How to study biological samples by FIB/SEM? - · PDF fileHow to study biological samples by FIB/SEM? ... SEM system for exposing a cell interior of metal storing cells and making a

How to study biological samples by FIB/SEM?

Marziale Milani 1, Damjana Drobne 2 and Francesco Tatti 3 1Materials Science Dept. and Lab. FIB/SEM “Bombay”, University of Milano-Bicocca, Via

Cozzi 53, I-20125 Milano, Italy 2Department of Biology, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia 3 Department of Biology, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia 1. Introduction

The focused ion beam (FIB)/scanning electron microscope (SEM) is a scanning microscope with an electron column and an ion column embedded in the same specimen chamber; both beams are aiming at the same point on the specimen surface. The FIB, generated by a Ga Liquid Metal Ion Source (LMIS), impacts the sample normal to the surface and can be focused to a spot as small as few nanometres. The FIB can be rastered in a user defined pattern over larger areas to selectively sputter and mill away the surface. By flooding the exposed surface with specific gases, during ion or electron bombardment new material can be deposited or some specific face can be removed faster (enhanced etching). The combination of both unselective ion milling and selective etching using reactive species creates a very powerful sample preparation tool. The focused ion beam operated at low beam currents is used for imaging, and high beam currents are used for site specific in situ sputtering or milling. The signal from the sputtered secondary ions or secondary electrons is collected to form an image. The FIB/SEM investigation can be applied on bulk samples, prepared for conventional SEM or on bulk resin-embedded specimens prepared for conventional TEM, at any chosen site.

Modern Research and Educational Topics in Microscopy. A. Méndez-Vilas and J. Díaz (Eds.) ©FORMATEX 2007

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Elementary interactions and ion-beam driven processes are summarized in the preceding drawings: ion beam – sample primary interactions (surface, sub-surface and bulk) (above), ion beam driven operations (below). The focused ion beam system for microscopy and nanomachining is widely utilized in semiconductor technology. Up to now, the FIB/SEM was applied on a variety of biological samples, however there are still many questions left opened which have to be answered before FIB/SEM will be widely applied in structural research in life sciences. The preparation of biological specimens for scanning or transmission electron microscopy starts with primary fixation, which is followed by washing, secondary fixation and dehydration. For SEM, it is continued by drying, mounting on a metal specimen stub, and coating a specimen with a thin, electrically conductive layer. For TEM, it is proceeded by infiltration of specimen with transitional solvent, infiltration with resin, embedding, curing and cutting. Now, although some variations exist in sample preparation among laboratories, most techniques are standardized and may be found in specialized textbooks on electron microscopy methodology. Here the application of FIB/SEM on yeast cells and an epithelial tissue is presented. The advantages of FIB/SEM over conventional SEM or TEM are discussed and some future perspectives of FIB/SEM for biological samples are exposed. The aim is to encourage the SEM microscopists to broaden the applicability of SEM toward subsurface imaging and to use the FIB/SEM to prepare biological samples for analytical microscopy.

2. The FIB/SEM application on unprepared cells

Introduction: Here the investigation on yeast cells is presented, which was performed with the aim to demonstrate the potentiality of the FIB/SEM technique. Major attention was devoted to the feasibility and quality evaluation of different operations on air dried yeast cells. Aim: To show that informative high magnification imaging and FIB sectioning can be performed in biological systems in the absence of chemical fixation, thus speeding up information acquisition, a feature of relevance in biomedical analysis. Materials and Methods: Liophylized Saccharomyces cerevisiae cells were hydrated in deionized water (and glucose) at room temperature. A drop of the suspension at selected cell concentration was deposited onto a silicon dice, dried at room temperature, inserted into the working chamber and brought to high vacuum. Results and discussion: The results of this work reveal that yeast cells sustain very high vacuum without visible damage, they sustain ion beam milling and imaging and moreover, ion milling revealed many internal features of cells at the submicron scale (Figs. 1 a-c). This is a proof that the FIB/SEM operating in high vacuum environment can be applied also on dried biological material, that is not

©FORMATEX 2007Modern Research and Educational Topics in Microscopy. A. Méndez-Vilas and J. Díaz (Eds.)

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prepared following standard procedure. Moreover it opens the path to the investigation on physical basis of cell response to high vacuum conditions.

Fig.1 a-b. Secondary electron images of FIB milled yeast cells obtained from a primary ion beam. Internal structures, membranes and morphological stability and integrity can be assessed; moreover the regions underlying the bud scare, i.e. the site were cell division, can be observed.

