Hawkes P.W., Spence J.C.H. (Eds.) Science of Microscopy. V.1 and 2

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Figure 12–10. Images of a wetted Al

matrix featuring z-oriented holes (Whatman plc, Brentford,

UK) with a spin cast of a dyed (JA 26) polymethyl methacrylate solution. The rings formed in this way

are ∼250 nm in diameter and are barely resolved in confocal mode. (a–d) The confocal image (a) and

STED images with two depleting beams perpendicularly polarized and aberrated by “1D” phase-plates

(b–d). The excitation PSF (g) and the STED PSF for y polarization (h) and x polarization (i) are shown

on the right. The STED intensity was chosen at the spots marked in the saturation curve (f). The smaller

effective spot size also results in an extended OTF as seen in the second column. Here, the insets show

the 2D Fourier transformation of the images in the left and the graphs show a proﬁ le along the x direc-

tion. Note the logarithmic scales. The Fourier transform of the image is given by the product of OTF

and the Fourier transform of the object [Eq. (2)]. For such regular structures, an estimate for the

modulus of the OTF can therefore be gained by estimating the latter and solving for the OTF. The

dashed line shows the Fourier transform of a ring with a diameter of 275 nm and a width of 50 nm and

the estimated OTF is presented in (e). (f) The suppression of ﬂ uorescence resulting from stimulated

emission. The phase-plates were removed and the ratio of ﬂ uorescence without STED light (F

) and

with the STED beams switched on (F) was recorded. The intensities are pulse intensities per beam at

the global maximum.

Plane

mirror

Bending

Magnet

Condenser

zone plate

Objective

zone plate

Pinhole

X-ray

sensitive

CCD

Sample

stage

National Synchrotron Light Source

X-ray Ring

X1 undulator

Monochromator

Zone plate

Order sorting

aperture

Detector

Specimen

ft x

ray

2.8 GeV electrons

Figure 13–15. Schematic of the main components of

a transmission X-ray microscope or TXM (top: cour-

tesy of D. Attwood, Lawrence Berkeley National Lab-

oratory) and a scanning transmission X-ray microscope

or STXM (bottom: courtesy of Y. Wang, then of Stony

Brook.)

400

800

1200

1600

01234

Z / µm

Counts

Z / µm

FWHM

53nm

01234

Confocal

STED-4Pi

25nm

60nm

Immunolabeled

microtubule

Overview

Monolayer

Microtubules

Monolayer

Microtubules

Figure 12–11. Subdiffraction immu-

noﬂ uorescence imaging with STED-4Pi

microscopy. (a) Overview image (xy) of

the microtubular network of an HEK

cell. (b) Sketch of typical dimensions of

a labeled microtubule ﬂ uorescently

decorated via a secondary antibody. (c)

and (d) Standard confocal and STED-

4Pi xz image recorded at the same site

of the cell; the straight line close to the

cell stems from a monomolecular ﬂ uo-

rescent layer attached to the adjacent

coverslip. In both images, the pixel size

was 95 × 9.8 nm in the x and z direction,

respectively; the dwell time per pixel

was 2 ms. Note the fundamentally

improved clarity in (d). The STED-4Pi

microscope’s PSF features two low side

lobes caused by the secondary minima

STED intensity distribution. These

lobes are <25% and were removed in

the STED-4Pi image using linear ﬁ lter-

ing as outlined in the text [see Eq.

(16ff)]. (e) and (f) Corresponding pro-

ﬁ les of the image data along the dashed

lines in (b) and (c) quantify the

improved axial resolution of the STED-

4Pi microscopy mode (f) over the con-

focal benchmark. Peaks 1, 2, and 3 due

to microtubules are broader than the

response to the monolayer. Note the

ability of the STED-4Pi microscope to

distinguish adjacent features.

2 µm

Abs. coeff.

µ (µm

-1

)

0.5

Flagella

Flagellar roots

and neuromotor

Nuclear

membrane

Cell wall

Nucleolus

Figure 13–30. 3D rendering (left) and reconstruction slices (right) of the algae Chlamydomonas rein-

hardtii viewed by soft X-ray tomography at the BESSY I synchrotron. This alga was plunge-frozen in

liquid ethane, and imaged over 180º rotation sequence. The reconstruction is given in terms of the

permission from Elsevier.)

Figure 13–27. Near-carbon-edge

absorption spectra of several amino

acids, showing the effects of various

molecular bonds in the absorption

spectrum. These resonances can

be used for chemical contrast in X-

ray microscopy. (Reprinted from

mission from American Chemical

Society.)

