Kiryl D. Piatkevich

Massachusetts Institute of Technology | MIT · MIT Media Laboratory

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivativ...

Spatially resolved proteomics is an emerging approach for mapping proteome heterogeneity of biological samples, however, it remains technically challenging due to the complexity of the tissue microsampling techniques and mass spectrometry analysis of nanoscale specimen volumes. Here, we describe a spatially resolved proteomics method based on the c...

Engineered light, oxygen, and voltage (LOV)-based proteins are able to fluoresce without oxygen requirement due to the autocatalytic incorporation of exogenous flavin as a chromophore thus allowing for live cell imaging under hypoxic and anaerobic conditions. They were also discovered to have high sensitivity to transition metal ions and physiologi...

Red fluorescent proteins with a large Stokes’ shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP...

Spectrally diverse fluorescent proteins (FPs) provide straightforward means for multiplexed imaging of biological systems. Among FPs fitting standard color channels, blue FPs (BFPs) are characterized by lower brightness compared to other spectral counterparts. Furthermore, available BFPs were not systematically characterized for imaging in cultured...

Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1 (K+ ratiometric indicator for optical...

Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is a promising material because of its favorable electrical and mechanical properties, stability in ambient environments, and biocompatibility. It finds broad application in energy storage, flexible electronics, and bioelectronics. Additive manufacturing opens a plethora of new ave...

Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process ex...

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up...

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the r...

Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1, based on the insertion of a potassium...

Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo. To perform high speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes wi...

Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessm...

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up...

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up...

Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging using conventional microscopes. Current ExM methods permeate specimens with free-radical-chain-growth-polymerized polyacrylate hydrogels, whose network structure limits the local isotropy of expansion as well as the preservation of morphology...

Nanoscale imaging of all anatomical structures over whole vertebrates is needed for a systematic understanding of human diseases, but this has not yet been achieved. Here, we demonstrate whole-ExM, which enables nanoscale imaging of all anatomical structures of whole zebrafish larvae by labeling the proteins of the larvae with fluorophores and expa...

Near-infrared (NIR) genetically encoded calcium ion (Ca ²⁺ ) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previ...

Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and art...

Near-infrared (NIR) genetically-encoded calcium ion (Ca ²⁺ ) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross-talk with visible-light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previ...

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has highe...

Cortical layer 1 (L1) interneurons have been proposed as a hub for attentional modulation of underlying cortex, but the transformations that this circuit implements are not known. We combined genetically targeted voltage imaging with optogenetic activation and silencing to study the mechanisms underlying sensory processing in mouse barrel cortex L1...

2020 The Author(s) In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random...

Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging on conventional microscopes. Current ExM methods permeate biological specimens with free radical-polymerized polyacrylate hydrogels, whose network structure limits the microscopy resolution enabled by ExM. Here we report that ExM is possible...

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, sig...

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell...

We, the authors, wish to make the following corrections to our paper [...]

A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phy...

Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H2O2 dynamics in live cells within a limited field of view. The visualization of H2O2 within a whole organism with a single cell resolution would benefit from...

The stability of neural dynamics arises through a tight coupling of excitatory (E) and inhibitory (I) signals. Genetically encoded voltage indicators (GEVIs) can report both spikes and subthreshold dynamics in vivo , but voltage only reveals the combined effects of E and I synaptic inputs, not their separate contributions individually. Here we comb...

A longstanding goal in neuroscience has been to image membrane voltage, with high temporal precision and sensitivity, in awake behaving mammals. Here, we report a genetically encoded voltage indicator, SomArchon, which exhibits millisecond response times and compatibility with optogenetic control, and which increases the sensitivity, signal-to-nois...

Our ability to investigate the brain is limited by available technologies that can record biological processes in vivo with suitable spatiotemporal resolution. Advances in optogenetics now enable optical recording and perturbation of central physiological processes within the intact brains of model organisms. By monitoring key signaling molecules n...

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca²⁺) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca²⁺ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently availab...

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing...

In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.

In the supplementary information originally posted online, Supplementary Tables 1-5 and the Supplementary Note were missing. The error has been corrected online.

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of...

Background: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ions while maintaining a higher fluorescence brightness at the basal l...

Optogenetic control of individual neurons with high temporal precision within intact mammalian brain circuitry would enable powerful explorations of how neural circuits operate. Two-photon computer-generated holography enables precise sculpting of light and could in principle enable simultaneous illumination of many neurons in a network, with the r...

Figure S1. An N-terminal domain of kainate receptor subunit 2 is retained in the soma. (a) A hippocampal neuron expressing GFP. (b) A hippocampal neuron expressing KA2-GFP. (c) A hippocampal neuron expressing KA2(1–150)-GFP. Scale bars for a–c: 50 µm. (d, e, f) Box-and-whiskers plots of GFP brightness versus position along a neurite, normalized to...

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FP...

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired int...

Excel file containing values of all data points presented in this manuscript. (XLSX)

File containing all supporting Figures (A-O) and Tables (A-C). Figure A in S1 Supporting information. Alignment of the amino acid sequences for CaMs and peptides from CaM-dependent kinases and calcineurins found in different species. Figure B in S1 Supporting information. Alignment of the amino acid sequences for original library and FGCaMP calcium...

Lightsheet ratiometric calcium imaging with FGCaMP during 4-AP induced neuronal activity in paralyzed larvae embedded in ultra-low-melting agarose gel. (AVI)

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two- and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 μm into fixed scattering brain tissue is achieved, approximately...