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

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Figure 7–3. Single particle investigation of the giant protein complex TPP II from Drosophila mela-

nogaster embedded in vitriﬁ ed ice. In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial compo-

nent of the protein degradation pathway. The 150-kDa subunits of Drosophila TPPII assemble into a

giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and

twisted strands that form a spindle-shaped structure (length 56 nm, width 24 nm). a) Cryo-electron

micrograph of isolated TPP II complexes illustrating the very weak image contrast and the high level

of noise. b) Averaging and classiﬁ cation of a large number of equivalent projections of separate mole-

cules. Once a large set of views is available, a preliminary 3D reconstruction can be computed and

reﬁ ned iteratively. c) The 3D model obtained by cryo-electron microscopy, reveals details of the

molecular architecture and, in conjunction with biochemical data, provides insight into the assembly

mechanism. The building blocks of this complex are apparently dimers, within which the 150 kDa

monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single

strands, two of which comprise the fully assembled spindle (Rockel et al., 2002 and 2005).

Figure 7–4. Cellular cryo-electron tomography of the magnetotactic microorganism Magnetospirillum

griphiswaldense. The entire bacterium is oriented like a compass needle inside the magnetic ﬁ eld in its

search for optimal living conditions. The miniature cellular compass is made by a chain of single

nano-magnets, called magnetosomes (the scale bar represents 200 nm). a) The two-dimensional image

represents one projection (at 0°) from an angular tilt-series. b) x–y slices along the z axis through a

typical three-dimensional reconstruction (tomogram). c) Surface-rendered representation of the inside

of the cell showing the membrane (blue), vesicles (yellow), magnetite crystals (red) and a ﬁ lamentous

structure (green). Until now, it was not clear how the cells organise magnetosomes into a stable chain,

against their physical tendency to collapse by magnetic attraction. However, the biochemical analysis

revealed a protein responsible for the chain formation and the 3D investigation a cytoskeletal struc-

ture, which aligns the magnetosomes like pearls on a string (Scheffel et al., 2005).

Figure 7–5. First electron tomographic investigation of a eukaryotic cell; the slime mould Dictyostel-

ium discoideum embedded in vitriﬁ ed ice. a) Phase contrast image and corresponding ﬂ uorescence

image (inset) of cells on TEM grids. b) Cryo-electron micrograph at 0° tilt (conventional 2D

projection) of a ∼200 nm thin peripheral region of the cell. c) Tomographic reconstruction from a

complete tilt-series (120 images) and d) visualization by segmentation. Large macromolecular com-

plexes, e.g. Ribosomes are shown in a green color, the actin ﬁ lament network in orange-red and the

cells’ membrane in blue. Cryo-tomograms of Dictyostelium discoideum cells grown directly on carbon

support ﬁ lms have provided unprecedented insights into the organization of actin ﬁ laments in an

unperturbed cellular environment. The tomograms show, on the level of individual ﬁ laments, their

modes of interaction (isotropic networks, bundles, etc.), they allow us to determine the branching

angles precisely (in 3-D), and they reveal the structure of membrane attachment sites (Medalia

et al., 2002).

Figure 7–6. CET in combination with the single particle approach of transport-competent Dictyostel-

ium discoideum nuclei. a) Three-dimensional reconstruction of the peripheral rim of an intact nucleus.

X-y slice of 10 nm thickness along the z axis through a typical tomogram. Side views of nuclear pore

complexes (NPCs) are indicated by arrows. Ribosomes connected to the outer nuclear membrane are

visible (arrowheads). Inset displays a phase-contrast image and the corresponding ﬂ uorescence image.

b) Surface rendered representation of a segment of nuclear envelope (NPCs in blue, membranes in

yellow). c) Structure of the Dictyostelium NPC after classiﬁ cation and averaging of subtomograms.

