Focused ion beam for microscopy and in situ sample preparation: application on a crustacean digestive system

Drobne, Damjana; Milani, M.; Ballerini, Monica; Zrimec, Alexis; Berden-Zrimec, Maja; Tatti, Francesco; Drašlar, Kazimir

doi:10.1117/1.1803846

Cited by 22 publications

(22 citation statements)

References 19 publications

(20 reference statements)

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“…The main advantage of FIB-SEM imaging is permitting faster visualization of serial ultra-thin sections, which optimizes the adjustments needed for identification of the target structures (23,24). As a result, we confirmed that chondrocytes in the proliferative and the hypertrophic zones extend their short cytoplasmic processes to the cartilaginous fibrils that parallel the longitudinal axis of the growth plate, and that vesicular structures are present in the intercolumnar septa -even at the proliferative zone (Figs.…”

Section: Discussionsupporting

confidence: 70%

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

Hasegawa

Endo

Tsuchiya

et al. 2017

Journal of Oral Biosciences

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Objectives: The aim of this study is a biological application of focused ion beam-scanning electron microscopy (FIB-SEM) to demonstrate serial sectional images of skeletal tissues, here presenting the ultrastructure of 1) cartilaginous extracellular fibrils and 2) osteoblastic cytoplasmic processes.Methods: Seven weeks-old female wild-type mice were fixed with half-Karnovsky solution and subsequent OsO4, and the tibiae were extracted for block staining prior to observation under transmission electron microscope (TEM) and FIB-SEM.Results: TEM showed the fine fibrillar, but somewhat amorphous ultrastructure of the intercolumnar septa in the growth plate cartilage. Alternatively, FIB-SEM revealed bundles of stout fibrils at regular intervals paralleling the septa's longitudinal axis, as well as vesicular structures embedded in the cartilaginous matrix of the proliferative zone. In the primary trabeculae, both TEM and FIB-SEM showed several osteoblastic cytoplasmic processes on the osteoid, with numbers higher than those seen in the bone matrix. FIB-SEM revealed the agglomeration of cytoplasmic processes beneath the osteoblasts, which formed a tubular continuum extending from those cells. Based on these findings, we postulated that osteoblasts not only extend their cytoplasmic processes through to the bone matrix, but also stack these cell processes on the osteoid of the primary trabeculae. Conclusion:Taken together, it is likely that FIB-SEM imaging strategy on serial sections may successfully deliver new insights on the ultrastructure of cartilage and bone tissues. words

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Section: Discussionsupporting

confidence: 70%

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

Hasegawa

Endo

Tsuchiya

et al. 2017

Journal of Oral Biosciences

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“…Other investigators have developed protocols to study conventionally prepared biological samples with a FIB/SEM system (Drobne et al 2004(Drobne et al , 2005aHing et al 2003;Milani et al 2004a;Moulders 2003;Young 1993). Since the FIB/SEM system allows in situ observation of ultrastructural features, we questioned whether it is possible to observe unprepared, in situ FIB milled cells by an FIB/SEM system.…”

Section: Introductionmentioning

confidence: 97%

Focused ion beam manipulation and ultramicroscopy of unprepared cells

Milani

Drobne

2006

Scanning

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Summary:The focused ion beam (FIB) technique of nanomachining combined with simultaneous scanning electron microscopy (SEM) was used for submicron manipulation and imaging of unprepared (fresh) cells to demonstrate the potentiality of the FIB/SEM technique for ultramicroscopic studies. Sectioning at the nanoscale level was successfully performed by means of ion beamdriven milling operations that reveal the ultrastructure of fresh yeast cells. The FIB/SEM has many advantages over other ultramicroscopy techniques already applied for unprepared/fresh biological samples.

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“…The low ion beam current polishing (typically 100 to 300 pA) can be used to reduce the damaged layer (Drobne et al, 2004(Drobne et al, , 2005aIshitani et al, 1995). Otherwise, not much is known about the surface damage induced by FIB.…”

Section: Introductionmentioning

confidence: 97%

“…The FIB is widely used in semiconductor industry and material sciences, and most recently it has entered into life sciences (Ballerini et al, 1997;Drobne et al, 2004Drobne et al, , 2005aIshitani et al, 1995;Milani et al, 2006a,b).…”

Section: Introductionmentioning

confidence: 98%

“…Recently, the FIB/SEM system proved the applicability for site-specific exposing of subsurface structures of biological samples prepared either for conventional scanning electron microscopy (Drobne et al, 2004(Drobne et al, , 2005a, and of unprepared cells (Milani and Drobne, 2006). A challenge of the present time remains to differentiate the true structure from artifacts and to control them and to assess the potential of FIB/SEM in life sciences.…”

Section: Introductionmentioning

confidence: 98%

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Surface damage induced by FIB milling and imaging of biological samples is controllable

Drobne

Milani

Lešer

et al. 2007

Microscopy Res & Technique

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Focused ion beam (FIB) techniques are among the most important tools for the nanostructuring of surfaces. We used the FIB/SEM (scanning electron microscope) for milling and imaging of digestive gland cells. The aim of our study was to document the interactions of FIB with the surface of the biological sample during FIB investigation, to identify the classes of artifacts, and to test procedures that could induce the quality of FIB milled sections by reducing the artifacts. The digestive gland cells were prepared for conventional SEM. During FIB/SEM operation we induced and enhanced artifacts. The results show that FIB operation on biological tissue affected the area of the sample where ion beam was rastering. We describe the FIB-induced surface major artifacts as a melting-like effect, sweating-like effect, morphological deformations, and gallium (Ga(+)) implantation. The FIB induced surface artifacts caused by incident Ga(+) ions were reduced by the application of a protective platinum strip on the surface exposed to the beam and by a suitable selection of operation protocol. We recommend the same sample preparation methods, FIB protocol for milling and imaging to be used also for other biological samples.

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Focused ion beam for microscopy and in situ sample preparation: application on a crustacean digestive system

Cited by 22 publications

References 19 publications

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

Focused ion beam manipulation and ultramicroscopy of unprepared cells

Surface damage induced by FIB milling and imaging of biological samples is controllable

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