By AZoNano Editors
Table of Contents
Atomic Force Microscopy
Membranes and Membrane Proteins
The BioScope Catalyst along with optical microscopy provides life science researchers an opportunity to study the biological species on a broad range of size scales. The BioScope Catalyst has advanced engineering and mechanical stability, hence high resolution three-dimensional images of single biomolecules and biomolecular complex structures can be obtained. The imaging conducted using the BioScope Catalyst provides data at single cell level by characterization into nucleic acids and proteins and membranes, etc.
Atomic Force Microscopy
The Atomic Force Microscopy (AFM) technique provides high-resolution imaging at nanoscale resolution of a three- dimensional structure without staining or coating the sample. Thus, AFM scores over many other techniques by allowing studies of biomolecules and parameters of biological processes at the process site itself and taking real time readings. The BioScope Catalyst AFM can be used with light microscopy techniques to provide optically guided navigation of the probe and creating clear images correlating the AFM and optical image data. Live cells were studied by using the BioScope Catalyst along with the largest closed loop, X-Y scan range; the results were highly accurate. The results are illustrated in Figure 1.
Figure 1. (A) Registered image overlay of a two-channel confocal laser scanning fluorescence microscopy image and AFM topography image of fibroblast cells labelled with Alexa Fluo 546 Phalloidin (red) and DAPI (blue). The BioScope Catalyst MIRO software enables registration of an optical field of view to the AFM scan area. Optical images can then be used to navigate the AFM probe to a region of interest to perform high resolution AFM imaging and/or highly sensitive force measurements. (B) Region of interest obtained from the image overlay showing correlated AFM and fluorescence data channels. Confocal fluorescence images were obtained with a Leica SP5 confocal system and using a 40x oil immersion objective. AFM images were obtained with a BioScope Catalyst operated in contact mode in buffer solution using MLCT AFM probes (k ~0.01N/m).
The advanced mechanical stability and engineering of the BioScope Catalyst makes it suitable to study biomolecular species also. It provides consistent results even when used with an inverted optical microscope, as shown in Figure 2.
Figure 2. A 1ìm Phase Image of a C60H122 alkane. The C60H122 is spincast onto an HOPG substrate with the resulting ultra-thin alkane layer exhibiting a lamellar structure ~7.5nm in width and ~0.4nm in height. Images were acquired on a BioScope Catalyst AFM operated in Tapping Mode using FESP AFM probes (k ~3N/m).
Studying the structure and nature of deoxyribonucleic acid (DNA) is vital in understanding the stored genetic code which is extremely helpful in genetic-related disease research. AFM-based imaging is capable of providing intermolecular interaction data of the DNA in real time by creating a near physiological environment. For the imaging, the negative DNA strands are adsorbed by the freshly cut mica surface which is either charged by divalent cations (Ni++ or Mg++) or chemically altered by positively charged silane (APS-mica). The AFM images of the DNA molecules are shown in Figure 3. The small volume flow cell of the BioScope Catalyst provides a conducive environment for observing the molecules needing only small quantities of sample and inlet and outlet ports facilitate easy fluid exchange. The Peak Force Tapping technique along with ScanAsyst has further improved the imaging quality and providing consistent results with the Catalyst. Figure 3 shows data obtained using the PF Tapping method on the BioScope Catalyst.
Figure 3. Three-dimensional topography image of pUC plasmid DNA adsorbed onto a mica substrate. The individual DNA strands are clearly visible against the mica background. Images were acquired on a BioScope Catalyst AFM operated in PeakForce Tapping in buffer solution using ScanAsyst Fluid+ AFM probes (k ~0.7N/m). Image XY-Scale = 2ìm.
Protein molecules are important for regulating the biological processes, the direct observation of which throws light on the relation between the structure and function of biomolecules. Viruses essentially exist inside a protein shell called capsid, this covers the viral DNA. Once a host is found the virus DNA is released and it multiplies to spread the infection. Viruses are classified based on the capsid structure which can be studied in detail through AFM imaging. Figure 4B shows the structure of Herpes Simplex Virus obtained by AFM imaging done with the BioScope Catalyst.
Figure 4. (A) Transmission electron micrograph of a Herpes Simplex Virus capsid. Image courtesy of Wouter Roos, Vrije Universiteit, Amsterdam, Netherlands (Reprinted with permission. Source: Roos et al., Proc. Natl. Acad. Sci. USA, 2009, Vol. 106, 9673-78) (B) A 250nm AFM Topography image of a single herpes simplex virus capsid. The arrangement of protein molecules as 3-dimensional subunits on the surface of the capsid, known as capsomeres, is clearly visible in the AFM image. AFM images were obtained on the BioScope Catalyst operated in PeakForce Tapping mode in buffer conditions and using ScanAsyst Fluid+ AFM probes (k ~0.7N/m). Sample courtesy of Wouter Roos and Gijs Wuite, Vrije Universiteit, Amsterdam, Netherlands.
