Maria Smedh, Centre for Cellular Imaging. Maria Smedh, Centre for Cellular Imaging

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1 Nonlinear microscopy I: Two-photon fluorescence microscopy Multiphoton Microscopy What is multiphoton imaging? Applications Different imaging modes Advantages/disadvantages Scattering of light in thick tissue Absorption in biological tissue Incident collimated light Image-bearing ballistic light Incident collimated light Image-bearing ballistic light Major intracellular absorbers Human hand Detector (camera) Object Tissue Object Scattered diffuse light Adopted from P. French, Physics World 1999 K. König, Journal of Microscopy 200 (2000) From The Science of Photobiology, Ed. K. C. Smith, Plenum, 1977 Biological tissues both absorbs light and scatter light strongly, which makes high-resolution deep imaging impossible for traditional fluorescence imaging. The relatively high-transmission spectral region between ~ nm is often described as the "optical window" of biological tissue.

2 What is multiphoton microscopy? Theoretically it was shown already in 1931 by Maria Göppert- Mayer that two photons of lesser energy together can cause an excitation normally produced by the absorption of a single photon of higher energy: One-photon excitation Two-photon excitation Göppert-Mayer M., Űber Elementarakte mit zwei Quantensprüngen (On elementary processes with two quantum steps), Ann. Phys. 401 (1931) Her theory was experimentally validated in The first multiphoton images were presented in Science as late as 1990 (Denk et al). UV light is phototoxic! Two-photon excitation uses near-ir, i.e. is less harmful to living cells and tissue. The two-photon excitation (2PE) event depends on that the two photons are absorbed nearly simultaneously (~ s), resulting in a quadratic dependence on the light intensity rather than the linear dependence of conventional fluorescence Non-linear process!

3 In bright sunlight, a molecule of rhodamine B absorbs one photon every second absorbs a photon pair every 10 million years no 3-photon absorption is expected throughout the entire age of the universe! Excitation and bleaching in multiphoton microscopy 1P excitation 2P excitation From Molecular Expressions Optical Microscopy Primer From Molecular Expressions Optical Microscopy Primer Because the excitation only occurs in the focal plane there is no need for a pinhole in multiphoton microscopy Because the excitation only occurs in the focal plane there is no photobleaching in other parts of the sample

4 MP detection set-up MP detection set-up Descanned beam path: The emitted light returns along the same path as the excitation light, striking the scanning mirrors before passing through the confocal pinhole to the detector. From Molecular Expressions Optical Microscopy Primer Non-descanned (direct) beam path: A dichroic mirror can be placed immediately after the objective lens to reflect the emitted light directly to a detector. The multiphoton microscope The multiphoton laser Beam Conditioning Unit (BCU) Specimen Ti:S laser Diode pump laser Direct detectors To generate sufficient MP signal the excitation light has to be very intense. Therefore, pulsed lasers are used that emit ultrashort pulses with high peak intensities. Using a pulsed laser also keeps the average power relatively low, which is less harmful for the specimen. Scan head Z motor The most common MP laser is the mode-locked titanium sapphire (Ti:S) laser, which is tunable between ~ nm. Microscope

5 The multiphoton laser A pulsed laser with pulse width τ occuring at frequency f r increases the npe probability, compared to continuous-wave (CW) illumination, by a factor of: Imaging penetration depth can improve the imaging penetration depth by a factor of two or more in typical biological specimens that are highly light scattering. I 1 Pulsed = n ICW r ( τ f ) 1 The Ti:S laser produces about 80 million pulses per second (80 MHz), each with a pulse duration of about 100 fs. The 2PE probability is increased by a factor of ~ 10 5 by the pulsed laser. Absence of out-of-focus absorption allows more of the excitation light photons to reach the desired specimen level. A non-scattering rhodamine-stained polymer film, which contains a uniform distribution of a high fluorophore concentration. Imaging penetration depth Imaging penetration depth The near-infrared light employed in two-photon excitation undergoes less scattering than light that is bluer in color (shorter wavelengths), i.e. more excitation light reaches the focal plane. Simple approximation of Rayleigh scattering: Incident light I S Particles ~ λ 4 Scattered light 488 nm excitation light scatters approximately 7 times more than 800 nm excitation light! Light scattering affects two-photon microscopy less than confocal microscopy, i.e. more emission reaches the detector. Confocal Two-photon

