (c) PEEM image of recA-DNA revealing spatialresolution. Reprinted with permission from Birrell et al. (11).

Figure 2. A schematic of the PEEM instrument consisting of the mainchamber, lens column and detection center. Reprinted with permissionfrom Ade et al. (31).

Photochemistry and Photobiology, 2009, 85 9

sample. A variety of photon sources are available to excite theelectrons (X-ray, synchrotron radiation, or UV arc lamps andlasers). UV excitation is generally required to photoionize thevalence electrons of metals and biological materials, and so

this spectral region is the focus of this review. As a conse-quence of the instrumental design, shown in Fig. 2, the sampleof the surface must be illuminated with a photon beam at a

high incident angle. This is necessary because the maximalresolution of the instrument is achieved only when there is ashort working distance between the anode and the sample

(�4 mm).Typically, the sample is held at a negative potential and an

electric field is created between it and a grounded anode. This

potential accelerates the generated photoelectrons through aseries of electron optics. After passing through the lenscolumn, the emitted electrons are amplified by a microchannelplate and subsequently imaged onto a phosphor screen. A

CCD camera captures the data from the phosphor screen. Theelectron optics project the image plane of the sample onto themicrochannel plate, thus enabling the direct collection of a

spatially resolved image.For a geometrically flat sample, the current lateral instru-

mental resolution is about 10 nm (39). Samples with significant

topography introduce spherical aberrations as the emittedelectrons are ejected in various angles. Similarly, according toEinstein’s equation,

KE ¼ hv� /; ð1Þ

samples of multiple chemical components with different

characteristic photoionization energies, /, introduce chromaticaberrations as the emitted electrons are ejected at differentkinetic energies, KE, upon illumination with constant photon

energy, hv. In addition to the sample-dependent acceleratingfield aberrations, there is an objective lens spherical aberrationfor UV illumination that also affects the resolution. Reports

calculating the theoretical resolution for PEEM systems indi-cate that it could achieve 5 nm and propose that implemen-tation of aberration-corrected lenses can improve the

resolution (40,41). Currently, there are aberration correctiontechniques that have been developed and implemented

in PEEM systems to enhance the resolution for UV (42) andX-ray (43–45) photon excitations.

In addition to discussing the lateral resolution, it isnecessary to briefly address the depth of information of

PEEM. The depth of information is the distance between thesurface and a point at which information from the samplecontributes to the image at a specified resolution (46). To

generate photoelectrons, light that penetrates the sample isabsorbed, induces photoionization and the photoionizedelectrons are transported to the surface where they then

escape from the sample to the vacuum (8). The depth-dependent yield of photogenerated electrons is not known.Estimates for organic materials suggest that electrons are

generated within the first 10 nm (46). However, it is reasonableto assert that the yield will decrease with depth from thesurface and so while light may be absorbed deep in the sample,the volume relatively near the surface will comprise the

dominant contribution to the signal (5).

SPATIAL DEPENDENCE OF THE SIGNAL

As stated above, the incident light beam illuminates the sampleat a high incident angle with respect to the surface normal.Consequently, if the sample possesses significant morphology,

only a portion of the sample surface is illuminated, andaccordingly imaged. The curved side of the specimen facing theincident beam will have a high photoelectron flux whereas the

opposite side will be shadowed and dark. Given the geomet-rical considerations described above, spherical samples willhave an asymmetric photoemission image. As shown in Fig. 3

the intensity contour plot of the PEEM image of a 150 nmgold nanosphere reveals the photoemission image to beellipsoidal. The long axis is the axis that is not deformed by

the shadowing that results from the geometrical constraints ofthe PEEM and the incident light source (47).

The above point is important because the three-dimensionaltopography of biological specimens will also result in photo-

electron emission at various angles. Thus, ‘‘shapes’’ revealed inthe PEEM image may reflect the interplay between theillumination angle and the surface geometry, not simply the

Figure 3. PEEM image of a 150 nm gold nanosphere and its associated intensity contour plot. Asymmetric photoemission is visible in both images.

10 Dana N. Peles and John D. Simon

shape of the biomolecule. If the incident photon energy used togenerate the photoelectrons is near the sample’s photoioniza-tion threshold, then the emitted electrons will possess a lowkinetic energy. The large accelerating potential enables these

electrons to be collected with a minimum loss of lateralresolution—the electrons are emitted at various angles for acurved substrate and would then tend to spread laterally (3).

