Genetically-encoded fluorescent probes are used in neuroscience to visualize neurons, record action potentials, and follow communication between neurons in real time in vivo. Two- or three-photon excitation of these probes provide high spatial resolution and deeper imaging as implemented in multiphoton laser scanning microscopy. To select optimum laser wavelength and pick brighter and more photo-stable probe, characterization of nonlinear optical properties of these probes is necessary.

 

We provide the Resource for Multiphoton Characterization of Genetically-Encoded Probes for the BRAIN Initiative researchers and broader neuroscience community. We will be happy to help you with:

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• Measuring of 2-photon absorption spectra in absolute, cross section values;

• Measuring 3-photon absorption spectra and cross sections;

• Characterizing multiphoton photobleaching parameters, i.e.  quantum yields, nonlinear absorption cross sections     

• Measuring one-photon absorption, emission, and excitation spectra

• Measuring fluorescence quantum yields and lifetimes

• Evolving brighter two-photon probes

 

 

 

 
 
          

 

 

 

Measurement Methods and Protocols

Experimental setup, methods, and protocols for the two-photon absorption spectroscopy can be found at https://en.bio-protocol.org/e3498

Experimental setup and methods for evolving brighter two-photon probes are described on https://opg.optica.org/boe/fulltext.cfm?uri=boe-11-12-7192&id=442725

Experimental setup, methods, and protocols for measuring molecular parameters of multiphoton bleaching can be found at  https://www.mdpi.com/1422-0067/23/2/770/htm

 

Contact and shipping information

If you have created a new fluorescent probe (or use a known probe whose multiphoton properties are not known) and would like to know its multiphoton properties, such as optimum laser wavelength, brightness relative to other probes, photo-stability under femtosecond laser excitation, etc., please contact us.


Contact Mikhail (Misha) Drobizhev [email protected] (406)-994-7810

Shipping address:

Mikhail Drobizhev

Montana State University

109 Lewis Hall

Bozeman, MT, 59717, USA

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Annual Multiphoton Workshop

We organize annual summer workshops in Bozeman, Montana. There we discuss theoretical and experimental aspects of multiphoton absorption, its applications in multiphoton microscopy, and provide hands-on demonstration of the measurements methods.

Photo from the 2019 Multiphoton Workshop.  To see more, please visit the Google Photos album.  https://photos.app.goo.gl/VDW7mUksJt997xW66



 
 
 
 
 
Annual Workshop
 
 
 
 
Workshops agendas and materials:

https://docs.google.com/document/d/1Kn3HewZYnUBoBebKQcFcm4Lh-fac-zuM3YHC2NmqbUc/edit

Useful online resources for two-photon spectra of FPs

1. Fpbase is a free and open access, community-editable database for fluorescent proteins photophysical properties. Two-photon spectra of many FP variants and FP-sensors characterized by us during the years are uploaded there. https://www.fpbase.org/

2. Max Planck Institute Brain project runs the repository of excitation, emission, and 2P excitation of many small fluorophores and FPshttps://public.brain.mpg.de/shiny/apps/SpectraViewer/

Our Publications

https://scholar.google.com/citations?user=5u3vlSwAAAAJ&hl=en

Funding

Our Resource is supported by the NIH/NINDS BRAIN Initiative grant U24 NS109107.