Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders

Abstract

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.

Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein–Protein Interaction Analysis, and Protein Function Exploration

“…GFP and RFP nanobodies can also be used to study nuclear pore complex (NPC) and caveolae ultrastructure in detail. Unlike indirect antibody immunochemistry, nanobody staining resulted in a far better approximation of the actual dimensions of both structures (24, 25). …”

“…Several publications describe the use of anti-GFP and anti-RFP nanobodies for super-resolution microscopy. These nanobodies target genetically encoded fluorescent fusion proteins and are equipped with a strong organic dye, usually coupled to the nanobody by means of N -hydroxysuccinimide ester-labeling (see later in this section) (22–24). The first use of this technology was reported by Ries and coworkers (22).…”

“…Equipping the nanobodies with a C-terminal oligo-lysine stretch might divert NHS-labeling from intrinsic nanobody lysine residues (24). However, modification of multiple lysines can create hydrophobic patches that increase unspecific binding and thus background staining (27).…”

Nanoscopic Stoichiometry and Single‐Molecule Counting

“…CECAMs are intimately involved in binding interactions between cells and are implicated in a variety of pathogenic processes related to cellular growth and differentiation . Cells expressing the monomeric variant CEACAM1‐4L fused to eYFP were immunolabeled by targeting the fluorescent protein tag with Alexa‐647 anti‐GFP nanobodies . Direct STORM imaging was performed on four cells within one sample chamber, and clearly separated foci within the diffraction‐limited image were selected as candidate molecules.…”

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

“…С помощью этого подхода ис-следователям удается достигать нанометрового пространственного разрешения с минимальной погрешностью при анализе живых клеток [34]. Использование наноантител в этом случае дает большое преимущество, поскольку обеспечивает максимальную точность при определении лока-лизации единичных молекул ввиду много мень-шего размера наноантител по сравнению с клас-сическими антителами [60,90]. Уже доказано, что «chromobodies» способны обнаруживать антиге-ны в хроматине, репликационных комплексах, цитоскелете, и их использование позволяет ви-зуализировать динамические изменения во вре-мя клеточного цикла в реальном времени [99].…”