However, the situation is certainly more complicated by acomplex three-dimensional surface, which will present topo-graphic contrast as the trajectories of the emitted electrons are

influenced by the high electric field of the specimen (48).Spreading or bunching of the electrons can also occur from thetopographical electric field variations. As a result, the image

may have darker or brighter regions according to the topo-graphic features of the sample. An aperture located in the lenscolumn is used to prevent deflected electrons from reaching thedetection area. Deflection of the electrons typically occurs

from the sides of a protrusion whereas the electrons arisingfrom surrounding flat regions and the top regions of protru-sions pass through the electron-optics system undeflected.

Thus, the top regions of protrusions and surrounding flat areasappear brighter whereas the sides of protrusions are darker.

Finally, a two-dimensional image detection (the CCD serves

as a two-dimensional image plane, with each pixel having thesame area) creates a nonlinear distance scale for the image of athree-dimensional object. Consider the projection image of asphere (Fig. 4a). The region of a curved three-dimensional

surface imaged onto an individual pixel increases with the

angle from the surface normal. Therefore, a curved three-dimensional shape will affect the resolution achieved by theinstrument. With increasing angle from the surface normal, thephotoejected electrons will initially have increased velocity

projections in the plane of the substrate, and therefore will fanout as they undergo different degrees of curvature to beaccelerated toward the lens column. As a result, if the

accelerating potential is reduced, the size of the image willincrease. Figure 4b displays the length of the long axis of theimage of a �150 nm spherical Sepia eumelanin granule (recall

that spherical structures appear as ellipsoids) with respect tothe accelerating voltage. A linear dependence is observed forthe acceleration potential examined. Nepijko et al. (47) devel-

oped a theoretical analysis of such an experiment and providedfunctional relationships for determining the actual size of thethree-dimensional spherical particle from such data.

All of the above-mentioned spatial considerations are

important when imaging samples with significant structure,as is the case with biological specimens. The interpretation ofthe generated images may be more complicated than what one

would expect with flat surfaces as in surface and materialssciences because the resulting image is dependent on themorphology and topography of the specimen. While a

challenge to disentangle these features, it also presents theopportunity and potential power of PEEM to biologicalsystems; the data will reflect the three-dimensional propertiesof the system being imaged.

POLARIZATION DEPENDENCEOF THE SIGNAL

As a consequence of the PEEM geometry the photon beamused to illuminate the sample must come in at a high incident

angle (�77� from the surface normal). This angle of incidenceis critical because for many materials it is quite close toBrewster’s angle. Recall that Brewster’s angle is defined as theangle, measured from the surface normal, at which s-polarized

light is reflected and p-polarized light is absorbed (49). (Thedirection of polarization is perpendicular and parallel to theplane of incidence for s- and p-polarized light, respectively.)

Considering the PEEM geometry the incident plane isdescribed by the propagation direction of the incident photonbeam and the surface normal. Due to the high incident angle of

the light source, the linearly polarized vectors are oriented in aplane that is tilted 13� from the surface normal. Thereforeunder PEEM geometry, s-polarized light is parallel to theplane of the substrate, whereas p-polarized light is 13� from the

surface normal, as shown in Fig. 5.PEEM imaging requires a conductive substrate for contin-

ual electron replenishment during photoemission. Most of the

conductive substrates are reflective and reflect light from theincident photon beam. For a biological specimen characterizedwith a curved three-dimensional geometry, the total electric

field at the sample surface will not just include the incominglight but also include the light that is reflected off the substrate(Fig. 6a). A curved biological specimen is susceptible to this

interaction because the reflected beam is able to strike thesurface along the curved region. However, a disk or a box-likestructure has vertical edges that block the whole object fromthis reflection (Fig. 6b), whereas a film covers the substrate

and prevents it entirely. It is important to understand this

Figure 4. (a) A three-dimensional sphere projected onto a two-dimensional surface. (b) A plot showing the dependence of the imagediameter of a �150 nm Sepia eumelanin granule on the acceleratingpotential of the instrument.

Photochemistry and Photobiology, 2009, 85 11

substrate effect for the analysis of three-dimensional biologicalsamples in PEEM.