Cytoplasmic face of the NPC (upper left); the cytoplasmic ﬁ laments are arranged around the central

channel. Nuclear face of the NPC (upper right); the distal ring of the basket is connected to the nuclear

ring by the nuclear ﬁ laments. Cross sectional view of the NPC (bottom). The dimensions of the main

features are indicated. All views are surface-rendered (nuclear basket in brown; Beck et al. 2004).

Figure 7–7. Plunge freezing instrumentation. a) Typical arrangement of cells cultured on TEM grid just

prior to plunging. The schematic indicates the dimensions. b) CAD (computer aide d design) image of

a home-build plunge freezing apparatus (Images courtesy of R. Gatz, MPI of Biochemistry, Martinsried

(near Munich), Germany). The small reservoir (yellow) in the middle of the dewar (green) contains the

liquid ethane, which is chilled by a surrounding bath of liquid nitrogen. The forceps, which hold the

grid are attached to a weighted arm. When the arm is released by means of a foot-trigger, the grid is

gravity-plunged into the ethane. The cross-sectional scetch (left part of image B) illustrates the ‘guillo-

tine-like’ arrangement. c) The Vitrobot (FEI company, Eindhoven, The Netherlands), a ‘robot’ for vitri-

ﬁ cation, allows the control of environmental and all necessary processing parameters.

Figure 7–22. Detection and identiﬁ cation of individual macromolecules in cellular tomograms is

based on their structural signature. Because of the crowded nature of the cytoplasm and ‘contamina-

tion’ with noise, an interactive segmentation and feature extraction is not feasible. It requires sophis-

ticated pattern recognition techniques to exploit the information contained in the tomograms. A

volume rendered presentation of a 3D image is presented on the left. Even though some high-density

features may be visible, an unambiguous identiﬁ cation of individual structures would be difﬁ cult if

not impossible given the residual noise. An approach, which has proven to work, is based on template

matching. Templates of the macromolecules under scrutiny are obtained by a high- or medium resolu-

tion technique (X-ray crystallography, NMR, electron crystallography or single particle analysis).

These templates (4 times magniﬁ ed in this ﬁ gure; 20S proteasome and thermosome) are then used

to search the entire volume of the tomogram systematically for matching structures by three-

dimensional cross-correlation and the result is reﬁ ned by multivariate statistical analysis. In principle

the 3D image has to be scanned for all possible Eulerian angles ϕ,ψ and θ around three different axes,

with templates of all the different protein structures one is interested in e.g. the thermosome (blue)

and proteasome (yellow). The search procedure is computationally very demanding but can be paral-

lelized with respect to the different angular combinations in a highly efﬁ cient manner. Finally, the

position and orientation of the different complexes can be mapped directly in the 3D image.

Figure 7–18. a) Geometric model of specimen rotation around a tilt axis. b) Automated data collection

scheme for the ‘full-tracking/ full-focusing’ case. For tracking, focusing and the ﬁ nal image acquisi-

tion the same magniﬁ cation is used and the beam is adjusted in a way that the areas where tracking,

focusing a and exposure is done do not overlapp (blue and green circle). Using very short exposure

times in combination with high binning values (4 × 8 or even 8 × 8) the exposure to the sample in

auto-tracking and auto-focusing can be minimized.

change tilt angle

‚image shift‘ correction

Focus correction

image acquisition

Tracking

Focus

Exposure

Figure 7–23. Mapping ribosomes in an intact S. melliferum cell. a) x–y slice from the corresponding

tomogram. Image analysis of this portion of the cell is displayed in subsequent panels. b) Locations

and orientation of all ribosomes detected by template matching. Each 70S ribosome is represented by

the averaged density (see Fig. 26) derived from the tomogram. The colour coding indicates the detec-

tion ﬁ delity; green is high, yellow is intermediate, red is low and probably represents false positives.

The brightness of the colour corresponds to correlation peaks heights. c) Final ribosome atlas after

removal of putative false positives (images courtesy of J. Ortiz, MPI of Biochemistry, Martinsried,

Germany).

