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4 Far-Field Optical Microscopy Beyond the Diffraction Limit

4 Far-Field Optical Microscopy Beyond the Diffraction Limit

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developed far-field optical microscopy techniques such as stimulated emission

depletion (STED) microscopy [2.7], stochastic optical reconstruction microscopy

(STORM) [2.8], or photoactivated localization microscopy (PALM) [2.57] can substantially surpass the Abbé diffraction limit down to spatial resolutions of ∼ 10 nm

and potentially to molecular sizes [2.8, 2.9]. In addition, they can be used for 3D

imaging [2.58, 2.59] and for video-rate imaging of fast nanoscale processes in living cells [2.9]. In these techniques, photoswitching of fluorophore molecules [2.60]

plays an important role.



2.4.1 Stimulated Emission Depletion (STED) Optical Microscopy

This far-field fluorescence optical microscopy [2.61, 2.62] yields a focal plane

resolution of 15–20 nm in biological samples which is a 20–40-fold increase in

resolution beyond the diffraction limit. STED microscopy typically uses a scanning

excitation illumination spot that is overlapped with a doughnut-shaped counterpart

(see Fig. 2.17) for deexcitation of fluorophores by light, a phenomenon referred

to as stimulated emission depletion. Oversaturation in the deexcitation suppresses

emission in the doughnut and squeezes the fluorescence spot to sub-diffraction

dimensions so that superresolved optical images emerge by scanning this spot

through the object.

Making use of the 20 nm resolution provided by STED, optical studies of the

nucleus of a fixed but otherwise intact mammalian cell can be performed. This

appears to be suitable to bring further critical insight into how nuclear organization

ensures regulated gene expression.

Figure 2.18 displays the protein-heavy subunit of neurofilaments in the

human neuroblastoma cell line SH-SY5Y (retinoic acid–BDNF–differentiated),

which establishes cross-links to organize and stabilize neurofilaments in axons

(long process of a nerve fiber that conducts impulses to the nerve cell).

Neurofilaments play an essential role in many neurodegenerative diseases, such as



Fig. 2.17 Stimulated emission depletion (STED) microscopy. ExsPSF: measured focal spot for

excitation (wave length 470 nm; blue; FWHM 190 nm); STED PSF:STED excitation, doughnut

max

shape; (603 nm; orange); Eff PSF: the final spot yields a 22 nm effective size; Iˆ STED : crest intensity

of the STED beam. (Reprinted with permission from [2.61]. © 2006 National Academy of Sciences

U.S.A)



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Far-Field Optical Microscopy Beyond the Diffraction Limit



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Fig. 2.18 Imaging nanofilaments in human neuroblastoma. (a inset) Low-magnification confocal

image indicates the site of recording. (a–d) Contrary to the confocal recording (a), stimulated emission depletion (STED) recording (b) displays details <30 nm, as also highlighted by the comparison

of image sub-regions shown in (c) and (d) bordered by dashed lines in (a) and (b) respectively.

LD – linear deconvolution; (i) and (ii): measurements of line intensities for the determination of

the imaging resolution. (Reprinted with permission from [2.61]. © 2006 National Academy of

Sciences U.S.A)



Parkinson’s disease. In contrast to the confocal image, the STED image identifies

neurofilamental substructures of 20–30 nm.

In addition to the imaging of biomaterials, STED can image the position of

color centers (atomic defects) in diamond with an accuracy of 0.15 nm and a resolution 28-fold smaller (8 nm; see Fig. 2.19) than can conventional fluorescence

microscopy. This enables the addressing of the diamond color centers in much

higher concentrations which may be of interest in quantum computing or cryptography. Fluorescent nanodiamonds could also be favorably used as biological markers

because of their bleaching resistance [2.63].



2.4.2 Stochastic Optical Reconstruction Microscopy (2D-STORM)

In each cycle of this technique (see [2.8]), only a fraction of each of the fluorophores in the field of view is switched on, such that no images of the active

fluorophores are overlapping. This allows the position of these separate fluorophores

to be determined with high accuracy by fitting the point-spread function (PSF) of



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Fig. 2.19 High-resolution

stimulated emission depletion

(STED) optical microscopy

for imaging of vacancynitrogen color centers in

diamond with a resolution of

8 nm, 28-fold narrower than

with conventional fluorescence microscopy which

yields a focal spot of 223 nm

in diameter. (Reprinted with

permission from [2.63].