The reflection coefficients that define the reflectivity of thesubstrate for a specific polarization are calculated through

Fresnel equations (49), Eq. (2)

Rs ¼nvacCos½hi� �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffin2sil � nvacðSin½hi�Þ2

q

nvacCos½hi� þffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffin2sil � nvacðSin½hi�Þ2

q

Rp ¼ �n2silCos½hi� � nvac

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffin2sil � n2vacðSin½hi�Þ

2q

n2silCos½hi� þ nvac

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffin2sil � n2vacðSin½hi�Þ

2q

ð2Þ

where nsil and nvac are the refractive indices of silicon and

vacuum, respectively, and hi is the angle of incidence. Therefractive index of a vacuum is 1, but the refractive index of thesubstrate is dependent on the wavelength of the incident light

and the temperature of the substrate. For the purposes of thisreview, we will focus on silicon as the conductive substrate, asit is a common substrate, and reasonably assume a tempera-ture of 25�C. As for the wavelength, we will examine 207.0,

243.5 and 310.5 nm (5.99, 5.09 and 3.99 eV, respectively),because typical threshold scans are obtained by scanning overthe region from 4 to 6 eV. At 25�C and these incident wave-

lengths, the refractive index of silicon is 1.2215, 1.7385 and4.5401 (50), respectively. Figure 7 shows the reflection coeffi-cients for s- and p-polarized light as a function of the angle of

incidence, measured from the surface normal, of the incominglight. The Brewster angle of silicon for 207.0, 243.5 and310.5 nm is 50.7�, 60.1� and 77.6�, respectively. Consequently,considering the incident angle of PEEM, �77�, polarization-dependent reflection effects must be considered in analyzingthe images.

The total electric field, for each polarization, at a point on

the surface of a curved biological sample consists of theincoming wave plus that of the reflected wave, Eq. (3).

Etot ¼ E0ðeikincqÞ þ E0RðeikrefqÞ ð3Þ

In this equation, R is the reflection coefficient characteristic ofthe substrate (Eq. 2) q is the vector location and kinc and krefare the incident and reflected k-vectors. For simplicity, the

electric field contribution from the laser, E0, is factored outand normalized to 1—this value only affects the magnitude of

the total electric field. The transverse component of the k-vectors is conserved after reflection and therefore can also befactored out. Equation (4) then describes the propagation ofthe light wave in the direction of the surface normal for both p-

and s-polarized light.

Etots ¼ Eo eðikzzÞ þ Rse

ð�ikzzÞ� �

Etotp ¼ Eo eðikzzÞ þ Rpe

ð�ikzzÞ� �

:ð4Þ

In the above expression, z is the distance from the substrateand the z-component of the k-vector is defined askz ¼ ð2p=kÞ cos½hi� with k the wavelength of the incident light.Thus, as shown in Eq. (4), the total electric field at a particular

wavelength is dependent upon the angle of incidence and thedistance above the substrate. Figure 8 shows the total electricfield as a function of height at the PEEM incident angle of 77�and k = 207.0, 243.5 and 310.5 nm. The curves shown inFig. 8 are for pure s- and p-polarized light sources, butcombinations of these two light polarizations can describe

virtually any light source. For biologically relevant heights, nmrange, the total electric field between s- and p-polarized lightvaries significantly at the relative wavelengths used in a

threshold scan. Nonuniform emission resulting from variationsin the total electric field will be present along the curved

Figure 5. Orientation of s- and p-polarized light under PEEM geom-etry. The surface normal doubles as the imaging axis. p-Polarized light(black solid line) is tilted 13� from the surface normal. s-Polarized light(red dashed line) lies in the plane of the substrate.

Figure 6. (a) The total electric field at the surface of a spherical sampleincludes both the incoming light and the polarized light that is reflectedoff of the substrate. (b) The total electric field at the surface of a disk-like sample only includes the incoming light as the vertical edgesprevent the reflection from interacting with the surface.

12 Dana N. Peles and John D. Simon

surface of a biological specimen with significant height due tothis effect at all wavelengths.

It is important to note that in addition to the fluctuation of

the total electric field along a curved surface (due to thesubstrate reflection), the sample specimen is also characterizedwith its own unique dielectric properties and utilization of a

particular polarization could enhance the emission of photo-electrons. Materials with larger dielectric constants are more

sensitive to polarization (39) and signals could be greatlyenhanced or inhibited by the polarized light source. Thus,disk or box-like samples will exhibit a fluctuation of the

electric field that is a consequence of the dielectrics of thesample whereas curved samples will experience a fluctuationdue to the dielectrics of both the sample and the substrate.

Figure 7. Reflection coefficients for s- (solid) and p-polarized (dashed)light as a function of the angle of incidence, measured from the surfacenormal, of the incoming light for (a) 207.0 nm, (b) 243.5 nm and (c)310.5 nm.

Figure 8. Total electric field as a function of height at the PEEMincident angle of 77� at (a) 207.0 nm, (b) 243.5 nm and (c) 310.5 nmfor s- (solid) and p-polarized (dashed) light.

