A Novel Method for‘‘Wet’’SEM · A Novel Method for‘‘Wet’’SEM Iris Barshack MD Juri...
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A Novel Method for ‘‘Wet’’ SEM Iris Barshack MD Juri Kopolovic MD Department of Pathology, The Chain Sheba Medical Center, Tel-Hashomer, A⁄liated to the Tel-Aviv University, Sackler School of Medicine, Israel Yehuda Chowers MD* Department of Gastroenterology, The Chain Sheba Medical Center, Tel-Hashomer, A⁄liated to the Tel-Aviv University, Sackler School of Medicine, Israel Opher Gileadi PhD Anya Vainshtein BA Ory Zik PhD Vered Behar PhD Quantomix Ltd., Rehovot, Israel Progress in the processing of wet tissues, without the need of fixation and complex preparation procedures, may facilitate the microscopic examination of tissues and cells. Microscopic examination of tissues is a central tool in clinical diagnosis as well as in diverse areas of research. The authors present the application of Wet SEM, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). The technique is based on 2 principles. First, samples are imaged in sealed specimen capsules and are separated from the evacuated interior of the electron microscope by a thin, electron-transparent partition membrane that is strong enough to sustain a 1-atm pressure difference. Second, imaging is done in a SEM, based on detection of backscattered electrons, which penetrate a few microns into the specimen and thus give information on the cellular level. Keywords colon, inflammatory bowel disease, microscopy, SEM Progress in the processing of wet tissues, without the need of fixation and complex preparation procedures, may facilitate the microscopic examination of tissues and cells for clinical diagnosis as well as diverse areas of research in the life sciences. Microscopic examin- ation of tissues is a central tool in clinical diagnosis as well as in diverse areas of research. We present here the application of Wet SEM, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). Light microscopy (LM) is performed with thin sections of tissue (several microns), stained with chemicals, antibodies, and=or enzymes to reveal general tissue architecture, to identify normal or abnormal cell types, and to localize specified molecules. Transmission electron microscopy (TEM) requires specially prepared ultrathin sections (typically 50 nm), and reveals a wealth of subcellular information. Each of the aforementioned techniques has limitations: the resolution of light microscopy is limited by diffraction to 0.25 mm; the use of TEM is encumbered by extensive processing of the sample, which may alter FIG. 1 Wet SEM specimen capsule. Received 7 October 2003; accepted 6 November 2003. Address correspondence to Dr. Iris Barshack, Department of Pathology, Sheba Medical Center, Tel-Hashomer, 52621, Israel. E-mail: barshack@ sheba.health.gov.il Ultrastructural Pathology, 28:29–31, 2004 Copyright # Taylor & Francis Inc. ISSN: 0191-3123 print/1521-0758 online DOI: 10.1080/01913120490275222 29
Transcript of A Novel Method for‘‘Wet’’SEM · A Novel Method for‘‘Wet’’SEM Iris Barshack MD Juri...
A Novel Method for ‘‘Wet’’ SEM
Iris Barshack MDJuri Kopolovic MD
Department of Pathology, The Chain ShebaMedical Center,Tel-Hashomer, A⁄liated to the Tel-Aviv University,Sackler School of Medicine, Israel
Yehuda Chowers MD*
Department of Gastroenterology, The Chain Sheba MedicalCenter, Tel-Hashomer, A⁄liated to the Tel-Aviv University,Sackler School of Medicine, Israel
Opher Gileadi PhDAnya Vainshtein BAOry Zik PhDVered Behar PhD
Quantomix Ltd., Rehovot, Israel
Progress in the processing of wet tissues, without the need of fixationand complex preparation procedures, may facilitate the microscopicexamination of tissues and cells. Microscopic examination of tissuesis a central tool in clinical diagnosis as well as in diverse areas ofresearch. The authors present the application of Wet SEM, atechnology for imaging fully hydrated samples at atmosphericpressure in a scanning electron microscope (SEM). The technique isbased on 2 principles. First, samples are imaged in sealed specimencapsules and are separated from the evacuated interior of the electronmicroscope by a thin, electron-transparent partition membrane that isstrong enough to sustain a 1-atm pressure difference. Second,imaging is done in a SEM, based on detection of backscatteredelectrons, which penetrate a few microns into the specimen and thusgive information on the cellular level.
Keywords colon, inflammatory bowel disease, microscopy, SEM
Progress in the processing of wet tissues, without theneed of fixation and complex preparation procedures,may facilitate the microscopic examination of tissuesand cells for clinical diagnosis as well as diverse areasof research in the life sciences. Microscopic examin-ation of tissues is a central tool in clinical diagnosis aswell as in diverse areas of research. We present here theapplication of Wet SEM, a technology for imaging fullyhydrated samples at atmospheric pressure in a scanningelectron microscope (SEM).
Light microscopy (LM) is performed with thinsections of tissue (several microns), stained withchemicals, antibodies, and=or enzymes to revealgeneral tissue architecture, to identify normal orabnormal cell types, and to localize specifiedmolecules. Transmission electron microscopy (TEM)requires specially prepared ultrathin sections (typically50nm), and reveals a wealth of subcellular information.Each of the aforementioned techniques has limitations:the resolution of light microscopy is limited bydiffraction to 0.25mm; the use of TEM is encumberedby extensive processing of the sample, which may alter
FIG. 1 Wet SEM specimen capsule.
Received 7 October 2003; accepted 6 November 2003.
