AAV Biosensors

Biosensors are genetically engineered fluorescent proteins (FP) attached to an additional protein sequence which makes them sensitive to small biomolecules or other physiological intracellular processes (e.g. Ca²⁺). These biosensors are introduced into cells, tissues or organisms to detect changes by fluorescent microscopy or spectrum changes. Many biosensors permit long-term imaging and can be engineered to specifically target cellular compartments or organelles. Additionally, biosensors permit signaling pathway exploration or allow the measurement of a biomolecule. They do all of this while preserving both spatial and temporal cellular processes.

CaMPARI or Calcium Modulated Photoactivatable Ratiometric Integrator is a photoconvertible protein construct. CaMPARI enables imaging of integrated calcium activity of large populations of cells over defined time windows.

CaMPARI is based on EosFP, a fluorescent protein whose emission changes from green to red upon irradiation with UV-light (~400 nm). By engineering libraries of EosFP variants, researchers were able to develop a protein that undergoes significantly faster green-to-red photoconversion (PC) only when calcium is present while PC light is applied. This permanent conversion provides the ability to record calcium activity for all areas illuminated by PC light. The red fluorescence intensity correlates with calcium activity. CaMPARI complements existing calcium indicators by allowing total calcium activity measurement over large areas of cells and tissues.

• Not restricted to the field of view of a microscope, as during real-time calcium imaging
• Enables higher-throughput calcium assays with cultured cells

• Calcium activity imaging across large cell populations and/or tissues
• Labeling of ‘active’ cells within a tissue (such as brain) during stimulus or behavior in model organisms
• Tracing of neurons based on their calcium activity level
• Integration of subcellularly localized calcium activity when targeted to specific subcellular locations

RCaMP is a GECI which was generated from a fusion of the red fluorescent protein (RFP), calmodulin, and M13, a peptide sequence from myosin light chain kinase. Upon the binding of GECI to Ca²⁺ there is an induction of a change in fluorescence signal. It is this change in signal, which allows for measuring the action potentials, and other receptor activation events, that trigger Ca²⁺ fluxes.

• Provides new color channels for single-wavelength functional imaging
• Minimal bleed through into green channel, unlike other biosensors
• Compatible with optogenetic activation/silencing via tools such as channelrhodopsin-2, facilitating “read-write” optogenetics
• Tunable affinity under control of various promoters
• Less auto-fluorescence than green GECIs and dyes

• Multifunctional imaging for drug screening
• Imaging cellular activity in cells and organisms that already express GFP

RCaMP is a genetically encoded calcium indicator (GECI) which was generated from a fusion of the red fluorescent protein (RFP), calmodulin, and M13, a peptide sequence from myosin light chain kinase. Upon the binding of GECI to Ca2+ there is an induction of a change in fluorescence signal. It is this change in signal which allows for measuring the action potentials, and other receptor activation events, that trigger Ca2+ fluxes.

• Provides new color channels for single-wavelength functional imaging.
• Minimal bleed through into green channel, unlike other biosensors.
• Compatible with optogenetic activation/silencing via tools such as channel rhodopsin-2, facilitating “read/write” optogenetics.
• Tuneable affinity under control of various promoters.
• Detection using standard optical and nano technologies.

jRGECO1a and jRCaMP1b offer improved red GECIs based on mRuby and mApple, respectively - with sensitivity comparable to GCaMP6.

jRGECO1a and jRCaMP1b offer improved red GECIs based on mRuby and mApple, respectively - with sensitivity comparable to GCaMP6.

• Increased red fluorescence sensitivity, comparable to GCaMP6
• Best performer for tracking sensitive stimuli; has advantages in response speed
• Facilitates deep-tissue imaging, dual color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging

• In vivo imaging
• Deep tissue imaging
• Dual color imaging