Name: atomic force microscopy for surface mapping
Uploaded: 2023-04-30T12:00:37.000Z
Duration: 1 min 8 s
Channel: JoVE

מיקרוסקופ כוח אטומי או AFM יוצר תמונה באמצעות ננו-גשושית כדי לספק מידע טופוגרפי על דגימה ברזולוציה ננומטרית.
בעוד שמיקרוסקופ אופטי יכול להגדיל עד 1000X, פוטנציאל ההגדלה של AFM הוא עד 1,000,000X.
AFM יכול ליצור תמונות של דגימות קבועות וחיות, מה שמאפשר לו ללכוד תהליכים תאיים דינמיים כגון דינמיקה של אקטין (actin dynamics).
ננו-גשושית AFM מחוברת לקצה של מיכל גמיש, ויחד הם סורקים את הדגימה. הגשושית עוקבת אחר קווי המתאר של משטח הדגימה, נעה למעלה ולמטה, מה שמחליף את הקנטילבר.
בסוג אחד של AFM, קרן לייזר מכוונת אל הקנטיליבר, ותוך כדי תנועה, גם השתקפות הלייזר נעה.
גלאי אור רגיש למיקום מתעד את הסטייה של קרן הלייזר.
הנתונים נשלחים למחשב שבו התוכנה יכולה לעבד אותם כדי ליצור תמונה תלת מימדית של משטח הדגימה.

<ol>
	<li>קאסוסו, איגנסיו, פליקס ריקו וסימון שירינג. "AFM ביולוגי: מאיפה אנחנו באים – איפה אנחנו נמצאים – לאן אנחנו יכולים ללכת". כתב העת להכרה מולקולרית 24.3 (2011): 406-413.</li>
	<li>גרסיה, ריקרדו ורובן פרז. "שיטות מיקרוסקופ כוח אטומי דינמי". דוחות מדע פני השטח 47.6-8 (2002): 197-301.</li>
</ol>

מיקרוסקופיה של כוח אטומי

Atomic Force Microscopy

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מיקרוסקופיה של כוח אטומי (AFM) היא סוג של מיקרוסקופ בדיקה סריקה שיכולה לנתח פרטים טופוגרפיים של דגימות שונות כמו קרמיקה, זכוכית, פולימרים ודגימות ביולוגיות. AFM מציעה רזולוציה גבוהה פי 1000 ממערכת ההדמיה האופטית. תמונות שנוצרו מ-AFM הן פרופילי משטח תלת מימדיים, המציעים יתרון על פני התמונות הדו-ממדיות השטוחות מטכניקות הדמיה אחרות.

חקירת AFM

הגשושית נחשבת ללב של כל מערך AFM וכולל את המכלול והחיישן. גשושיות הן החלק הנפוץ ביותר המוחלף בסוג זה של מיקרוסקופ מכיוון שהאינטראקציה המתמדת עם הדגימות שוחקת את הקצה. לכן, בחירת החומר לבדיקה תלויה בתכונות הדגימה. בדיקות סיליקון, המשמשות לניתוח דגימות קשות, הן נוקשות וחדות יותר מבדיקות סיליקון ניטריד, שמתאימות יותר לסריקת דגימות רכות יותר. החיישנים החדים הללו מיוצרים באמצעות תחריט אלקטרוכימי או ננו-צינוריות פחמן לניתוח דיוק גבוה יותר.

מצבי הדמיה של AFM

ב-AFM, טופוגרפיה של פני השטח נחקרת באמצעות האינטראקציה בין קצה הבדיקה למשטח הדגימה. ישנם שני מצבי הדמיה עיקריים - מצב סטטי, המכונה גם מצב מגע, ומצב דינמי.

במצב סטטי או מגע, חיישן הגשושית נמצא במגע רציף עם משטח הדגימה. כשהחיישן נגרר על פני השטח, כוחות הדחייה בין הדגימה לקצה מביאים לכיפוף המכלול, שמתועד. כל משטח הדגימה נסרק קדימה ואחורה בשני צירי x ו-y, הנקראת סריקת רסטר, בעוד שהתנועה האנכית של המכלול מתעדת את ציר ה-Z, וכך מייצרת תמונה תלת מימדית.