Photochemistry and Photobiology, 2009, 85 13

Experimental fits of polarization curves for a gold nanosphere

and a gold nanodisk are shown in Fig. 9. The curves areobtained by fitting the measured integrated brightness of thePEEM image as a function of polarization. Despite being of

the same composition, the curve for the nanosphere issignificantly shifted from the curve of the nanodisk. This isexpected to be primarily a result of the added substrate effect

for the gold nanosphere. Hence, when analyzing biologicalmaterials with larger dielectric constants and curved surfaces,polarized photon beams should be an important consider-ation.

DETERMINATION OF THRESHOLDPOTENTIALS

To acquire the photoionization threshold of a specimen, UVwavelengths are scanned and the integrated brightness of theimage is analyzed as a function of the excitation energy. The

measured brightness of the photoelectron image is taken to beproportional to the photocurrent as the emission currentcannot be accessed in a standard PEEM. An image is obtained

for each wavelength and its integrated brightness is measuredby a summation of all of the pixel gray scale depths in theselected region. The minimum and maximum gray scale values

are regarded as black or white saturated, respectively. Imageswith a large fraction of the pixel counts located at these valuesresult in a loss of the information regarding the brightness of a

granule and the photoelectron current is no longer propor-tional to the integrated brightness.

In addition to the previously discussed considerations ofspatial and polarized signal dependence for three-dimensional

biological materials, other standard data corrections areneeded for threshold measurements. The voltage on themultichannel plate and ⁄ or the software gain of the imaging

program is adjusted to ensure the intensity histogram of theimage spans a distribution across the pixel gray scale depths toavoid black and white saturation. Also because of adsorption,

the photon flux of the incident wavelength changes as onescans through the UV region and hence it is necessary tomeasure and compensate for this fluctuation.

To obtain the photoionization potential of the sample,threshold curves are functionally fit with early theoreticalmodels proposed to explain photoelectric curves. In 1931,Fowler (51) described the photoelectric curve of solid metallic

surfaces with an expression that is a function of temperatureand the frequency of the incident light. The integratedbrightness of the sample, S, proportional to the photocurrent,

I, is in proportion to the product of the square of thetemperature of the sample, T, and a function f(u) given by:

S / Iðv0 � hvÞ1=2 / T2fhv� vkBT

� �; ð5Þ

fðuÞ ¼ eu � e2u

22þ e3u

32� . . . ðu � 0Þ

¼ p2

6þ u2

2� e�u � e�2u

22þ e�3u

32� . . .

� �ðu � 0Þ:

ð6Þ

In the above expressions, u� hv�vkBT

, v is the frequency of the

incident light, kB is the Boltzmann constant, h is Planck’sconstant, v0 is the threshold potential and v is the thermionicwork function (v = v0 ) e*, where e* is the energy of the

highest occupied molecular orbital). The threshold can thus bedetermined by functionally fitting the threshold curves of S ⁄T2

vs hv ⁄ kBT. The Fowler equation has been used to determine

the surface photoionization threshold potentials of a variety ofhuman pigments (33–38) and will be discussed later.

A simplified model, the Fowler-Nordheim law (52), is alsoused to determine photoionization potentials. The Fowler-

Nordheim law describes the photoelectric current by:

I1=2 ¼ C� ðhv� vÞ ð7Þ

where C is a constant dependent on the sample, v is thethreshold photoionization potential and hv is the photon

energy. The photoionization potential is obtained by extra-polating the square root of the integrated brightness of thePEEM image, S1 ⁄ 2, to zero on a plot of S1 ⁄ 2 vs the photon

energy. This model may be simpler, but it implicitly assumesthe measurement is being made at extremely low temperaturesas it cannot account for the ‘‘tail’’ that appears in the photo-

ionization threshold curve of room-temperature (or heated)samples, which originates from a thermalized (Boltzmann)density of states serving as the ground state from which pho-

toionization occurs. This can lead to misinterpretation of datacollected at room temperature. Although the temperature-related emission has a much smaller contribution than theactual threshold emission at room temperature, uncertainty

from the extrapolation method may still exist due to thethermal energies of the electrons (53).

In 1962, Kane (54) proposed a model for the photoelectric

emission of semiconductors that involved direct and indirectprocesses with yields proportional to E ) v and (E ) v)5 ⁄ 2,respectively. Following Kane’s theory for semiconductors,

Kochi et al. (55) established a cubic power model to explainphotoemission from organic crystals. These models, like theFowler-Nordheim model, do not include the effect of temper-

ature and therefore do not account for the thermal tail of thethreshold curve. The above theoretical models are based onphotoelectric curves of metallic, semiconductor or organic

Figure 9. Plot of polarization-dependent signal for a gold nanosphere(solid) and a nanodisk (dashed). Plots reveal the presence (goldnanosphere) and absence (gold nanodisk) of substrate effects.