Fig.1 c FIB sectioning of a yeast cell that has been incubated for months in a reactor with glucose and metabolites

3. Linking gross morphology to cell ultrastrucure

Introduction: Currently there are numerous microscopy methods available for providing subcellular structural information at resolution down to 1–2 nm. However still, simultaneous imaging of intracellular


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structures and tissue gross morphology remains a challenge in structural investigation of biological samples. The FIB/SEM offers attractive possibility to expand the sample surface investigation by the subsurface structural research at any selected location. The introduction of FIB for in situ exposing subsurface structures has a potential to advance new structural information on biological samples which is missed while switching from surface investigation performed by conventional SEM to TEM ultrastructural research. Aim: To present the application of FIB/SEM system for simultaneous research of the digestive gland epithelium gross morphology and the cell ultrastructure. Materials and Methods: Terrestrial isopods, Porcellio scaber (Latreille 1809) (, Crustacea), were collected under concrete blocks and pieces of decaying wood (Ljubljana, Slovenia). Digestive gland tubes were isolated and fixed in 0.1 % glutaraldehyde and 0.4 % paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2.5 hours at room temperature. The chemically fixed samples were followed by OTOTO conductively staining (OsO4 / thiocarbohydrazide / OsO4 / thiocarbohydrazide / OsO4) and dehydration, or chemically fixed samples were directly dehydrated. The conductive staining introduced metals into the sample in order to increase its conductivity. After dehydration in a graded series of ethanol, the samples were dried at the critical point (Balzers Critical Point Dryer 030) and gold coated (Sputter coater SCD 050, BAL-TEC, Germany). The samples were fixed on brass holders with silver paint (High purity silver paint, SPI), mounted on the sample holder into the specimen chamber (5-axis eucentric stage) of a Dual Beam system for FIB / SEM operation (FEI Strata DB 235 M). The rough milling conditions to open a trench employed ion currents of 5 to 7 nA, at 30 kV. Lower beam currents of 100 to 300 pA were used to polish the cross section. Spot size in the case of rough milling was approximately 150–100 nm of diameter, and for polishing it ranged from 20 nm to 35 nm of diameter. Secondary electron detectors were: Everhardt Thornely Detector (ETD), Continuous Dynode Electron Multiplier (CDEM) and Back-Scattered Electron, secondary ion detector was a CDEM. Dwell time for milling was 1 µs and the overlap was 50 %. The SEM imaging was performed by means of the FEG electron column available in the same system with a resolution of 1 nm at 30 kV. The spot size in the case of SEM was up to 0.5 nm in diameter. Results and discussion: It is observed a connection between digestive gland gross morphological characteristics (extruding and non-extruding phase of a cell) and cell ultrastructure (Figs 2 a-c, 3 a, b). How the structural variability of lipid bodies and laminated cellular structures is related to the function of hepatopancreatic cells remains to be investigated. However, the FIB/SEM appears to be a very suitable tool for such type of structural research.


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Fig. 2 a-c. Secondary electron images of OTOTO prepared digestive gland tube. a) Mechanically opened digestive gland tube. b) A cell is extruding its content into the gland lumen. Lipid droplets are spherical of different size. c) FIB exposed apical part of a cell and the extruding material. d) Interior of FIB exposed cell.

Fig. 3 a, b. Secondary electron images of OTOTO prepared digestive gland tube. a) FIB milled trench on the apical part of a cell that is covered by microvilli. b) Inner part of FIB exposed cell.


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Fig. 4 a - c. Sample is chemically fixed, but post-fixation and conductive staining are omitted. a) FIB milled trench on the apical part of a cell that is then imaged by SE or BSE. b) Secondary electron image of FIB exposed cell interior. Bright round shaped regions are composed of high atomic numbered elements. c) Backscattered image of the same FIB exposed region show that high atomic numbered elements appear significantly brighter on BSE image when compared to SE image. The FIB/SEM investigation of biological cells and tissue was conducted also by different authors. The FIB/SEM was preferably used on embedded biological samples, but we want to encourage the use the FIB/SEM also on biological samples prepared for conventional SEM. The aim of such approach is to allow to establish a link between larger scale tissue morphology and cellular and subcellular structures. We believe that the ability to reveal specific intracellular structural details and to link them to the gross morphology of the tissue or organ is at the moment among most promising and beneficial applications of FIB/SEM in life sciences. This is of particular interest when cells or cellular inclusions have a dynamic nature due to normal, stress or pathologic conditions.