Arginine:

C=N

π*

Alanine:

aliphatic

Cysteine: side chain -SH

Glutamine: -NH2

Tyrosine: aromatic

292291290289288287

Photon Energy (eV)

292291290289288287 292290288286284

Mass Absorption Coefficient (10

/g)

292291290289288287 292291290289288287

Figure 13–29. Human ﬁ broblast with immunogold

labeling for tubulin. This is a composite of two

images: a bright ﬁ eld image (gray tones) to image

overall mass, and a dark ﬁ eld image (red tones) to

selectively imaging the silver-enhanced gold labels.

This whole-mount cell was ﬁ xed and then permeabo-

lized to allow for introduction of the immunogold

labels, after which it was air dried. (From Chapman

of America.)

light epi

Ca Mn

10 µm

Figure 13–35. Visible light and epiﬂ uorescence micrographs, and false color X-ray ﬂ uorescence

element maps of a centric diatom collected from the southern Paciﬁ c. In this region of the ocean, iron

availability is a biolimiter with an impact on oceanic uptake of carbon dioxide from the atmosphere.

X-ray microprobes allow one to study iron content on a protist-speciﬁ c basis. (Reprinted from Twining

Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5

525 530 535 540 545 550

Photon energy (eV)

2 µm

0.0

0.5

1.0

1.5

2.0

Optical Density

Figure 13–34. Cluster analysis in a spectromicroscopy study of lutetium in hematite. Lutetium is

serving as a homologue to americium in an investigation of the uptake and transport of nuclear waste

products in groundwater colloids. By using a pattern recognition algorithm to search for pixels with

spectroscopic similarities, a set of signature spectra is automatically recovered from the data (shown

here in a color-coded classiﬁ cation map) and thickness maps can be formed based on these signature

spectra. Analysis at the oxygen edge reveals two different phases of reactivity for lutetium with hema-

tite. Analysis by Lerotic (2004), from a study by T. Schäfer, INE Karlsruhe.

Intensity (a.u)

166 165 164 163 162 161 160

Binding Energy (eV)

S 2p

MoS

tips

sidewalls

base

Figure 13–42. In Electron Spectroscopy for Chemical Analysis or ESCA microscopy, a monochromatic

beam is used to illuminate a region several micrometers across; electron optics are then used to image

a tunable electron ejection energy to reveal surface chemistry. Though this does not involve zone plate

imaging, we include it here due to its widespread use with tunable X rays. In this case a 90-nm resolu-

tion ESCA microscope was used to locate aligned MoS

nanotube bundles and select certain areas

along the axes of the tubes for detailed examination. The image at left was acquired using Mo 3d

electrons, while S 2p, Mo 3d, and valence band spectra taken at the tips and sidewalls and the growth

base from the Si wafer appear strongly affected by the low dimensionality of the nanotubes and

differ signiﬁ cantly from the corresponding spectra taken on a reference MoS

crystal. (Reprinted from

Figure 14–13. Transport imaging

in polycrystalline ZnO. (a) Surface

topography. (b and c) SSPM images

under lateral bias exhibit potential

drops at grain boundaries, indica-

tive of grain boundary resistive

behavior. Note that the direction of

potential drops inverts with bias.

(d) Current maps for positive and

negative bias polarity. (Partially

reproduced from Kalinin and

Bonnell, Zeitschrift fur Metallkunde,

90, 983–989, 1999.)

Figure 13–43. Scanning photoemission microscope (SPEM) study of a plasma display cell. In this micro-

scope the specimen is scanned through the zone plate focus while photoelectrons are collected by an electron

spectrometer. This ﬁ gure shows a SPEM image, a scanning electron micrograph, and photoelectron spectra

from several regions of the sample. In a plasma display cell, light of the appropriate color emerges through a

front glass window which is protected from plasma damage by a composite insulating layer including MgO.

The photoelectron spectra show aging in the Mg(OH)

component of the layer over the life of the display cell.

SEM Image

SPEM image

Relative B.E. (eV) Relative B.E. (eV)

Degree of ageing

Relative B.E. (eV)

MgCO

MgO

Mg 2p, 2

MgCO

MgO

Mg(OH)

Mg 2p,1

Intensity (a.u.)

MgCO

MgO

Mg 2p, 3

5 0 -5 5 0 -5 5 0 -5

Figure 14–22. (A) Images of the proﬁ les of peri-

odic dielectric constant (on the left) and ferro-

electric domain boundaries (on the right). (B)

Line proﬁ les of frequency (top) and quality

factor (bottom) images.

Figure 14–23. Atomic resolution NC-AFM, STM,

and KFM on Ge/Si (105). [Courtesy of Eguchi et al.

Reproduced with permission from Physical Review

Letters, 93(26), 2004.]

