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Two-photon Excitation Spectra of Fluorochromes

Spectra Database

The Spectra Database hosted at the University of Arizona is a comprehensive database for many many dyes. This database uses flash, so you either have to install the Adobe Flash Player or use a browser that comes with in-built flash support, like the Chrome Browser. Adobe Flash Player had security issues in the past, so if installed it should be regularly updated. Unfortunately the update process is not very convenient. This may argue for using Chrome for this occasion.

Most dyes in the database have only one-photon excitation spectra. For some, however, in addition a 2P excitation spectrum is available. To only list those, select '2-Photon Cross-Sections' from the drop down menu. Unfortunately, also all one-photon spectra for the same dyes are shown in the list, so you may have to search a bit. The 2P-spectrum is sometimes included with the one-photon excitation (e.g. Cy5, Cy5.5). 

Usually, however, the two photon spectrum is indicated with -2P behind the fluorochrome's name. A few examples: Alexa Fluors 350, 488, 568, 594, 610, 647, 660, 680, DAPI, DiI, various fluorescent proteins from the Drobizhev paper (see below), calcium indicators (incl. Fura-2, Indo-1., Fluo3).

Published Articles

There are many published articles on this topic. The following are listed because they contain substantial numbers of interesting fluorochromes.

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

F. Bestvater, E. Spiess, G. Stobrawa, M. Hacker, T. Feurer, T. Porwol, U. Berchner-Pfannschmidt, C. Wotzlaw and H. Acker. Journal of Microscopy 208 (2),108–115 (2002). doi: 10.1046/j.1365-2818.2002.01074.x

This article shows two-photon absorbtion (excitation) spectra between ~600 - 1150 nm for the following fluorescent dyes: Quinacrine, Hoechst 33342, DAPI, AMC, Rhod123, PI, PhenGreen, SNARF, dcf-2, Bodipy D, Bodipy-TR, Acridine orange, Alexa 488, 546 and 594, Cy2, Cy3, Rhodamin RedX, Lissamin Rhodamin, Texas REd, DTAF, FITC, ER-Tracker White/Blue, MItoTracker REd, LysoTracker Yellow and Red.

Two-photon absorption properties of fluorescent proteins.

Mikhail Drobizhev, Nikolay S Makarov, Shane E Tillo, Thomas E Hughes & Aleksander Rebane. Nature Methods 8, 393–399 (2011). doi:10.1038/nmeth.1596

This article shows one- and two-photon absorbtion (excitation) spectra between ~600 - 1200 nm for the following fluorescent proteins: Fig. 1: EBFP2.0, EGFP, mBlueberry1, mAmetrine, Citrine, DsRed2, ECFP, mOrange, TagRFP. Fig. 3: tdTomato, DsRed2, mBanana, mRFP, mCherry, mStrawberry. Fig 5: tdTomato, tdKatushka2.

Excitation spectra and brightness optimization of two-photon excited probes.

Jörg Mütze, Vijay Iye, John J. Macklin, Jennifer Colonell, Bill Karsh, Zdeněk Petrášek, Petra Schwille, Loren L. Looger, Luke D. Lavis, Timothy D. Harris. Biophysical Journal 102 (4), 934–944, (2012). doi:10.1016/j.bpj.2011.12.056. 

This article shows two-photon excitation spectra between ~700 - 1000 nm for the following fluorescent dyes (AlexaFluor abbreviated as A): Fig 2, Alexa dyes: A350, A555, A647, A610, A633, A546, A568, A594, A430, A488, A314. Fig 3, rhodamine dyes: tetramethylrhodamine, 5-carboxy-tetramethylrhodamine, Q-rhodamine, rhodamine 101, 110, 575 and sulforhodamine 101. Fig 4, Calcium ion indidators: eGFP (for comparison), GCaMP2, GCaMP3, CalciumGreen, CalciumOrange, CalciumCrimson, XRhod1, Rhod4, Rhod2, Fluo8, Fluo3, Fluo4, OGB1, OGB1 APO, OGB5N, OGB5N Apo. Fig 5: Fluorescin, Bodipy492/515, BodipyTR, Resorufin.