14 Dana N. Peles and John D. Simon

surfaces, and such systems differ from biological specimens. Asdiscussed below, the applicability of the Fowler analysis tobiological and molecular systems remains to be rigorouslyvalidated.

Wilson et al. (56) obtained the ionization energy ofbiological nanoparticles by analyzing the photoelectronkinetic energies with use of a velocity-map imaging photo-

electron spectrometer. Extrapolation to zero of the linearrelationship between the maximum kinetic energy release andthe incident photon energy provides the ionization energy

according to Einstein’s equation. Wilson et al. confirmed theionization energy of the biological nanoparticles with theFowler-Nordheim model and a plot of the total electron yield

vs the incident photon energy. The same value was obtainedupon utilization of both techniques. Additionally, studies ofblack human hair eumelanosomes using PEEM in theabsence of heating revealed similar results, within experimen-

tal uncertainty, between the Fowler model (33,36) and thetemperature-independent Fowler-Nordheim model (37,38).Recently, threshold measurements of the biological protein

fibrinogen were analyzed according to the Fowler model, theFowler-Nordheim model and the cubic power model (32). Acomparison of the photoionization potentials obtained from

all three theoretical models revealed no significant deviationsand a conclusion that all can be equally used to analyze athreshold curve (in the absence of heating) was drawn. Fromthese results, photoionization potentials of biological mate-

rials can be obtained with models established on nonbiolog-ical systems.

The connection between the vacuum threshold potential

and the electrochemical potential vs NHE has been explored ingreat depth (57–61). The relationship between the two scaleshas largely been derived by comparing electrochemical and

work function data on Hg. The IUPAC-recommended rela-tionship is that 0 V vs NHE corresponds to )4.44 V invacuum. However, it is worth mentioning that the studies cited

above report values corresponding to 0 V vs NHE between)4.4 V and �)4.8 V. Generally, measurements incorporatingthe contact potential difference of the uncharged metal and thework function of the clean surface result in higher values in

this range whereas work on immersed electrodes, which arebelieved to retain their interfacial region, result in lower values.

The analysis of wavelength-dependent PEEM images isuniquely capable of providing electrochemical data for thesurface of the intact human pigments, which cannot bedetermined by conventional approaches.

PEEM STUDIES OF HUMAN PIGMENTS

In this section, the application of PEEM to biologicalproblems is exemplified by examining some of our recentwork on human melanosomes. Melanosomes are organellesfound in several regions of the human body (skin, eye, hair,

inner ear and a related structure in the brain), whosefunctions include photoprotection, mitigation of the effectsof reactive oxygen species and ⁄ or metal chelation. The

dominant constituent of the melanosome is the pigmentmelanin. In general, the functions ascribed to melanosomesare enabled by the melanins they contain. Melanin is

generally classified into two major types, eumelanin andpheomelanin, reflecting different molecular precursors inmelanogenesis. In the last decade, there has been significant

progress in understanding melanins and their impact onhuman health. Studies designed to probe the surface prop-erties of melanosomes are few, yet the physical and chemicalproperties of the surface of the melanosome are inextricably

linked to function (62).First, we examine work demonstrating how PEEM uniquely

enables determination of the surface electrochemical properties

of different types of human melanosomes. The relevance of thethreshold potential data collected under high vacuum condi-tions to that exhibited by pigments in physiologically relevant

buffer solutions is established by comparing results fromdifferent experimental methodologies. Figure 10 shows theexperimental data for melanosomes isolated from human

black and red hair (36,37). The fit of these data to the Fowlerequation reveals that black and red hair melanosomes bothhave a surface photoionization threshold of 4.4 ± 0.2 eV(282 nm). To accurately fit the data for red hair melanosomes,

a second threshold potential of lower energy 3.8 ± 0.2 eV(326 nm) is required.