Figure 7–25. a) Averaged structure of the 70S ribosome derived from the dataset above (average from

300 individual particles to ∼4 nm resolution). The map highlights the 30S subunit in yellow. b) Docking

of high resolution structures of the 70S ribosome into the map shown in A. c) “Crown view” of B

(images courtesy of J. Ortiz, MPI of Biochemistry, Martinsried, Germany).

Figure 10–13. Electrostatic correction of chromatic aberration. (a) Rays in a quadrupole quadruplet

or sextuplet. (b) Match between the potentials needed to satisfy Scherzer’s condition and those in the

corrector.

5.2 eV 6.4 eV 9.6 eV 11.4 eV

Figure 8–26. Quantum size contrast between regions with different thickness of an Fe ﬁ lm on W(110),

taken with different electron energies, that is wavelengths. The images in the top row show the inten-

sity and those at the bottom the magnetic signal (exchange asymmetry). Blue and red correspond to

opposite magnetization directions.

Figure 8–18. The three fundamental operation modes of a SPELEEM system. The various sections of

the instrument are shown folded into one plane. In imaging and diffraction the energy selection slit

is inserted in the dispersive plane DP and the image/diffraction pattern behind DP is imaged with

the projector. The intermediate lens IL is used to switch between imaging and diffraction, simultane-

ously with the exchange of the contrast aperture in FPI and the ﬁ eld-limiting aperture in IIP. For fast

spectroscopy both apertures are inserted, the energy selection slit is removed, and the dispersive plane

is imaged by the projector.

-3E+08

-2E+08

-1E+08

0E+00

1E+08

2E+08

3E+08

-15 -10 -5 0 5 10 15

Z [mm]

Quadrupole field [V/m/m]

Scherzer's condition

3D Quadrupole

Figure 11–12. Multiple excitation of three ﬂ uorescent dyes using 740 nm under a TPE regime. The

conventional excitation would have required the utilization of 360 or 405 nm, 488 nm, and 543 nm laser

lines. The ﬁ nal image (lower right quadrant) is realized by merging the three subsets. (This image has

been acquired by students of the Biotechnology School during the course of Advanced Microscopy

Techniques activated at the University of Genoa, academic year 2005. Advisors: Grazia Tagliaﬁ erro

and Alberto Diaspro.)

Rhodamine B

Bodipy Fl

Dil

Coumarin

Lucifer Yellow

Fluorescein

Cascade Blue

Pyrene

Bis-MSB

Dansyl

DAPI

600 700 800

Two photon Excitation

Cross Sections (GM)

Wavelength (nm)

Ti:Sapphlie

SHG of Cr:YAG

SHG of Cr:Forsterite

Cr:LiSGAF

Cr:LiSAF

Nd:YLF or Nd:glass

–1

–2

–3

900 1000 1100

Figure 11– 6. Two-photon cross-sections for popular

ﬂ uorescent molecules as a function of the excitation

wavelength. Red bars indicate the emission range of

some common laser sources utilized in TPE micro-

scopy and spectroscopy.

Figure 11–2. Comparison between conventional

(1P) and two-photon (2P) excitation with respect to

image formation. When focusing on the actual

focal plane under 1P, a contribution from adjacent

planes that are physically excluded in the 2P process

is obtained, as happens in a confocal setup. (From

Giuseppe Vicidomini, LAMBS, MicroScoBio, Uni-

versity of Genoa.)

4% 100%

PSF OTFPSF OTF

Widefield Confocal SWM I

M 4Pi C

TPE 4Pi A TPE 4Pi C

PSF OTF

det

Figure 12–4. Overview of the excitation (bottom), detection (center), and effective (top) OTFs’ modulus

and the corresponding PSFs of the wide-ﬁ eld, confocal, standing wave (SWM), I

M, 4Pi confocal type

C, TPE 4Pi confocal type A, and TPE 4Pi confocal type C microscopes. The color look-up table (LUT)

has been designed to emphasize the important weak OTF regions. The OTFs are shown in the squares

