Fluorophores and Chromophores for Two-Photon Excitation

The evolving of fluorescent probes has kept pace with the development of new microscopy techniques to allow the visualization of molecules, cell-signalling events, subcellular features and whole cells. Some fluorescent probes must be loaded into individual cells and have been used mainly for in vitro studies (for example, fura-2 and other Ca²⁺ indicators). Other probes, such as the CellTracker™ probes from Molecular Probes (Invitrogen) are stable and have a variety of applications ranging from cell viability assays, cell adhesion, cell migration and cell-cell fusion studies.

A breakthrough development in the use of fluorescent probes for biological studies has been the development of the use of naturally fluorescent proteins as fluorescent probes. The jellyfish Aequorea victoria produces a naturally fluorescent protein known as green fluorescent protein (GFP). The gene for this protein has been cloned and can be transfected into other organisms. This is a very powerful tool for localizing regions in which a particular gene is expressed in an organism, or in identifying the location of a particular protein. Surprisingly, in many cases these chimeric proteins preserve their original function. It is therefore often possible to use this technique to visualize the intracellular distribution of a cytoskeletal protein. The beauty of the GFP technique is that living, unstained samples can be observed. There are presently several variants of GFP which provide spectrally separable emission colors¹.

2PE Focus It is difficult to predict 2PE spectra from the one-photon spectra because different quantum mechanical selection rules apply. However, • The optimal wavelength for two-photon excitation is usually shorter than twice the one-photon excitation maximum • The two-photon excitation spectrum is usually broader than the corresponding one photon spectrum • The emission spectrum is generally the same for one- and two-photon excitation

Fluorescent probes have been developed which change their optical properties in response to changes in specific aspects of their environment. For instance, probes such as Fura-2, Indo-1, and Fluo-3 exhibit a spectral shift in its fluorescent emission when bound to calcium ions. Such molecules are known as indicator molecules as they may be used to monitor the concentration of the molecule to which they are sensitized. Fluorescent indicators currently exist for calcium, pH, ATP, membrane potential, and several neurotransmitters. When cells are preloaded with an indicator for a physiologically significant molecule, fluorescence microscopy may be used to measure the intracellular distribution of that molecule. This technique is very powerful in that it can allow dynamic signaling events (e.g. calcium dynamic imaging) to be visualized in living tissue. An exciting recent development has been the development of a free-calcium reporter based on GFP. Two color variants of GFP have been engineered into a single chimeric molecule that contains calmodulin, a calcium binding protein². The molecule is named Cameleon and, upon binding calcium, it undergoes fluorescence resonance energy transfer (FRET); the emission from the shorter wavelength fluorescent peptide is quenched and energy is transferred to excite the longer wavelength peptide thus changing the ratio of the two emitted wavelengths. The result is a probe-concentration insensitive calcium indicator. The advantage of this technique is that the indicator does not have to be loaded into the cell under study. Instead, the organism is transfected with the gene so all cells within the organism (for which the gene promoter is appropriate) will express the calcium indicator.

Common fluorophores and chromophores for two-photon microscopy3,4
Probe	Ex (2PE), nm	Em, nm
Long-term tracking of living cells CellTracker™ Green CMFDA (CFSE) CellTracker™ Orange CMTMR SNARF-1	780 820 700-810	516 566 580/640
Calcium Indicators Fluo -3, -4, -5F, 4FF Oregon Green BAPTA -1, -2 Calcium green-1 + Ca2+;Calcium green-1 2 Ca2+ Fura-2 + Ca2+; Fura-2 2 Ca2+ Indo-1 + Ca2+; Indo-1 2 Ca2+	810 810 820 800 700	520-530 520 530 505 400
Quantum dots	broad	variable
Fluorescent Proteins eCFP eGFP eYFP mRFP, mCherry	800-900 900-1000 930-1000 1030	505 510 530 610
Photoswitchable fluorescent proteins paGFP Kaede KFP1 Dronpa psCFP PA-mRFP KikGR Dendra mEosFP	750 730 1120 780,1010 800 760 760 960 780	515 520/580 600 520 470/510 605 520/590 505/575 520/580
Caged glutamate MNI-glutamate	730
Caged calcium DM-nitrophen Azid-1 NDBF-EGTA	730 700 710
Visualization of organelles Dil (plasma membrane) Rhodamine 123 (mitochondria) DAPI (nucleus) Hoechst (nucleus)	700 780-860 700 780>820	565 550 455 478

 

References:

1. Heim and Tsien, 1996. Curr Biol 6(2):178.
2. Miyawaki et al., 1997 Nature 388(6645):882.
3. Michael D. Cahalan, Ian Parker, Sindy H.We* and Mark J.Miller, Two-Photon Tissue Imaging: seeing the immune system in a fresh light, Nature 872, november 2002, vol. 2.
4. Karel Svoboda and Ryohei Yasuda, Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience, Neuron 50, 823–839, June 15, 2006 2006 Elsevier Inc.