A common ionization potential for the two pigments is not

surprising. The human red hair melanosomes are not ‘‘pure’’pheomelanin; chemical analysis reveals they are �25%

Figure 10. The integrated brightness of the PEEM image for black (left) and red (right) hair melanosomes divided by the square of the sampletemperature, I ⁄T2 are plotted as a function of the excitation energy (hm ⁄ kBT). The solid lines are the best fit of the nonlinear Fowler equation to thedata points. For black hair melanosomes, the data can be described by a single ionization threshold of �4.4 ± 0.2 eV (282 nm). To fit the data forred hair melanosomes require two ionization thresholds of �4.4 ± 0.2 and 3.8 ± 0.2 eV (282 and 326 nm). Reprinted with permission fromYe et al. (36).

Photochemistry and Photobiology, 2009, 85 15

pheomelanin, 75% eumelanin (63). Thus, they should revealthe threshold signature exhibited by the black hair melano-somes, which are pure eumelanin. The additional lowerionization threshold observed for red hair melanosomes is

therefore attributed to pheomelanin.It is important to establish that the threshold potential of

melanosomes observed in isolation under high vacuum (the

conditions of the PEEM experiment) are relevant to how theorganelle behaves under physiologically relevant conditions.While the intact organelle is not amenable to traditional

electrochemical techniques, there are related experiments thatcan be performed to address this issue. Specifically, weexamined femtosecond transient absorption spectroscopy and

electron paramagnetic resonance oximetry experiments ofsynthetic pheomelanins to determine the threshold for photo-ionization of pure pheomelanin in solution at physiological pH(36). Figure 11 shows these data. These model experiments

were designed based on the report by Chedekel et al. that UVexcitation of synthetic pheomelanin in solution resulted in theformation of the superoxide radical anion, being formed by a

mechanism where the first step is photoionization of thepigment (64–66):

Pheomelanin þ hv! e�aq þ pheomelanin radical ð8Þ

e�aq þO2 ! O��

2 ð9Þ

Figure 11 shows the absorption spectrum of pheomelaninand the action spectrum of Chedekel et al. for the formation of

the superoxide radical anion (64). We attribute the constantrate of superoxide formation reported by Chedekel et al. in theregion of k > 400 nm to a dark reaction (36), and so oncecorrected for this dark reaction, these data would represent the

action spectrum represented by the dashed line, where photo-generation of superoxide now becomes appreciable when k <330 nm. Data from our studies using femtosecond absorption,electron paramagnetic resonance oximetry and PEEM are also

indicated in this plot. The femtosecond transient spectroscopyexperiments were designed to detect the presence of solvatedelectrons following photoexcitation. Such signals are observed

following excitation at 303 nm, but are not found forexcitation at 350 nm, revealing that the threshold lies betweenthese two wavelengths (36). A detailed action spectrum using

this approach was not collected due to the complexity of theexperimental technique. Electron spin resonance oximetryexperiments narrow this range, and were significantly easier

to implement as excitation is achieved using a conventionallamp and narrow bandpass optical filters. These data showthat the threshold for the synthetic pigment lies between 338and 323 nm (36). Finally, we recall that PEEM reveals a

threshold potential of 326 nm (36,64). There is remarkableconsistency between these experiments. More importantly,there is excellent agreement between the synthetic pigment in

buffer solution and the intact human melanosome under theisolated high vacuum conditions characteristic of PEEM.

Having now established that the threshold potential deter-

mined by PEEM is relevant to pheomelanin under physiolog-ical conditions, it is of interest to consider the implications ofthe value obtained. Specifically, consider the solar radiationimpinging on the surface of the Earth. Figure 12 shows the

solar irradiance at the Earth’s surface for several differentsolar zenith angles (36,67), clearly revealing that humans areexposed to the wavelengths of light needed to ionize pheomel-

anin, but not those needed to photoionize eumelanin. As aresult, the lower ionization potential observed for pheomelanincould be a part of the explanation for the greater incidence rate

of UV-induced skin cancers in the populations whose melaninscontain increased concentrations of pheomelanin (68,69).

As a second example of the application of PEEM to human

pigments, we consider the spatially resolved images on humanneuromelanin. Neuromelanin is composed of both eumelaninand pheomelanin, with pheomelanin constituting about 25%of the total pigment (70,71). The spatial distribution of these

two different pigments within the organelle remains animportant problem to solve, especially because pheomelaninhas been shown to be able to induce oxidative stress, whereas

eumelanin mitigates such stress (72). The above discussiondemonstrates that the threshold potentials of pheomelanin andeumelanin can be distinguished by PEEM, and so the spatial

analysis of data on human neuromelanin granules wouldprovide information on the pigment present on the surface ofthe granule.

