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© JPK Instruments AG - all rights reserved NanoWizard, LightWizard and CellHesion are registered trademarks of JPK Instruments AG - www.jpk.com

Dr Kate Poole tel.: +49 30 5331 12545 [email protected]

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.

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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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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.

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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.

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