AFM imaging is particularly useful in the study of viruses exhibiting abnormal assembly or aggregation. This provides vital information for research related to Alzheimer’s and Parkinson’s disease. Figure 5 shows how AFM imaging results with the BioScope Catalyst on A-â fibers relating to Alzheimer’s disease, providing details on the structure and nanomechanical properties of the fiber. Researchers are specifically looking for the interaction of the amyloid proteins (shown in Figure 5C) in living cells.
Figure 5. PeakForce QNM images of amyloid fibers adsorbed onto a freshly cleaved mica surface. (A) Topography images reveal some of the fibers to have a twisted structure (blue arrows) while others do not (red arrows). (B) The Modulus data and the (C) deformation data channels are obtained simultaneously to the topography image. These images indicate the amyloid to have a lower modulus (darker color scale) and correspondingly, a higher degree of deformation (lighter color scale) than the underlying mica substrate. It is also observed that the twisted amyloid fibers have a slightly lower modulus value as compared to those fibers that are not twisted (i.e. twisted fibers appear slight darker than the straight fibers in the modulus image). Images were obtained on a BioScope Catalyst operated in PeakForce Tapping mode using ScanAsyst AFM probes (k ~0.4N/m). Sample courtesy of Xingfei Zhou, Ningbo University, China.
Figure 6 shows how the perfusion stage incubator (PSI) with the BioScope Catalyst provides the right environment for experiments. The BioScope Catalyst AFM, the PSI and the MIRO software provide comprehensive data for amyloid fiber analysis.
Figure 6. Setup of the BioScope Catalyst Perfusion Stage Incubator (PSI). (1) The PSI supports standard glass bottom Petri dishes for compatibility with high NA objectives. (2) The flow diffuser directs liquid flow in laminar fashion across the sample area, ensuring even fluid exchange and isolating noise from the liquid inlet and outlet. (3) The perfusion clamp stabilizes the Petri dish and contained stainless steel inlet and outlet tubes that are in thermal contact with the heating stage to preheat the incoming liquid and gas. (4) A silicone baffle seals between the Petri dish and probe holder, reducing evaporation and allowing control of the gas space above the liquid. (5) The specialized PSI probe holder contains a temperature sensor for local monitoring of temperature. The right-side image shows the PSI fully assembled together with the sample heating stage on the BioScope Catalyst AFM.
Membranes and Membrane Proteins
Cell membranes encapsulate the cells acting as a separating layer between cells. They have many functions such as keeping the cytoskeleton in place, providing shape to the cells and facilitate material transportation from and into the cells. The study of membranes is a bit complicated due to the hydrophobic domains present throughout the membrane. The challenge lies in studying the membrane proteins without disturbing the native membrane environment. AFM addresses this challenge by which studies can be conducted on the fluid under conditions similar to physiological conditions. AFM can provide images of live cells giving high-resolution images of cell membranes and the structure. Figure 7A shows the AFM images of bacterial membrane on a mica substrate. A two-dimensional crystalline lattice structure called the S-Layer is shown; this layer gives mechanical and chemical protection to the cell. The AFM image also exhibits small periodicities in the S layer lattice, which is useful for biometric and nanotechnological applications.
Figure 7. (A) AFM phase image of bacterial S-layers from E. coli. The bacterial membranes were excised from the cells and the membrane patches immobilized onto a freshly cleaved mica surface. The lattice pattern formed by the S -layer proteins is clearly evident in the phase image and is observed to have a periodicity of ~18nm. (B) High-resolution image of the S-layer lattice periodicity observed on a single membrane patch. Images were obtained on a BioScope Catalyst operated in TappingMode in buffer conditions using SNL AFM probes (k ~0.32N/m). Sample courtesy of Hans Oberleithner, Institute for Physiology II, University of Muenster, Germany.
AFM is useful in providing high-resolution images of molecules at the single cell level in biomolecular research. The BioScope Catalyst along with the AFM provides an opportunity to study DNA, proteins and cell membranes.
Bruker Nano Surfaces provides Atomic Force Microscope/Scanning Probe Microscope (AFM/SPM) products that stand out from other commercially available systems for their robust design and ease-of-use, whilst maintaining the highest resolution. The NANOS measuring head, which is part of all our instruments, employs a unique fiber-optic interferometer for measuring the cantilever deflection, which makes the setup so compact that it is no larger than a standard research microscope objective.
This information has been sourced, reviewed and adapted from materials provided by Bruker Nano Surfaces.
For more information on this source please visit Bruker Nano Surfaces.