Figure 13 shows a plot of the integrated intensity of the

PEEM image of pigment granules isolated from the substantianigra of a human brain as a function of wavelength, and theassociated fit of the Fowler equation (34). The fit of the

theoretical expression to the experimental data is excellent, andreveals a photoionization threshold potential of 4.5 ± 0.2 eV(282 nm) (34), which is within experimental error of the

oxidation potential measured for human eumelanosomes.There is no indication in the data that pheomelanin is on ornear the surface of the granule. Detailed studies of the kinetic

studies on the early chemical steps of melanogenesis show thatin the case of pigments containing a mixture of pheomelanin

Figure 11. The absorption (solid line) and action spectrum (squares)reported by Chedekel et al. for the photogeneration of superoxideradical anion by synthetic pheomelanin is reproduced. Also indicatedon the plot is the threshold for photoionization determined by PEEMmeasurements on human pheomelanosomes, femtosecond absorptionspectroscopic detection of solvated electrons and electron para-magnetic resonance oximetry on synthetic pigment. Reprinted withpermission from Ye et al. (36).

16 Dana N. Peles and John D. Simon

and eumelanin, pheomelanin formation occurs first with

eumelanin formation predominantly occurring only aftercysteine levels are depleted (73). Such a kinetic model predictsa structural motif with pheomelanin at the core and eumelaninat the surface. This ‘‘casing’’ model was originally proposed in

1982 by Rorsman (74) based on biochemical evidence and hasreceived indirect support from several other studies (75–77).The PEEM data provide definitive data in support of such a

structural model.This structural model has significant implications for the role

of neuromelanin. Dopamine, the precursor to neuromelanin, is

cytotoxic to neuronal cells through oxidation to dopaminequi-none (78). The formation of cysteinyldopamines is hypothesizedto be a protective mechanism, preventing formation of the

neurotoxic dopaminequinone. A casing model for the structure

of neuromelanin therefore provides a mechanism to capturedopaminequinone as part of the pheomelanic core, removingthis neurotoxic precursor, and then shielding it from the neuronby encasing it in a eumelanin coat.

It is also interesting to consider this result in terms of thepathology of Parkinson’s disease (PD). In PD, there is aselective loss of pigmented neurons in the substantia nigra (79).

Unlike the case with pheomelanin, a surface composed ofeumelanin is not sufficiently reductive to generate a high levelof oxidative stress. Therefore, neuromelanin’s surface electro-

chemical potential is less reductive than might be anticipatedgiven neuromelanin’s complicated but unresolved role in theselective loss of pigmented neurons of the substantia nigra

associated with PD. However, degradation of neuromelaninwould expose the pheomelanic core, which would then causeoxidative stress. Thus a slow loss of pigment could actuallyresult in a constant level of inflammation.

CHALLENGES IN ELUCIDATING MOLECULARINFORMATION ON BIOLOGICAL SYSTEMSUSING PEEM

The previous section, along with the earlier work by Griffith,clearly reveals that PEEM provides important insights into

biological systems. The technology has matured and there areinstruments available for use at most of the light sourcefacilities around the world (for example, see user list at http://

www.elmitec-gmbh.com/). Advancements in the applicationsof this technique require interplay between the development oftheoretical analyses and models and experiments designed to

understand the effect of morphology on the images obtained.Different classes of materials (e.g. lipids, proteins) havedifferent ionization properties. In addition, because the photo-

current is describable theoretically by a Golden rule formula,the photocurrent is dependent on the overlap of the directionsof the incident electric field and the transition moment of themolecule. This latter effect affords the ability to look at

molecular orientation and alignment, which contributes to theuniqueness of PEEM among electron microscopies. While suchexperiments are currently feasible—in principle any system that

can be studied by SEM can be examined by PEEM—there arechallenging theoretical issues in extracting detailed molecularinformation from the images obtained. The remainder of this

section examines some of these issues, and research in theseareas is required for PEEM to achieve its maximal potential asan imaging method.

Establishing the validity of the Fowler and related

theoretical models to ‘‘molecular’’ systems or developing

molecular-based theories

As stated above, the current models for elucidating threshold

potentials, and hence oxidation potentials, rely on fittingwavelength-dependent PEEM intensities to either the originalFowler equation, or an expression derived from a similar set of

starting principles. In these theoretical derivations, the numberof electrons available for photoionization are modeled inaccord with the distribution law of Sommerfeld’s theory of

metals. Whether this model can be applied to accuratelydetermine thresholds for biological materials has not beenestablished. However, in order to assess the surface oxidative

Figure 12. The incident solar radiation at the surface of the Earth isplotted as a function of wavelength, solar zenith angles (SZA) andozone concentration (in Dobson units, DU). The four curves corre-spond to (____) SZA = 0, 100 DU, (- – -) SZA = 0, 400 DU, (– – –)SZA = 75, 100 DU, (- - -) SZA = 75, 400 DU. The ionizationthresholds for the different melanins suggest an increased photoreac-tivity of pheomelanosomes under normal solar exposure. Reprintedwith permission from Ye et al. (36).