4. Biological sample preparation for backscattered (BSE) imaging

Introduction: Secondary electron (SE) imaging is the main method to understand structural characteristics of specimens (with limitation to depths ranging from 5 to 50 nm depending on the energy of primary electrons). However, the backscattered (BSE) image probably reflects the true situation more than the secondary electron image, which contains more than just morphological information. BSE are


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elastically scattered electrons of high energy. They may escape from great depths in the specimen and exit some distance from the entry point of the beam electrons. If one quantifies the yield of secondary and backscattered electrons in various atomic elements, it will be observed that the yield of both secondary and backscattered electrons increase as the atomic number increases; this is because large atomic numbered elements have more orbital electrons available to interact with beam electrons. Interestingly, the ratio of backscattered electrons to secondary ones increases with the atomic number. A main hindrance of BSE imaging of biological samples is that a flat surface is not (or very rarely) the case in biological samples: actually the specimen should be flat in order to reduce interfering topographic contrast. Aim: To use of FIB/SEM system for exposing a cell interior of metal storing cells and making a flat surface of biological sample suitable for BSE imaging. Materials and Methods: The sample preparation was similar as described above. Differences in sample preparation were related to conductive staining and coating. When preparing a sample for BSE imaging, no metals were introduced during sample preparation. Here, the samples were only glutaraldehyde and paraformaldehyde fixed and then dehydrated. Secondary fixation and conductive staining were omitted. After critical point drying, the samples were coated with carbon and not with gold. Results and discussion: Secondary electrons are used to generate an image based on topographic contrast, whereas backscattered electrons are not normally used to study topographies. In BSE imaging contrast is based on detecting areas of different atomic numbered elements (Z-contrast). There are two characteristics of biological samples: first, most of the elements present in specimens are of relatively low atomic number and yield few BSE; and second, biological specimens are not flat. The results confirmed that the higher atomic numbered elements appear slightly brighter than lower atomic numbered elements in a secondary electron detector (Figs 3 a, b) and significantly brighter in a backscattered electron detector (Fig 3 c). In this work the evidence that the FIB/SEM can be used to make a flat surface for both SE and BSE imaging is provided. The cells investigated in our study store metals in metal granules, which can easily be identified by BSE. The BSE imaging can be used when investigating those biological samples where not only low atomic numbered elements are present, but also high atomic numbered elements are expected (metal storing tissue, skeleton etc.).

5. Conclusion and future perspectives of FIB/SEM in life sciences

There is a general belief that scanning electron microscopes are not suited for subsurface investigations and researchers should switch from using SEM to TEM. This however may result in losing important information. Perhaps the upgraded SEM that allows in situ controlled and precise sample manipulation is probably the optimal choice for many imaging needs in biology. Besides, the FIB/SEM system appears to be a suitable tool to produce flat, arbitrarily oriented and shaped surfaces of a biological samples for BSE and also for other analytical electron microscopy techniques. Therefore FIB/SEM offers a range of new possibilities in electron microscopy of biological samples that are worthwhile to be tested.

References

[1] Bozzola J J, Russell L D. Electron Microscopy: Principles and Techniques for Biologists. Jones and Bartlett Publishers, 1998

[2] Drobne D, Milani M, Ballerini M, Zrimec A, Berden Zrimec M, Tatti F, Drašlar K. Focused ion beam for microscopy and in situ sample preparation: application on a crustacean digestive system. J Biomed Opt 9:1238-1243, 2004

[3] Drobne D, Milani M, Zrimec A, Lešer V, Berden Zrimec M. Electron and ion imaging of gland cells using the FIB/SEM system. J Microsc 219:29-35, 2005


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[4] Hayat M A. Principles and Techniques of Electron Microscopy: Biological Applications. Cambridge University Press, 2000

[5] Milani M, Drobne D. Focused ion beam manipulation and ultramicroscopy of unprepared cells. SCANNING 28:148-154, 2006

[6] Milani M., Drobne D, Drobne S, Tatti F. An atlas of FIB/SEM in soft materials and life sciences. Aracne Editrice, 2006

[7] Milani M., Simone S, Tatti F. FIB/SEM for soft matter and life sciences. GIT Imaging and Microscopy 3:38-4, 2006

[8] Milani M., Drobne D, Tatti F. FIB/SEM for biological data mining. Focused Ion Beam (FIB) cell sectioning for X-ray analysis - Part I&II GIT Imaging and Microscopy 2007. In press

[9] Hopkin S P. Ecophysiology of Metals in Terrestrial Invertebrates (Pollution Monitoring Series). Elsevier, 1989


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How to study biological samples by FIB/SEM? - · PDF fileHow to study biological samples by FIB/SEM? ... SEM system for exposing a cell interior of metal storing cells and making a