Figure 14–15. Experimental images (outside) and theoretical simulations (inside) of the ﬂ ow of elec-

tron waves through a quantum point contact. Fringes spaced by half the Fermi wavelength demon-

strate coherence in the ﬂ ow. [Courtesy of Westervelt et al. Reproduced with permission from Physica

E, 24, 63–69, 2004.]

Figure 15–13. (a) Bias-dependent STM on GaAs(110): selective imaging of Ga and As sublattices at

by the Armeucion Physics Socuity). (b) Compound STM image of the InP(110) surface, assembled from

Elsevier).

Figure 15–16. I(V) tunneling spectroscopy on C

/C-nanotube “peapods” (Reprinted with Permission

nanotube “peapod.” Sample bias voltage is plotted on the horizontal axis and displacement along the

tube on the vertical axis. (B) Representative dI/dV spectra at selected positions along the tube. Large

conductance peaks are found at positions of embedded C

molecules. (C) Variation of tunneling con-

ductance along the tube axis. (D) Reference spectroscopic map on an empty C-nanotube section

without embedded C

, in which no strong modulation of the tunneling conductance is observed.

Figure 15–18. Spin-polarized STM on a Gd(0001) sample with an exchange-split surface state and a

magnetic Fe tip with constant spin polarization close to E

. (a) Due to the spin-valve effect the tun-

neling current of the surface state spin component parallel to the tip magnetization is enhanced. (b)

Illustration of the reversal in the dI/dV signal at the surface state peak position upon switching the

sample magnetically. (c) Experimental observation of this reversal in tunneling into an isolated Gd

Figure 16–1. STM topographies of Rec-DNA complex (left) and an aberrant bacteriophage capside

(right). In the upper right corner, a hole has been created applying a voltage pulse between the tip and

the sample. Both images immediately reveal the molecular arrangement of the respective complex

structure. (Left, from Amrein et al., 1988, reproduced with permission. Right, from Amrein et al., 1998b,

reproduced with permission.)

Figure 15–37. Identiﬁ cation of

the molecular conformation of

Cu-DTBPP on Cu(211). (a) Molec-

ular model of Cu-DTBPP, with the

four-lobed pattern observed by

STM marked in yellow. (b–e) STM

contrast calculation for different

angles between the four legs and

the substrate: (b) 60°, (c) 45°, (d)

30°, and (e) 10°. (f) Experimental

STM image of the molecule.

(Reprinted from Moresco et al.,

Elsevier.)

Figure 16–3. Neuronal cell, cultured on an electronic

chip. The chip is designed such as to pick up an action

potential of the cell. The image demonstrates proper

tracing of the cell surface. In a future application, an

appropriately designed stylus might be used as an

additional electrode to excite or record an action

potential at any location of the cell body or a process

of the neuronal cell.

Figure 16–18. Images of a ﬁ xed 3T3 cell in buffer, on a coverslip. Left to right: Optical (differential

interference contrast, DIC) and simultaneous AFM (JPK Instruments, contact mode) topography and

feedback signal.

Figure 17–36. Bond for-

mation induced with

STM tip. A sequence of

STM constant-current

images at 13 K showing

the formation of Fe–CO

bonds by vertical manip-

ulation. Fe atoms are

imaged as protrusions

and CO molecules as

depressions. The white

arrows indicate the pair

of adsorbates involved

in each bond formation

step. In (B) and (C) a CO

molecule has been

picked up and bonded

to an Fe atom to form

Fe(CO). In (D) a second

CO molecule has been

bonded to Fe(CO) to

form Fe(CO)

. (From Lee

and Ho, 1999.)

Figure 17–43. Atomic resolution magnetic imagi ng

with SP-STM. (A) Constant-current image of one

monolayer of Mn on W(110) recorded with a non-

magnetic W tip at 16 K. (B) Image recorded with a

magnetic Fe tip showing an antiferromagnetic con-

ﬁ guration as predicted by theory. The colored

insets show calculated STM images. (C) Experi-

mental and theoretical line sections from (A) and

(B). The image size is 2.7 nm by 2.2 nm. (From

Heinze et al., 2000.)

Figure 18–9. (a) Low magniﬁ cation bright-

ﬁ eld image of self-assembled Co nano-

particle rings and chains deposited onto an

amorphous carbon support ﬁ lm. Each Co par-

ticle has a diameter of between 20 and 30 nm.

(b–e) Magnetic phase contours (128× ampliﬁ -

cation; 0.049 radian spacing), formed from the

magnetic contribution to the measured phase

shift, in four different nano particle rings. The

outlines of the nano particles are marked in

white, while the direction of the measured

magnetic induction is indicated both using

arrows and according to the color wheel shown

in (f) (red = right, yellow = down, green = left,

blue = up). (Reprinted from Dunin-Borkowski

et al., 2004b.)