© 2009 Eva Rittweger and

Stefan W. Hell,

Max-Planck-Institut für

Biophysikalische Chemie)



the emission by a Gaussian which gives the position of the PSF centroid with high

precision. Repeating this process for multiple cycles, each causing a stochastically

different subset of fluorophores to be turned on, allows the position of many fluorophores to be determined and thus an overall image to be reconstructed. Imaging

resolutions of approximately 20 nm can be achieved by using a fluorescence

microscope, low-power continuous-wave lasers, and a photo switchable cyanine

dye [2.8].



2.4.3 Three-Dimensional Far-Field Optical Nanoimaging of Cells

Three-dimensional STED imaging deep inside a cell can be achieved by generating a

spherical focal spot of ∼ 45 nm (λ/16) in diameter by making use of two lasers. This

gives rise to a spherical (isotropic) fluorescence spot (isoSTED) with essentially a

diameter of

d≈λ



(nπ Im Is



where n is the refractive index of the sample, Im the peak intensity of the doughnut beam (Fig. 2.17), and Is the characteristic intensity for the quenching of the

dye used [2.58]. By this isoSTED technique the distribution of mitochondrial

proteins in mammalian cells has been studied [2.58]. Mitochondria are spherical or tubular organelles with inner and outer membranes. They contain genetic

material and enzymes important for cell metabolism. In these organelles with diameters of 200–400 nm the protein distribution cannot be visualized by conventional

light microscopy. The translocase of the mitochondrial outer membrane (TOM

complex) serves as the mitochondrial entry gate for nuclear-encoded protein

precursors. Acting as an import receptor, Tom20 is a subunit of TOM. For



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Far-Field Optical Microscopy Beyond the Diffraction Limit



a



71



b



c



Fig. 2.20 (a–b) Stimulated emission depletion (isoSTED) fluorescence microscopy optical sections through the center (a) and the top (b) plane of a mitochondrion by focusing light into the

interior of a mammalian cell with imaging the distribution of the Tom20 protein clusters. The x–y

image through the center plane shows that the clusters are located at the rim of the organelle; scale

bar, 1 μm. (c) Two-color isoSTED imaging of a mitochondrion of a mammalian cell with the

outer membrane protein Tom20 labeled with NK51 (red) and the matrix protein mtHsp70 labeled

with DY-485XL (green). Scale bar, 1 μm. (Reprinted with permission from [2.58]. © 2008 Nature

Publishing Group)



visualization, Tom20 has been labeled with the orange-emitting fluorophore

NK51 (see [2.58]). Two optical sections (x–y images) through the center and

the top of mitochondrion inside a mammalian cell in Fig. 2.20a, b show that

Tom20 forms distinct clusters at the boundary of the organelle. The isotropic

3D sub-diffraction resolution is essential to identify the peripheral localization

of Tom20 [2.58]. As a demonstration of multiprotein imaging on the nanoscale,

additionally the matrix protein mtHsp70, a component of the mitochondrial

import motor conjugated with the fluorophore DY-485XL, has been visualized

(see Fig. 2.20c).

Three-dimensional (3D) far-field optical imaging can also be achieved by

stochastic optical reconstruction microscopy (STORM) with nanoscale resolution



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Fig. 2.21 Three-dimensional stochastic optical reconstruction microscopy (STORM) imaging of a

clathrin-coated pit (CCP) in a monkey kidney epithelial (BS-C-1) cell. (a) Serial x–y cross-sections

(each 50 nm thick in z) and (b) x–z cross-sections (each 50 nm thick in y) of a CCP and (c) an x–y

and x–z cross-sections presented in a 3D perspective, showing the half-spherical cage-like structure

of the pit. (Reprinted with permission from [2.59]. © 2008 AAAS)



making use of photoswitchable cyanine dyes [2.59]. In Fig. 2.21 x–y and x–z

sections and a 3D rendering of clathrin-coated pits (CCPs) in monkey kidney

epithelial (BS-C-1) cells are visualized by STORM. CCPs are spherical cage-like

structures, about 200 nm in size, assembled on the cytoplasmic side of the cell

membrane to facilitate endocytosis. The half-spherical cage-like morphology of the

nanoscopic CCP structure can only be observed with 3D imaging and not with 2D

images.



