Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

Abstract

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

Interferometric Scattering Microscopy: Seeing Single Nanoparticles and Molecules via Rayleigh Scattering

“…Nevertheless, lateral vibrations might compromise ultrasensitive measurements because even a few nanometers of motion suffice to change the differential contrast that results from imperfect background subtraction. 27…”

Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution

“…18 The interference between the scattered and reflected light enhances sensitivity by producing a signal that scales linearly with particle polarizability, which may be interpreted as mass for objects with fixed composition. 19–21 Our initial study demonstrated trapping of gold nanoparticles as small as 20 nm, and that polymer nanoparticles down to 50 nm diameter could be held for more than 30 seconds. Fluorescently labeled particles could also be trapped far beyond the time of photobleaching.…”

Interferometric Scattering (iSCAT) Microscopy and Related Techniques

“…3). Nevertheless, lateral vibrations might cause problems since even a few nanometers of motion could translate into fluctuations of the contrast when the background is subtracted (see section below) [135].…”

“…Another recent application of label-free single-protein detection was showcased in the context of real-time investigation of cellular secretion [135,166], shown in Fig. 12c.…”