Roadmap on Recent Progress in FINCH Technology.

Fresnel incoherent correlation holography (FINCH) was a milestone in incoherent holography. In this roadmap, two pathways, namely the development of FINCH and applications of FINCH explored by many prominent research groups, are discussed. The current state-of-the-art FINCH technology, challenges, and future perspectives of FINCH technology as recognized by a diverse group of researchers contributing to different facets of research in FINCH have been presented.

Imaging of In Vitro and In Vivo Neurons in Drosophila Using Stochastic Optical Reconstruction Microscopy.

The Drosophila melanogaster brain comprises different neuronal cell types that interconnect with precise patterns of synaptic connections. These patterns are essential for the normal function of the brain. To understand the connectivity patterns requires characterizing them at single-cell resolution, for which a fluorescence microscope becomes an indispensable tool. Additionally, because the neurons connect at the nanoscale, the investigation often demands super-resolution microscopy. Here, we adopt one super-resolution microscopy technique, called stochastic optical reconstruction microscopy (STORM), improving the lateral and axial resolution to ∼20 nm. This article extensively describes our methods along with considerations for sample preparation of neurons in vitro and in vivo, conjugation of dyes to antibodies, immunofluorescence labeling, and acquisition and processing of STORM data. With these tools and techniques, we open up the potential to investigate cell-cell interactions using STORM in the Drosophila nervous system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Drosophila primary neuronal culture and embryonic fillets Basic Protocol 2: Immunofluorescence labeling of samples Basic Protocol 3: Single-molecule fluorescence imaging Basic Protocol 4: Localization and visualization of single-molecule data Supporting Protocol: Conjugation of antibodies with STORM-compatible dyes.

Cell-type-specific, multicolor labeling of endogenous proteins with split fluorescent protein tags in Drosophila.

The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type-specific manner in Drosophila A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.

Fundamental precision bounds for three-dimensional optical localization microscopy using self-interference digital holography.

Localization based microscopy using self-interference digital holography (SIDH) provides three-dimensional (3D) positional information about point sources with nanometer scale precision. To understand the performance limits of SIDH, here we calculate the theoretical limit to localization precision for SIDH when designed with two different configurations. One configuration creates the hologram using a plane wave and a spherical wave while the second configuration creates the hologram using two spherical waves. We further compare the calculated precision bounds to the 3D single molecule localization precision from different Point Spread Functions. SIDH results in almost constant localization precision in all three dimensions for a 20 µm thick depth of field. For high signal-to-background ratio (SBR), SIDH on average achieves better localization precision. For lower SBR values, the large size of the hologram on the detector becomes a problem, and PSF models perform better.

Three-dimensional nanoscale localization of point-like objects using self-interference digital holography.

We propose localizing point-like fluorescent emitters in three dimensions with nanometer precision throughout large volumes using self-interference digital holography (SIDH). SIDH enables imaging of incoherently emitting objects over large axial ranges without refocusing, and single molecule localization techniques allow sub-50 nm resolution in the lateral and axial dimensions. We demonstrate three-dimensional localization with SIDH by imaging 100 and 40 nm fluorescent nanospheres. With 49,000 photons detected, SIDH achieves a localization precision of 5 nm laterally and 40 nm axially. We are able to detect the nanospheres from as few as 13,000 detected photons.

Charge-Directed Immobilization of Bacteriophage on Nanostructured Electrode for Whole-Cell Electrochemical Biosensors.

A new type of carbon nanotube (CNT)-based impedimetric biosensing method has been developed for rapid and selective detection of live bacterial cells. A proof-of-concept study was conducted using T2 bacteriophage-based biosensors for electrochemical detection of Escherichia coli B. The T2 bacteriophage (virus) served as the biorecognition element, which was immobilized on polyethylenimine (PEI)-functionalized carbon nanotube transducer on glassy carbon electrode. Charge-directed, orientated immobilization of bacteriophage particles on carbon nanotubes was achieved through covalent linkage of phage capsid onto the carbon nanotubes. The presence of the immobilized phage on carbon nanotube-modified electrode was confirmed by fluorescence microscopy. Electrochemical impedance spectroscopy (EIS) was used to monitor the changes in the interfacial impedance due to the binding of E. coli B to T2 phage on the CNT-modified electrode. The detection was highly selective toward the B strain of E. coli as no signal was observed for the nonhost K strain of E. coli. The present achievable detection limit of the biosensor is 103 CFU/mL.