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Far-Field Optical Microscopy Beyond the Diffraction Limit



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2.4.4 Video-Rate Far-Field Nanooptical Observation of Synaptic

Vesicle Movement

In addition to nanoscale structural studies in cells, the investigation of fast physiological phenomena in vivo is of particular interest. Recently, the movement

of synaptic vesicles in cultured hippocampal neurons (brain cells) has been

recorded by video-rate (28 frames per second) STED microscopy with a focal

spot size of 62 nm [2.9]. Vesicle movement has been difficult to study by conventional optical microscopy because of the small size of the ∼40 nm diameter

vesicles which are housed in presynaptic nerve terminals of ∼1 μm in diameter, referred to as synaptic boutons. By video-rate STED, the vesicle movement

with a speed peaking at 2 nm/ms (see Fig. 2.22) can be studied, showing that

drugs can reduce vesicle mobility which indicates that active transport plays a

role in vesicle traffic in axons. This study [2.9] demonstrates the emerging ability of optical microscopy to investigate intracellular nanoscale processes in real

time.



Fig. 2.22 Characteristics of synaptic vesicle movement. Successive frames of stimulated emission

depletion (STED) microscopy. The arrowheads indicate three vesicles, which were tracked in all

frames, localized in a sub-diffraction space. The inset in frame #26 shows an intensity profile along

the dotted white line. (Reprinted with permission from [2.9]. © 2009 AAAS)



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2.5 Magnetic Scanning Probe Techniques

The magnetic properties of solids, which arise due to the spin alignment of electrons,

can be studied by scanning probe microscopy such as magnetic force microscopy

(MFM; see [2.20, 2.64]) or, with atomic resolution, by spin-polarized scanning tunneling microscopy (SP-STM; see [2.10]). In addition to the brief survey given here,

a more detailed discussion of magnetic nano-imaging will be given in Sect. 8.1.



2.5.1 Magnetic Force Microscopy (MFM)

The MFM non-contact imaging mode is sensitive to the magnetostatic dipole–dipole

interaction between tip and sample. If a ferromagnetic tip approaches a magnetic

sample surface within a distance of typically 10–50 nm, the tip interacts magnetically with the stray field emanating from the sample [2.65]. The long-range

magnetic dipole interaction is usually probed by using the a. c. detection technique

and gradients rather than the magnetic dipole forces are measured.

For a magnetic domain in the tip, that is small compared to the extent of the

sample stray field Bs , the tip can be considered as a point dipole with the magnetic

moment m yielding [2.66] a force

F(dipole) = ∇ (m · Bs ) = (m · ∇) Bs .

Therefore, in the point-dipole limit, the MFM images are closely related to the

spatial distribution of the magnetic stray field gradient rather than the stray field.

Since magnetic forces Fmag may be either attractive or repulsive, problems with

the feedback loop stability may emerge. Therefore, an attractive electrostatic force

Fel via a bias voltage of 1–10 V between tip and specimen is applied. A separation

of topography and magnetic structure has been demonstrated by imaging a discretetrack magnetic recording sample with the magnetic structure of bits (see Fig. 2.23).



Fig. 2.23 Comparison of the different length scales involved in magnetic data storage. (a) and (b)

show magnetic force microscopy (MFM) images of bit tracks of a magnetic tape whereas (c) shows

a nanoscale magnetic medium where the imaging has been performed by spin-polarized tunneling

microscopy (SP-STM) which is directly sensitive to the local magnetization rather than to the

magnetic stray field sensed by MFM. (Reprinted with permission from [2.10]. © 2000 Wiley-VCH)



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