Mass Photometry (MP) — Physical Principles & Optical Setup

What is mass photometry and what molecular weight range can it detect?

A label-free single-molecule optical technique that measures the mass of individual biomolecules in solution across a range from 40 kDa to 5 MDa

What basic optical instrument configuration forms the core of a mass photometer?

An inverted microscope where biomolecules in a buffer drop land on a glass coverslip mounted on the stage and are illuminated from below

What is the physical mechanism of elastic light scattering used in mass photometry?

Off-resonance electromagnetic light waves interact with the electron distribution of biomolecules, setting up oscillating dipoles that re-emit light at the exact same wavelength

Which intrinsic physical properties of a molecule govern how much light it scatters?

Polarizability, refractive index increment, and the total amount of electron distribution, which directly correlates with molecular mass

While pure Rayleigh scattered light intensity scales with mass squared, why is interferometric detection linear with mass?

Scattered electric field amplitude Es is directly proportional to particle mass M, making the interference term 2 Er Es cos phi linearly proportional to mass

What mathematical equation describes the total detected light intensity Id in mass photometry?

Id equals the square of the sum of reflected field Er and scattered field Es, expanding to Ir plus Is plus 2 Er Es cos phi

What does each of the three terms in the interferometric intensity equation represent?

Ir is the large background reflected intensity from the interface, Is is negligible scattered intensity from one molecule, and 2 Er Es cos phi is the dominant interference term

What interface provides the reference reflected beam Er in mass photometry?

The glass-water interface between the glass coverslip and the aqueous sample buffer

What special optical component attenuates reflected light to amplify single-molecule contrast on the CMOS camera?

A spatial filter with a partial reflector that selectively reduces strong reflected background light relative to scattered light

What illumination scanning mechanism and frame rate are used in mass photometry?

Microsecond laser beam scanning driven by acousto-optic deflectors (AODs) with camera image acquisition averaging around 100 Hz frame rate

How are ratiometric images calculated during mass photometry movie processing?

Future time-averaged movie frames are divided by past background frames to subtract static glass roughness and leave only dynamic single-molecule events

What visual feature characterizes a single-molecule landing event in a ratiometric movie frame?

A circular diffraction-limited spot with a dark center and bright surrounding ring, reflecting negative ratiometric contrast

What distinguishes the optical signal of a landing molecule from a departing molecule?

Adsorption or landing onto the coverslip produces a dark center with negative contrast, whereas desorption or take-off produces a light center with positive contrast

How does interferometric contrast depend mathematically on the molecular mass of a landing protein?

Ratiometric contrast is strictly linearly proportional to molecular mass, where larger mass yields more negative contrast

How does single-molecule mass photometry differ fundamentally from dynamic and static light scattering?

DLS measures hydrodynamic size from Brownian motion, SLS measures time-averaged ensemble mass, whereas mass photometry resolves exact mass distributions of single molecules without ensemble averagin