above the corresponding PSFs; the zero frequency point is in the center and the largest frequency

displayed is 2π/80 nm

−1

. The circles represent the maximum possible carrier as explained in Figure

12–3. For TPE, the excitation OTFs slightly extend over these circles because the excitation wavelength

of 800 nm is less than double the one-photon excitation wavelength of 488 nm. While all these methods

extend the OTF along the axial direction, they fundamentally differ in contiguity and absolute strength

within the support region. For example, there are pronounced frequency gaps for the SWM and

depressions for the I

M. The rectangular images of the PSFs represent a region of 5 × 2.5 µm with the

geometric focus in the center.

e) f)

1 µm

a) b) c)

l(k

)

l(k

)

-1

FFT

confocal

4pi (raw)

4pi

FFT

Figure 12–5. Lobe removal and deconvolution in 4Pi microscopy. The ﬁ gure shows images of the same

pair of actin ﬁ bers in a ﬁ xed mouse ﬁ broblast cell recorded in the TPE confocal (a) and TPE 4Pi type

A (b and c) mode. The corresponding Fourier transform along the optic axis is also shown (d, e, and

f). The ﬁ ve-fold axial resolution increase (a vs. b) and the correspondingly extended OTF (d vs. e) are

immediately visible. The side lobes are well below 50% and the factorization of the PSF’s axial and

lateral dependence is possible in 4Pi microscopy. Therefore, an inverse discrete ﬁ lter can be found and

its application to the raw data (c) yields a valid and almost artifact-free image (b). Alternatively, lobe

removal can be performed in the frequency domain. Equation (16) indicates that the Fourier transforms

of the raw data (f) is given by the product of the Fourier transform of the lobe-free image (e) and the

lobe function



). Thus, (2) Fourier transforming the raw data, (3) multiplying with the inverse of

the lobe function’s Fourier transform,



−1

), and (4) Fourier backtransforming lead to almost the

same lobe-free image. This method can be applied even if the separation of axial and lateral depen-

dence is impossible for the PSF. The Fourier transforms along the axial direction (3 and 4) merely have

to be replaced by their 3D counterparts.

490nm

244nm

STED

source

EXCT.

source

DET

97nm

104nm

d) e)

fluorescent

non-fluorescent

450 650

λ[nm]

Excitation

STED

Detection

Figure 12–7. Stimulated emission depletion (STED) was the ﬁ rst implementation of the RESOLFT

principle. (a) Dye molecules are excited into the S

(state A) by an excitation laser pulse. (b) Fluorescence

is detected over most of the emission spectrum. How ever, molecules can be quenched back into the

ground state S

(state B) using stimulated emission before they ﬂ uoresce by irradiating them with a light

pulse at the edge of the emission spectrum shortly after the excitation pulse and before they are able to

emit a ﬂ uorescence photon. Saturation is realized by increasing the intensity of the depletion pulse and

consequently inhibiting ﬂ uorescence everywhere except at the “zero points” of the focal distribution of

the depletion light. (c) Schematic of a point-scanning STED microscope. Excitation and depletion beams

are combined using appropriate dichroic mirrors (DC). The excitation beam forms a diffraction-limited

excitation spot in the sample (inset in d) while the depletion beam is manipulated using a phase-plate

(PP) or any other device to tailor the wavefront in such a way that it forms an intensity distribution with

a nodal point in the excitation maximum (left inset in e). The third inlay shows the resulting quenching

probability when saturating the depletion process. (d) and (e) show an experimental comparison

between the confocal PSF and the effective PSF after switching on the depleting beam. Note the doubled

lateral and ﬁ ve-fold improved axial resolution. The reduction in dimensions (x, y, z) yields ultrasmall

volumes of subdiffraction size, here 0.67 al (Klar et al., 2000), corresponding to an 18-fold reduction

compared to its confocal counterpart. The spot size is not limited on principle grounds but by practical

circumstances such as the quality of the zero and the saturation factor of depletion.