Confocal and multi-photon microscopy

Both confocal and multiphoton microscopy are examples of far-field fluorescence microscopy.

Fluorescence microscopy involves the imaging of a sample by excitation of some fluorophore, either endogenous to the tissue or an applied label. The excitation source is generally a laser in the case of far field microscopy. One important feature of fluorescence is that the excitation light has a shorter wavelength than the emission light (the excitation light has more energy per

photon - E=hc/λ, where λ is the wavelength of the light). The graph below shows the excitation and emission spectra of a popular fluorophore used in microscopy. The excitation light is green, but the emitted light is red.

Another feature is that when a photon of excitation light interacts with a fluorophore, there is a delay before the emission fluorophore is released. The exact timing delay is random, but it there is an average time between excitation and emission, which is called the lifetime (τ) of the fluorophore.

Fluorescence

Confocal vs 2-photon microscopy

In far field fluorescence microscopy, the excitation light is focused down to the smallest possible point, the maximum resolution of the microscope being the result of how small that point is. The resolution is given by the equation R=0.61λ_ex/NA, where λ_ex is the wavelength of the excitation light and NA is the numerical aperture of the objective. Light is only gathered from a single point at a time, which is then scanned to make an image, in similar way to an old fashioned cathode ray TV set, if the scanner has a larger pixel size than the optical resolution, this limits the resolution of the microscope.

Not only must the light be focused in the 2-dimensional image plane (x,y), but also in the depth plane (z), allowing the imaging of a voxel of tissue in 3-dimensions.

Confocal and 2-photon microscopy differ in two basic ways; 1) The wavelength of light used to excite the fluorophore and 2) the method by which light from out of focus regions along the z-place are discarded.

For a confocal microscope, shown above, the focused light can be thought of as passing through a series of circles of decreasing size until it reaches the minimum resolvable spot size, and then expanding out again. Although the power through each level is increasing down to the focus, the photon flux is constant with respect to depth and so light is emitted from the entire excitation cone both entering and exciting the sample.

However, since the light focused onto the focal plane is parallel when it enters the back of the objective, the emitted light from the focal plane is parallel after it travels back through the objective. The in-focus light is then refocused through a pinhole. Light emitted from off the focal plane is not parallel out of the back aperture of the objective therefore does not pass through the pinhole.

2-photon microscopy uses excitation light that is twice the wavelength of the single photon excitation wavelength. The technology relies on the fact that if two photons with half the excitation energy required each arrive in close enough succession, excitation can occur. As a result, the level of excitation is dependant on the photon density, not the flux, as they pass through each level.

The likelihood of excitation is dependant on power, since power is greatest at the point of focus, 2-photon excitation has intrinsic optical sectioning and requires no pinhole.

An example microscope

* Light is emitted from a laser and bounced off at least 2 steering mirrors to align the beam so that it enters the center of the objective along the optical axis.

* The attenuation filter allows the user to control the laser intensity at the sample.

* The scan box scans beam so that the focal spot moves over the whole sample to allow an image to be reconstructed.

* The beam expander increases the width of the beam so that the back aperture is overfilled.

* The cleanup filter makes sure that only the required excitation light is present in the beam. The dichroic then allows the excitation light to pass through, when the spectrally shifted emission light returns, it is reflected and passed through an emission filter so that only the fluorophore of interest is seen.

* For a confocal scope, the light is focused through a pinhole. 2-photon scopes do not have a pinhole and so light is simply focused onto the detector.