Why does numerical aperture setting matter for IRM imaging of microtubules?

Setting the aperture diaphragm to about two-thirds of the objective's numerical aperture optimizes the signal-to-background noise ratio for microtubule visualization. This adjustment balances illumination intensity and contrast, which is critical for detecting label-free signals at high frame rates. Proper NA setting ensures reliable detection of microtubule dynamics without excessive background.

How does background subtraction improve quantitative measurement of microtubule length in IRM?

Background subtraction enhances contrast by removing uniform illumination and optical artifacts, allowing clearer delineation of microtubule edges. This improves precision in length measurement up to 20 nm, as demonstrated in the protocol. Averaging multiple background-subtracted images further reduces noise for accurate dynamic tracking.

What role does frame rate play in capturing microtubule growth and shrinkage events?

A frame rate of 10 Hz enables monitoring of microtubule dynamics with sufficient temporal resolution to capture growth and shrinkage events. For shrinkage, imaging at 100 frames per second is used to resolve rapid depolymerization kinetics. The frame rate is limited only by the camera, supporting scalable acquisition across experimental conditions.

Why is surface blocking with Poloxamer 407 necessary before microtubule imaging?

Blocking with 1% Poloxamer 407 in filtered BRB80 prevents non-specific binding of microtubules to the glass surface, ensuring specific adhesion via anti-rhodamine antibody. This step reduces background noise and improves signal-to-noise ratio during imaging. Proper blocking is essential for reproducible binding and accurate quantification of microtubule dynamics.

How does IRM compatibility with fluorescence imaging support target validation studies?

IRM can be combined with fluorescence imaging to study microtubule-binding proteins and their effects on dynamics without label interference. This correlative approach enables validation of targets by linking structural changes (via IRM) with molecular interactions (via fluorescence). The technique reduces photodamage, allowing extended co-imaging for mechanistic studies.

Implementacja mikroskopii interferencyjnej odbiciowej do bezznacznikowego, szybkiego obrazowania mikrotubul

10.6K wyświetleń

Cytowany 19 razy

09:45 min

8 sierpnia 2019

10.3791/59520-v

8 sierpnia 2019

10.6K wyświetleń

Mohammed Mahamdeh¹^,² , Jonathon Howard¹

¹Department of Molecular Biophysics and Biochemistry, Yale University, ²Harvard Medical School, Harvard University

Ten protokół jest przewodnikiem do implementacji mikroskopii odbicia interferencyjnego na standardowym mikroskopie fluorescencyjnym do bezznacznikowego, kontrastowego, szybkiego obrazowania mikrotubul przy użyciu testów powierzchni in vitro.