high resolution imaging with the nanowizard bioafm

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High resolution imaging with the NanoWizard® BioAFM

Since its invention, the atomic force microscope (AFM) has

been used to image a wide range of different samples.

When the AFM was modified such that it could image

samples in buffer it became possible to address biological

questions under physiological conditions with this

technique. The detail in atomic force images is unrivalled

by other microscopy techniques that can be used to image

samples in fluid, due to the signal to noise ratio of the

instrument. In addition, samples dried to preserve structure

do not need to be further treated to generate contrast.

The NanoWizard® BioAFM from JPK Instruments has a

number of features that enhance the capacity of this

technology for the highest resolution imaging of biological

samples. Namely, the JPK Nanowizard® is linearized in all

three dimensions. That is, there is a closed-loop feedback

that ensures precise positioning in the x and y axes as well

as in the z axis. Additionally, the Nanowizard® further

extends the applicability of atomic force microscopy (AFM)

imaging by enabling simultaneous AFM imaging with

additional optical microscopic techniques. Both of these

features can save the user time and resources when

striving for that perfect, high resolution image.

Atomic lattice of mica

Fig. 1 Mica imaged in contact mode. Scan size, 40 x 60 Å

Superior engineering and stability is required for the

acquisition of high resolution images. To demonstrate the

stability of the JPK Nanowizard® even when installed on an

inverted light microscope, freshly cleaved mica was

imaged in contact mode in air. The atomic lattice of mica

can clearly be seen (Fig 1.)

Hexagonally packed intermediate layer The hexagonally packed intermediate (HPI) layer of the

archaebacteria, Deinococcus radiodurans, has been

extensively studied using atomic force microscopy [1, 2].

Fig. 2 (A) HPI layer patch on mica, imaged in closed loop contact mode, in fluid. (B) High resolution image of HPI subunit pores. Red circle - example of a closed pore, blue circle – example of an open pore. Defects in the lattice are also evident, e.g. the missing suunit in the pore marked with a white arrow. Image (B) kindly provided by Dr. Patrick Frederix, University of Basel.

The HPI layer of D. radiodurans forms a surface layer,

presumed to act as a kind of molecular sieve to regulate

transport of nutrients and metabolites in and out of the cell.

Data has been generated on the structure and function of

the HPI layer using a variety of different techniques, from

biochemistry to electron microscopy. However, AFM

imaging of this sample can be carried out in fluid, at high

resolution, to follow dynamic changes in protein structure.

A

100 nm

B

10 nm

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The HPI layer is extracted from whole cells with detergent

and then adsorbed to a freshly cleaved mica surface. The

stable packing of the individual protein elements facilitates

the acquisition of high resolution images. The HPI layers

form patches on the mica surface, and overview images of

these patches already reveal the regular lattice-structure of

the HPI layer (Fig 2, A).

After the acquisition of an overview image of an HPI

membrane patch, a suitable region can be selected for

imaging at higher resolution (Fig 2, B). As the x-y

positioning of the JPK Nanowizard® is controlled by a

closed-loop feedback system the instrument will “zoom in”

to the selected region with high accuracy. This enables the

user to take fewer scans, reducing the likelihood of

contaminating the tip or damaging the membrane patch.

Nuclear pore complex The eukaryotic cell is organised into compartments called

organelles. Controlled transport across the membranes

surrounding each organelle allows the cell to

compartmentalize specific molecules, a process that

underlies cellular function. In the nuclear membrane, the

nuclear pore complex (NPC) is responsible for transport of

various molecules into and out of the nucleus.

Fig. 3 DIC image of NPC sample. The use of DIC clearly highlights debris.

Unlike the HPI layer of D. radiodurans, preparations of

NPC do not simply contain NPC condensed into a lattice.

The samples are prepared from whole nuclei, in this case

from Xenopus laevis, and can be quite heterogeneous [3].

As the life science version of the JPK Nanowizard® is fully

integrated into an inverted, light microscope, transmission

microscopy can be used to scan the sample for a region

that does not contain large amounts of debris, before

scanning. In such a way the user can, once again, reduce

the time required to find a suitable region for scanning, and

decrease the chance of contaminating the tip.

Fig. 4 Contact mode images of NPC on a glass coverslip. An overview image shows a mixture of NPC and contaminating material. The JPK Nanowizard® can then accurately zoom in on regions of interest for higher-detail scans.

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1 µm

200 nm

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NPC samples, on a glass coverslip, were imaged using

differential interference contrast (DIC) microscopy, clearly

highlighting debris that would be impossible to visualise

using bright field microscopy (Fig 3). The tip was then

positioned over an area with minimal debris and an

overview scan acquired (Fig 4). Again, the capacitively

controlled feedback then allows precise selection of an

area for a higher resolution scan.

DNA imaging Most of the data generated on the structure and function of

DNA has come from the field of molecular biology.

However, with the signal to noise ratio of AFM this

fundamentally important biological molecule can be studied

at high resolution in liquid and in air, to elucidate physical

structure and the interaction of DNA with DNA-binding

molecules. Under appropriate conditions, DNA can be

adsorbed to freshly cleaved mica and imaged in buffer.

Figure 5 shows Lambda phage DNA (ac mode in fluid).

Fig. 5 Topographs of Lambda phage DNA, imaged in intermittent contact mode in fluid. Colour scale 0-2 nm for both A and B.