במצב הדינמי, הגשושית מתנדנדת ממש מעל פני הדגימה, מתקרבת למשטח אך לא נוגעת במשטח. כוחות משיכה ודוחה קובעים את השונות במרחק בין החיישן לדגימה, ומשפיעים על המשרעת של תנודת המכלול. משוב זה נרשם כדי לבנות את הטופוגרפיה פני השטח של הדגימה.
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מיקרוסקופ כוח אטומי (AFM) הוא סוג של מיקרוסקופ בדיקה סריקה שיכול לנתח פרטים טופוגרפיים של דגימות שונות כמו קרמיקה, זכוכית, ...

Education

JoVE Core

Cell Biology

ביולוגיה של התא

Unit 1

יחידה 1

Visualizing Cells, Tissues, and Molecules

ויזואליזציה של תאים, רקמות ומולקולות

KEY TERMS AND CONCEPTS

מונחים ומושגים מרכזיים

atomic force microscopy for surface mapping

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images.

The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends on the sample being scanned. Silicon probes are stiffer and sharper, which makes them useful for hard samples. Silicon nitride probes are better for softer samples.

AFM tips can be made with electrochemical etching or with carbon nanotubes to improve accuracy. The microscope reads surface topography by tracking how the probe tip interacts with the sample surface. This interaction is recorded as the probe scans across the surface.

AFM uses two main imaging modes. In static mode, also called contact mode, the tip stays in continuous contact with the surface. As it drags across the sample, repulsive forces bend the cantilever, and that bending is recorded. The sample is scanned back and forth in the x- and y-axes, which is called raster scanning, while the vertical motion of the cantilever records the z-axis and builds a 3D image.

In dynamic mode, the probe oscillates just above the sample surface. It comes close to the surface without touching it. Attractive and repulsive forces change the distance between the tip and the sample and affect the oscillation amplitude. That feedback is then used to construct the surface topography.

Atomic Force Microscopy for Surface Mapping

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Microscope Slide Prep and Staining

Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures. A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes...

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Fluorescent Antibody Staining in Cells

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Antibody Staining in Cells and Tissues

Immunocytochemistry (ICC) and immunohistochemistry (IHC) use antibodies to detect specific proteins or antigens in samples. ICC focuses on individual cells, such as blood cells and stem cells. IHC is used for tissue samples. These methods were first published by Albert Coons in 1941, when they were used to detect pneumococcal antigen in mouse tissue sections from mice infected with Pneumococcus. Both techniques can produce either a colored signal or a fluorescent signal. Enzyme-conjugated...

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Confocal Microscopy for Sharp Cell Images

Confocal microscopy is an advanced imaging method used to capture sharp fluorescence images. Its main advantage is that it blocks out-of-focus light from the sample with pinholes. That helps produce high-resolution images with strong contrast. Unlike a standard optical microscope, a confocal microscope uses a focused laser beam to scan the sample surface at different z-planes. This makes it especially useful for thick specimens, such as biofilms. These samples can often be examined alive and...

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Fluorescent Methods for Protein Movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

total internal reflection fluorescence microscopy

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Surface-Only Imaging with TIRF Microscopy

Total internal reflection fluorescence microscopy, or TIRF, is a microscope method used to view fluorophores near a solid surface with a higher refractive index, such as a glass coverslip. It is useful when scientists want to study signals close to the cell surface. TIRF works when light from a medium with a lower refractive index, such as air, hits the glass coverslip at a critical angle. Instead of entering the glass, the light undergoes total internal reflection. This reflection creates an...

atomic force microscopy

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images. The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends...

super-resolution fluorescence microscopy

Sharper Cell Images with Fluorescence Microscopy

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Electron Microscopy: TEM vs SEM

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scanning electron microscopy

How SEM Reveals Surface Details

A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

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preparation of samples for electron microscopy

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Electron Microscopy Sample Prep Methods

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immunogold electron microscopy

Immunogold Labeling in Electron Microscopy

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions. The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins...

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Cryo-EM for Native Biomolecule Imaging

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3D Imaging with Electron Microscopy

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key terms and concepts

Learn how atomic force microscopy maps surfaces, why probe choice matters, and how contact and dynamic modes build detailed 3D images of samples.

Atomic Force Microscopy for Surface Mapping | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Atomic Force Microscopy for Surface Mapping

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