Leica TCS SP5 MP User Manual
Leica
TCS SP5 MP
Broadband Confocal and
Multiphoton Microscope
The Solution for Deep Imaging
1-photon excitation |
2-photon excitation |
focal plane
In 2-photon excitation fluorescence emission occurs only on the focal plane.
1-photon excitation |
2-photon excitation |
fluorescence |
fluorescence |
emission |
emission |
excitation
Energy diagram of fluorescence with 1-photon and 2-photon excitations
Deep Imaging
Since the advent of confocal microscopy, immense progresses have been made in cellular biology, neurosciences, medical research. Today, it is a major challenge to penetrate deeper into samples for improved studies of cells, organs or tissues. An efficient method to achieve a deep penetration into samples is two-photon and multiphoton excitation with laser scanning microscopes which are equipped with pulsed infrared lasers. Thanks to reduced absorption and scattering of the excitation light, two-photon and multiphoton confocal microscopes reach a penetration depth of about 400 µm.
In the case of two-photon excitation, the dye is excited by the simultaneous absorption of two photons. Due to the non-linearity nature of two-photon absorption, the excitation is limited to the focal volume and the photobleaching outside the focal plane is reduced. Only inside the confocal volume the photon density is sufficiently high to allow two photon absorption by the fluorophore.
Multiphoton excitation performance improves with pulsed laser excitation in the NIR spectra. Longer excitation wavelengths are scattered less in biological tissue allowing a deeper penetration in very thick specimen. Emission/Fluorescence signal is not degraded either by scattering from within the sample.
Advantages of multiphoton exitation:
•Greater penetration depth due to lower scattering
•Intrinsical optical sectioning properties – no need for a detection pinhole
•Bleaching restricted to focal plane – no volume bleaching
•Reduced phototoxicity due to spatial confinement, which is ideal for living cells.
•Uncaging, photoactivation or photobleaching in a diffractionlimited volume
The Leica TCS SP5 MP covers a wide range of imaging applications (multiphoton and one photon) by combining two technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time resolution.
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Applications
The invention of multiphoton microscopy in the 1990’s raised a tremendous interest and has become a widespread imaging method in the biological sciences since then. Meanwhile there is plethora of applications and publications involving multiphoton microscopy.
It is now established as the method of choice for non-invasive deep-penetration fluorescence microscopy of thick highly scattering samples and has been used for a diversity of specimen, e.g. lymphatic organs, kidney, heart, skin and brain (slices as well as intact organs).
Various research fields, e.g. immunology (lymphocyte tracking, embryology, cancer research and particularly neuroscience (e.g. for the study of calcium dynamics and neuronal plasticity) take the advantage of the deep in vivo imaging with multiphoton.
Top: hyppocampal region in mouse brain slice.
Courtesy of Dr. Michael E. Calhun, Hertie Institute, Tübingen, Germany. Middle: mouse embryo, detail of the heart.
Courtesy of Dr. Elisabeth Ehler, King’s College, London, UK.
Bottom: adult rat cardiomyocytes
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