The interaction of various proteins with DNA is

fundamental in the processes of replication and

transcription. One example is the association of DNA with

histones to form nucleosomes. This condensing of DNA

around the nucleosome core (consisiting of a histone

octamer) plays a role in the regulation of DNA replication,

and transcription, as the condensed DNA is not accessible

to other DNA-binding proteins.

Fig. 6 AC mode topograph of DNA-nucleosome complexes. The protein can clearly be distinguished bound along the length of the linearised pGEM plasmid. Image courtesy of Dr. Clemens Franz, Technical University of Dresden.

In this case, the linearized, 3 kb plasmid pGEM was

incubated with nucleosomes in a ratio of 1 mole of DNA to

20 moles of histone octamers. The pGEM plasmid has 20

putative nucleosome binding sites, however, it can be seen

that under the incubation conditions, nucleosomes did not

bind at all 20 binding sites (Fig 6).

Conclusions The signal to noise ratio, lack of requirement for staining or

pretreating and capability to function in liquid makes AFM

imaging an extremely powerful method for describing, at

high resolution, the structure of biological samples. The

design of the JPK Nanowizard® can facilitate such high

resolution studies. For instance, the accurate positioning in

x and y (due to closed-loop feedback) reduces the number

of scans that are required to “focus” at high resolution on a

region of interest. This decreases the likelihood of

damaging delicate samples and of contaminating the tip.

100 nm

A

20 nm

B

125 nm

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For samples prepared on glass, such as the nuclear pore

complex described above, contaminating debris can be

easily avoided by searching for a suitable area using

transmission light microscopy, again saving the user time.

The benefit of using AFM for such imaging studies lies in

the capability of AFM to image samples in liquid, under

physiological conditions. JPK Instruments manufactures

the Biocell™ that can allow the user to modify conditions

during scanning (Fig 7), such as controlled temperature

changes or the in situ addition of relevant molecules,

further enhancing the applicability of the JPK Nanowizard®

for the highest resolution imaging of biological samples.

Fig. 7 The JPK Biocell™. The Biocell™ is designed to enable optimal imaging conditions for both AFM and optical methods while allow rapid and precise temperature control from 20-60°C.

The JPK Nanowizard®, integrated into an inverted light

microscope, is also optimised for imaging of other

biological samples, from lipid bilayers [4] and biopolymers

such as collagen [5] to whole cells [6,7,8]. The AFM

imaging of such samples can be supplemented with

additional light microscopy techniques, such as laser

scanning confocal [4,6,8], epifluorescence [7,9] TIRF or

FRET. The JPK Nanowizard® has also been used to

quantify unbinding forces, from individual proteins to cell-

cell interactions [10,11]. As such, the JPK Nanowizard®

BioAFM is perfect for conducting imaging and force

measurements of biological samples from individual

proteins to whole cells, under controlled, physiological

conditions.

Acknowledgements Many thanks to all those who contributed samples and

images. The high resolution image of HPI was provided by

Dr. Patrick Frederix from the group of Prof. Engel,

University of Basel. The NPC sample was a kind gift from

Barbara Windoffer of the group of Prof. Dr. Oberleithner,

Universitätsklinikum Münster. The DNA-histone sample

was prepared by Dr. Dennis Merkel (Prof. Schwille’s

group) and imaged by Dr. Clemens Franz (Prof. Müller’s

group) both of the Dresden University of Technology.

Literature [1] Muller DJ, Baumeister W, Engel A. (1996) Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy. J Bacteriol. 178(11):3025-30. [2] Muller DJ, Baumeister W, Engel A. (1999) Controlled unzipping of a bacterial surface layer with atomic force microscopy. Proc Natl Acad Sci U S A 96(23):13170-4. [3] Rakowska A, Danker T, Schneider SW, Oberleithner H. (1998) ATP-Induced shape change of nuclear pores visualized with the atomic force microscope. J Membr Biol. 163(2):129-36 [4] Chiantia S, Kahya N, Schwille P. (2005) Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances. Langmuir. 21(14):6317-23. [5] Jiang F, Khairy K, Poole K, Howard J, Muller DJ. (2004)Creating nanoscopic collagen matrices using atomic force microscopy.Microsc Res Tech. Aug;64(5-6):435-40. [6] Poole K, Muller D. (2005) Flexible, actin-based ridges colocalise with the beta1 integrin on the surface of melanoma cells. Br J Cancer. 92(8):1499-505. [7] Sharma A, Anderson KI, Muller DJ. (2005) Actin microridges characterized by laser scanning confocal and atomic force microscopy FEBS Lett. 579(9):2001-8. [8] Poole K, Meder D, Simons K, Muller D. (2004) The effect of raft lipid depletion on microvilli formation in MDCK cells, visualized by atomic force microscopy. FEBS Lett. 565(1-3):53-8. [9] Franz CM, Muller DJ. (2005) Analyzing focal adhesion structure by atomic force microscopy. J Cell Sci. 118(Pt 22):5315-23. [10] Puech PH, Taubenberger A, Ulrich F, Krieg M, Muller DJ, Heisenberg CP. (2005) Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy. J Cell Sci. 118(Pt 18):4199-206. [11] Ulrich F, Krieg M, Schotz EM, Link V, Castanon I, Schnabel V, Taubenberger A, Mueller D, Puech PH, Heisenberg CP. (2005) Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin. Dev Cell.9(4):555-64.

high resolution imaging with the nanowizard bioafm

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