6 Comparison of imaging penetration depth Two-photon crossections The selection rules for absorption are different for 1PE and 2PE. Sometimes the 2P peak absorption is at twice the 1P peak, but often it is not. For symmetrical molecules, like Rhodamine, the single-photon excited states are two-photon forbidden. Three XZ profiles through the same acid-fucsin-stained monkey kidney sample imaged through a depth of 140 µm (scale bar, 20 µm). The imaging penetration depth with multiphoton (1047 nm, descanned, open pinhole) imaging is improved at least twofold relative to confocal (532 nm). Direct detection provides increased signal detection and even more increased depth of imaging. Coumarin Rhodamine Centonze and White, Biophys. J., 75 (1998) Two photon crossections Resolution in multiphoton microscopy 1P absorption 2P absorption DAPI fluorescein bodipy rhodamine PSF PSF PSF confocal = ( ) 2 illumination detection and PSF = PSF two-photon illumination The 2P absorption spectra are often very broad as opposed to the peak-like 1P absorption spectra. It is therefore possible to find one wavelength that excites two fluorophores equally well. Because of the longer excitation wavelengths (red to near-infrared, ~ 700 to 1200 nanometers) used in 2PE the point spread function is larger than for confocal microscopy.

7 Resolution in multiphoton microscopy Deep 2PE imaging in living animals 1PE 543 nm y z x x FWHM xy = 0.25 µm FWHM z = 0.50 µm scale bar = 0.50 µm scale bar = 0.50 µm 2PE 900 nm y z x x FWHM xy = 0.40 µm FWHM z = 1.1 µm There are different methods to reach deep into the tissue of living animals. For the study of the brain either the scull can be thinned down or completely removed and replaced with a coverslip. Other organs can be surgically exposed in a similar manner or be exteriorized. Orange bead ( 0.18 µm) imaged with 60x/1.4 oil objective Helmchen & Denk, Nature Methods 2 (2005) 932 Deep 2PE imaging in living animals 2PE imaging of human skin The neocortex of a transgenic mouse expressing Clomeleon, a genetically encoded chloride indicator. After removal of the scull nearly the entire depth of the six-layered brain tissue can be imaged! Helmchen & Denk, Nature Methods 2 (2005) 932 Potential endogenous two-photon excitable fluorophores are NADH, NADPH, flavins and collagen.

8 Different MPM imaging modes Two-photon excited (2PE) fluorescence is usually the primary signal source in MP microscopy, but three-photon excited (3PE) fluorescence, second- and third-harmonic generation (SHG, THG) can also be used for imaging. 3PE is an absorption process similar to the 2PE process, but three photons instead of two has to be absorbed at the same time. Energy Absorption processes 2PE 3PE Excited state Virtual state Ground state Different MPM imaging modes Second- and third- harmonic generation (SHG, THG) are scattering processes, not absorption with subsequent emission of fluorescence. Ordered structural protein assemblies like collagen fibres and microtubuli has the highest SHG signals. Energy Scattering processes SHG THG Excited state Virtual state Ground state 1 n E S ( nhg) = nei λs ( nhg) = λi Advantages Multi-photon pros and cons Ability to produce thin ( µm) optical sections deep into thick specimens. Less phototoxic wavelengths for living cells and tissue Disadvantages The high purchasing and operating cost of pulsed femtosecond IR lasers. Lab. demo 2a: Multiphoton microscopy Where? When? Who? Centre for Cellular Imaging Medicinareg. 7A, floor 1 (basement) Thursday 23 October 9:00 - Group 1 13:00 - Group 2 Carl Simonsson, Dermatochemistry