Two Photon Excitation Microscopy (TPE)   TPE is an optical process that uses multiphoton absorption to image live samples, and this technique results in less phototoxicity than conventional confocal microscopy. Two-photon microscopes use infrared excitation light to penetrate further into tissues and with minimal scatter compared to exciting at longer wavelengths. For each excitation of the fluorophore, two photons of infrared light are absorbed, and excitation is limited to a tiny focus volume, so there is less contribution from out-of-focus fluorescence.

Two-photon imaging has been applied to a myriad of live-cell applications including imaging of ion concentrations, cytoskeletal organization, reactive oxygen detection, and vesicle recycling.

Labels and bioconjugates

Molecular Probes® fluorophores have been tried and tested in TPE applications as conjugates of antibodies, proteins, dextrans, and other biomolecules. Use the list below to select the fluorophore with the best wavelength. The TPE excitation is shown in the table—citations are listed for each. Typically, Alexa Fluor® dyes have been used as bioconjugates for TPE in experiments to investigate cytoskeleton organization or neuronal tracing. Qdot® probes conjugated to antibodies have been used in immunohistochemical analysis. By following the links for each fluorophore you can also find all of the respective bioconjugates currently available to target your molecule of interest; the list ranges from antibodies and phalloidins to growth factors and lectins. If you don't find the conjugate you need on the list, the links will also show you reactive dye forms or optimized labeling kits to enable you to create your own bioconjugate probes.

  Alexa Fluor® 350 Alexa Fluor® 488 Qdot® 525 Alexa Fluor® 546 Alexa Fluor® 555
Target label/conjugate label/conjugate label/conjugate label/conjugate label/conjugate
Bibliography Citations Citations Citations Citations Citations
TPE excitation (nm) 720 720, 830 750 810 810
Laser line (nm) 350/405 488 350, 488 488 488
Standard filter set DAPI FITC FITC TRITC TRITC
Ex/Em (nm) 346/442 490/525 350–405/525 556/573 555/580
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  Alexa Fluor® 568 Qdot® 605 Probe Alexa Fluor® 594 Alexa Fluor® 647 Qdot® 655 Probe
Target label/conjugate label/conjugate label/conjugate label/conjugate label/conjugate
Bibliography Citations Citations Citations Citations Citations
TPE excitation (nm) 770 750 810 800 750
Laser line (nm) 561 350–594 594 594/633 350–615
Standard filter set RFP RFP Texas Red® Cy®5 Cy®5
Ex/Em (nm) 578/603 350–594/605 590/617 650/665 350–615/655
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Ion indicators

Ion Indicators for TPEA range of Molecular Probes® ion indicators have been used in TPE applications, primarily in neurons. Oregon Green® 488 BAPTA-1 has been used to image calcium signaling in the rat brain; fura-2 and X-rhod-1 have been used in Purkinje cells; and Fluo-5F has been referenced in publications on dendrites and dendritic spines. Other ions studied using TPE include sodium gradients in rat cardiomyocytes using SBFI and chlorine concentrations in olfactory neurons using MQAE.

The table below shows indicators used in TPE. You can also see a wider range of ion indicators organized by performance criteria in conventional imaging applications.

  Oregon Green® 488 BAPTA-1 Fura-2 Fluo-5F X-Rhod-1 SBFI MQAE
Target Ca++ Ca++ Ca++ Ca++ Na+ Cl
Bibliography Citations Citations Citations Citations Citations Citations
TPE excitation 880 780 810 900 760 750
Laser line 488 350 488 561 350 350
Standard filter set FITC FITC FITC Texas Red® dye Ratio Ratio
Ex/Em (nm) 494/523 340/510 494/516 580/602 340/500 350/460
Cat. No. O6806 F1201 F14222 X14210 S1262 E3101

Oxidative stress detection

TPE can be used to detect the localized release of reactive oxygen species and the redox state of the cell using a variety of indicators. Specifically, glutathione, H2O2, or generalized oxidative stress can be measured with a number of probes that have been shown to work well for TPE and offer a range of wavelength options. Citations are included for each probe as well as the TPE excitation wavelength used.

The table below shows indicators used in TPE. You can also see a wider range of reactive oxygen indicators organized by performance criteria in conventional imaging applications.

  Monochlorobimane Amplex® Red CM-H2DCFDA
Target Glutathione H2O2 Oxidative stress
Bibliography Citations Citations Citations
TPE excitation (nm) 780 750 or 800 740
Laser line (nm) 350 561 488
Standard filter set DAPI TRITC FITC
Ex/Em (nm) 394/490 571/585 495/597
Cat. No. M1381MP A12222 C6827

Cell structure probes

Cell structure probes exhibiting good results for TPE include nuclear stains, membrane stains, and cell-tracing dyes. Nuclear stains are available for use in both live (Hoechst 33342) and fixed (DAPI) cells, both with blue emission and a similar TPE excitation. Cell membrane probes offer a choice of red (FM® 1-43 dye) or far-red (DiD) emissions, and the cell-tracking dyes range from green through orange.

The table below shows the probes used in TPE, together with citations and TPE excitation. You can also see a wider range of cell structure probes organized by performance criteria in conventional imaging applications.

  DiD FM® 1-43 CFSE CellTracker™ Orange Lucifer Yellow DAPI Hoechst 33342
Target Cell membrane Cell membrane Cell tracing Cell tracing Cell tracing DNA DNA
Bibliography Citations Citations Citations Citations Citations Citations Citations
TPE excitation (nm) 817 840 820 820 850 740 740
Laser line (nm) 594/633 488 488 488 405 350/405 350/405
Standard filter set Cy®5 TRITC FITC TRITC TRITC DAPI DAPI
Ex/Em (nm) 644/665 479/598 490/525 541/565 428/536 350/470 350/461
Cat. No. D7757 T3163 C1157 C2927 L453 Find products H3570
For Research Use Only. Not for use in diagnostic procedures.