Address correspondence to Dr. Iris Barshack, Department of Pathology,
Sheba Medical Center, Tel-Hashomer, 52621, Israel. E-mail: barshack@
sheba.health.gov.il
Ultrastructural Pathology, 28:29–31, 2004
Copyright # Taylor & Francis Inc.
ISSN: 0191-3123 print/1521-0758 online
DOI: 10.1080/01913120490275222
29
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its structure significantly. Preparation of samples forstandard TEM also requires specific skills and takes atleast a few days to achieve. The very thin slices presenta very limited (and often arbitrary) portion of thesample, necessitating the imaging of multiple serialsections [1, 2]. SEM occupies a more restricted niche,usually limited to surface imaging. It is used toinvestigate surface topography, and imaging of intra-cellular structure by SEM requires specialized pre-paratory techniques such as fracturing and etching. Allthose techniques usually require fixation and complexmethods for preparation of the tissue. In markedcontrast to the above described methods, we present abrief preparation of fixed or unfixed tissues forevaluation by SEM.
Our technique is based on 2 principles. First,samples are imaged in sealed specimen capsules(Figure 1) and are separated from the evacuatedinterior of the electron microscope by a thin, electron-transparent partition membrane that is strong enoughto sustain a 1-atm pressure difference (QuantomiX,Israel). Second, imaging is done in an SEM, based ondetection of backscattered electrons (BSE). This setupresults in 3 unique characteristics:
1. Samples can be imaged in an electron microscope,at resolutions up to 20nm, in a fully wet state,without dehydration or coating.
2. Backscattered electrons result from interactions ofthe scanning electron beam with an internallayer of up to approximately 2mm into the sample;any material present beyond this layer is irrelevantto the imaging process. Consequently, thickspecimens, for example, tissue fragments of afew millimeters thickness, can be imaged withoutthin sectioning, embedding, or freezing.
3. Backscattering efficiency is sensitive to materialdifferences within the sample, allowing contrastmechanisms that are distinct from those operativein other microscopy methods. In particular,biological features may be distinguished withoutstaining, or with heavy metal staining, andgold-linked immunolabels are very clearly resolved.
For the evaluation of the method, we chose themucosal surface of the large bowel, comparing normalcolon to inflamed colon diagnosed as inflammatorybowel disease (IBD). Three cases of normal colonresection procedures and 3 cases of Crohn diseaseaffecting the colon were selected from the pathologyrepository of the Department of Pathology, ShebaMedical Center after taking samples for diagnosticpurposes. A few small fragments of a few millimeterseach were taken from each specimen. Tissue sampleswerewashed several times inwater, incubated for 5min
with 1% tannic acid, and stained with uranyl acetate,0.1%, pH 3.5, for 10min. Samples were placed in thewet SEM specimen capsule and visualized in the SEM.
The SEM was used in a few studies for theunderstanding of the morphology and evolution of IBDlesions throughout the gastrointestinal tract [3^5]. Inlesions such as ‘‘aphthoid’’ ulcers, the SEM demon-strated variety of villous abnormalities in regionsadjacent to ulcers [3]. In addition, the macroscopicallyunaffected mucosa was found to be distorted byirregularities in the normal polygonal units. The indi-vidual absorptive cells were found to lose theirpentagonal^hexagonal cell borders [4]. Moreover,quantitative techniques were used to develop moreobjective criteria for the diagnosis of dysplasia in IBDusing SEM [5]. In addition, the SEM was useful in thestudy of inflammatory processes, such as cell migrationin the lamina propria in samples of IBD [6].
By our method, using wet tissue, en face photo-graphs of colonic Crohn disease show marked irregu-larity of the cell borders with elongation of the cryptorifices and cell contours (Figure 2), similar to whatwas observed in previous studies [4].
This novel method offers a potential source of quickexamination of various tissues. A few preliminarystudies have already been performed in cultures of celllines [7], cytologic material, and human tissue biopsiesof thyroid, colon, and other organs. Tissues such asthyroid and liver are evaluated for quick examination forpurposes that may replace the frozen sections duringoperation. It will be important to evaluate the role thatthis method can play in quick diagnosis of additionalpathological specimens.
REFERENCES1. Rosai J, Rodriguez HA. Application of electron microscopy to the
differential diagnosis of tumors. Am J Clin Pathol.1968;50:535^562.
2. Williams MJ, Uzman BG. Uses and contributions of diagnosticelectron microscopy in surgical pathology: a study of 20 veteransadministration hospitals. Hum Pathol. 1984;15:738^745.
3. Rickerts RR, Carter HW. The ‘‘early’’ ulcerative lesion of Crohn’sdisease: correlative light- and scanning electron-microscopicstudies. J Clin Gastroenterol. 1980;2:11^19.
4. Myllarniemi H, Nickels J. Scanning electron microscopy ofCrohn’s disease and ulcerative colitis of the colon. Virchows ArchA Pathol Anat Histol. 1980;385:343^350.
5. Shields HM, Bates ML, Goldman H, et al. Scanning electronmicroscopic appearance of chronic ulcerative colitis with andwithout dysplasia. Gastroenterology. 1985;89:62^72.
6. McAlindon ME, Gray T, Galvin A, Sewell HF, Podolsky DK,Mahida YR. Differential lamina propria cell migration via basementmembrane pores of inflammatory bowel disease mucosa.Gastroenterology. 1998;115:841^848.
7. Thiberge S, Zik O, and Moses E. (submitted) An apparatus forimaging liquids, cells and other wet samples in the scanningelectron microscope.
A Novel Method for Wet SEM 31
