AAT Bioquest

Multi-Photon Microscopy

Astrocytes with Cal-590
Staining of astrocytes with Cal-590 in the in vivo mouse cortex. (A) Two-photon fluorescence image obtained in layer 2/3 of the visual cortex of an anesthetized mouse after bulk-loading with Cal-590 AM. The excitation wavelength was 1,050 nm. (B) Two-photon fluorescence image of the same optical section as in A after additional staining with sulforhodamine 101 (SR 101). The excitation wavelength was 900 nm. The red arrows indicate two labeled astrocytes. Source: Tischbirek, C., Birkner, A., Jia, H., Sakmann, B. & Konnerth, A. Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator. Proc. Natl. Acad. Sci. (2015).
In some cases, it may be necessary to visualize multiple ex/em wavelengths simultaneously, which cannot be done with basic fluorescence microscopy. Here multi-photon microscopy becomes advantageous, performed with very high powered, pulsed lasers that serve to package photons. Many times, red light is preferred over blue light as an excitation source because the longer wavelength penetrates deeper into tissues better than shorter wavelengths, and the absorption of high-energy blue light is more likely to damage cells. Either two-photon or three-photon microscopy are the most common variations of the technique.

Three-Photon Microscopy

Three-photon microscopy is similar in principle to two-photon, except three photons must interact and converge with the fluorophore simultaneously. The quantum mechanics of fluorescence absorption means that only, roughly, a 10-fold greater photon density is needed for three photon excitation than is required for two-photon absorption. Three-photon microscopy has extended fluorescent imaging into the realm of deep UV, where 720 nm light can be used to excite a fluorophore that normally absorbs at 240 nm.

Two-Photon Microscopy

Two-photon excitation occurs when two photons are simultaneously absorbed in a single quantized event. First, excitation is produced by a single pulsed laser focused through the microscope. As the laser beam focuses, photons become more crowded and the chance of simultaneous interaction of two photons on a single fluorophore increases. Excitation relies on simultaneous absorption, and the resultant emission is dependent on the square of the excitation intensity. Though two-photon microscopy is extremely expensive, this technique is superior for live cell imaging, particularly in thick, multicellular preparations like developing embryos or brain slices. Additionally, no background is present in produced images, this technique negates the need for pinholes or other light sources, and two-photon microscopy is extremely well-suited for 3D imaging.

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Product Ordering Information

Table 1. Ordering Info for Cal520 Calcium Indicators (Price Table) Products

Product Name
Unit Size
20410Cal-500™, potassium salt39050010x50 ug
20412Cal-500™ AM39050010x50 ug
20455Cal-670™, potassium salt65067010x50 ug
20456Cal-670™-Dextran Conjugate *MW 3,000*6506751 mg
20457Cal-670™-Dextran Conjugate *MW 10,000*6506751 mg
20460Cal-770™, potassium salt75077510x50 ug
20461Cal-770™-Dextran Conjugate *MW 3,000*7507751 mg
20462Cal-770™-Dextran Conjugate *MW 10,000*7507751 mg
20508Cal-590™-Dextran Conjugate *MW 3,000*5735881 mg
20509Cal-590™-Dextran Conjugate *MW 10,000*5735881 mg
20510Cal-590™ AM5735885x50 ug
20511Cal-590™ AM57358810x50 ug
20512Cal-590™ AM5735881 mg
20515Cal-590™, sodium salt5735885x50 ug
20518Cal-590™, potassium salt5735885x50 ug
20519Cal-590™, potassium salt5735881 mg
20530Cal-630™ AM6086265x50 ug
20531Cal-630™ AM60862610x50 ug
20532Cal-630™ AM6086261 mg
20535Cal-630™, sodium salt6086265x50 ug
20538Cal-630™, potassium salt6086265x50 ug
20546Cal-630™-Dextran Conjugate *MW 10,000*6086261 mg
20600Cal-520®-Dextran Conjugate *MW 3,000*4925141 mg
20601Cal-520®-Dextran Conjugate *MW 10,000*4925145 mg
20602Cal-520L®-Dextran Conjugate *MW 10,000*1 mg
20605Cal-520®-Biotin Conjugate4925145x50 ug
20606Cal-520®-Biocytin Conjugate4925145x50 ug
20609Cal-520® NHS Ester492514100 ug
20610Cal-520® maleimide492514100 ug
21130Cal-520®, AM49251410x50 ug
21131Cal-520®, AM4925141 mg
21135Cal-520®, sodium salt49251410x50 ug
21136Cal-520®, sodium salt4925141 mg
21140Cal-520®, potassium salt49251410x50 ug
21141Cal-520®, potassium salt4925141 mg
21142Cal-520FF™, AM4925141 mg
21143Cal-520FF™, AM49251410x50 ug
21144Cal-520FF™, potassium salt49251410x50 ug
21146Cal-520N™, AM49251410x50 ug
21147Cal-520N™, potassium salt49251410x50 ug