Figure 13. Plot of the integrated intensity of neuromelanin granules asa function of wavelength. The Fowler equation (Eq. 5) displays anexcellent fit with the threshold data and reveals a potential of4.5 ± 0.2 eV. Reprinted with permission from Bush et al. (34).

Photochemistry and Photobiology, 2009, 85 17

processes of biological organelles relative to other oxida-tion ⁄ reduction potentials of cellular processes, the accuracy ofthe potentials derived from the wavelength-dependent analysisneeds to be addressed.

Methods for determining the actual imaged region for a

three-dimensional object

As exemplified by gold spheres, the actual region imaged in the

PEEM depends on the experimental geometry and the shape ofthe material being imaged.While the analysis is straightforwardin the case of thin films, the situation is much more complex

when there is significant three-dimensional topography. Thisalso affects experimental protocols for obtaining images. Whenconsidering the imaging of spherical objects in the PEEM, the

image is not expected to be round because of the incident angleto the light beam. However, because of an expectation that aspherical object should give a round image, the experimentalistmay adjust the focusing conditions to make the image round,

which actually introduces a distortion to the image.

Depth profiling—electrons can originate from below the surface

While PEEM is a ‘‘surface-sensitive’’ microscopy, it is clear that

ionization occurs from atoms (in metallic films) and molecules(in organic and biological systems) that lie below the surface.The depths from which electrons are generated depend on

many variables. The penetration of light into the materialdepends on the oscillator strength. The escape yield depends onhow rapidly the surrounding material dissipates the kineticenergy of the photogenerated electron and the rate constants

for various electron-scavenging processes that can occur,especially in complex media such as biological systems. As alimiting example, one could image a complex system in which a

molecule (or material) is encased by materials of higherionization potential, which are transparent to the wavelengthof light corresponding to the threshold potential of the encased

molecule (or material). Such situations could include specifictypes of layered quantum dots, or the neuromelanin granulespresent in the human brain (34) where pheomelanin (threshold

potential of 3.8 eV) is encased in the black eumelanin (thresh-old potential of 4.4 eV). In these cases, one may observe thelower threshold material in the PEEM images for the corre-sponding wavelengths, but the signals are generated from

within the systems being imaged, not from their surface. Thus,with the application of this technique to complex media thatcontain molecules with varying oxidation potentials, there is

the need to develop methods for distinguishing surface pro-cesses from those occurring inside the samples of interest.

Understanding the factors that control the spatial intensity

distribution in a PEEM image

Because of the size of biological systems, the substrate effectsdiscussed above contribute to the spatial intensity of thePEEM image. In addition, the magnitude of these effects

changes with wavelength because both the reflection from thesurface and the height dependence of the resulting opticalinterference effects are wavelength dependent. Thus, in order

to develop insights about the biological (or materials) systemsof interest from the spatial PEEM image, the substrate effects

must be well understood so that attention is focused on themolecular properties of the system being imaged. In addition,there are instrumental effects that must be considered. Theinstrument is designed to image the plane of the sample

directly onto the microchannel plate ⁄CCD detector withconstant pixel dimension over the image. However, if there iscurvature in the sample, the actual surface area of the material

of interest can be quite different from pixel-to-pixel. Thus,caution must be exercised in interpreting the effects of spatiallydependent intensity maps. Reliable methods for disaggregating

all of these effects are required, and this necessitates thedevelopment of a standard set of control experiments forstudying biological assemblies.

Acknowledgements—This work was supported by Duke University.

We especially thank Professor Robert Nemanich, who has collabo-

rated with our group on many projects, for allowing us to continue to

use his PEEM instrument located at the Free Electron Laser Lab at

Duke. We thank Professor David Smith, Professor David Brady,

Dr. Jack Mock, Jonah Gollub, Professor Jack Rowe, Xianhua Kong,

Professor Glenn Edwards and Professor Ying Wu for helpful

discussions on various aspects of this work. We further acknowledge

those researchers that have been involved in our PEEM studies on

biological systems: Alexander Samokhvalov, Yan Liu, Lian Hong,

Jacob Garguilo, William Bush and Luigi Zecca.

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20 Dana N. Peles and John D. Simon