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Multiphoton Microscopy – a Satisfied Wish List

The colorful picture shows colon tumor cells, fluorescently labelled and lineage traced from a multicolor tracer. The gray color codes for the second harmonic generation (SHG) signal from Collagen 1. Lineage traced tumor cells are shown in magenta, blue, green, yellow and red. All channels were recorded with two-photon excitation, using the SP8 DIVE by Leica Microsystems. Sample and image were kindly provided by J. van Rheenen, H. Snippert, Utrecht (the Nederlands,) and I. Steinmetz, Leica Microsystems Mannheim.

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Biological specimens usually represent a very complex amalgam of tiny structures with differing refractive indices and absorbances. Even if the definition and sensitivity of the microscope are at the maximum, the focal plane is surrounded by these (optically) detrimental structures. Without appropriate measures, we would have a hard time to see anything in ordinary biological samples. Manufacturing thin slices of the sample, usually accomplished by use of microtomes, provides sufficiently clear images, but is not compatible with living objects.

Optical approaches are confocal microscopy and light sheet microscopy. In light sheet microscopy the sample is illuminated by a thin layer orthogonal to the observation, resembling an optical microtome cut. The confocal microscope is based on point-scanning the sample with a diffraction-limited illumination spot and sensing the signal with a diffraction-limited detection spot. The overlay of the two leads to spatial filtering off the extrafocal information.

Another optical option is nonlinear illumination, initially described as two-photon excitation [1] by M. Göppert-Mayer. Because of the square dependence on light intensity, excitation is constrained to a layer where the photon density is high enough to cause a significant signal: the slice is generated by selective excitation in the focal plane. For fluorescence, all we need to do is to illuminate with wavelengths roughly double the wavelength required for single-photon excitation. As a very beneficial side-effect, scattering is inversely proportional to the fourth power of the wavelength, allowing details deep inside the subject to be imaged with the longer wavelengths [2].

Illumination for Multiparameter Multiphoton Excitation

A central requirement in biological microscopy is correlation of different signals, for example differently stained structures or metabolic signals. In many cases, two or three fluorescent stains are employed and in some cases even more. Often, different excitation wavelengths are necessary to satisfactorily excite the applied fluorochromes. Although lasers for multiphoton excitation are tunable, simultaneous recording demands a multitude of laser lines injected simultaneously into the beam path. Therefore, it would be desirable to have three or even four independent ports for coupling lasers into the instrument. The TCS SP8 DIVE by Leica Microsystems offers coupling of up to four infrared (IR) lines.

The illumination in a microscope is – in the first place – always designed to utilize the full resolution of the objective lens. That translates into homogeneous illumination of the back focal plane. As lasers feature Gaussian beam profiles, the laser beam must be expanded to ensure the required illumination pattern. A drop in intensity at the borders – like with a Gauss profile – corresponds to a smaller pupil diameter and, consequently, a lower definition. As pupil diameters of different lenses may vary, it would be useful to have a variable beam expander that adapts to these different diameters.

On the other hand, when expanding a laser beam to “overfill” the lens’ pupil diameter, we sacrifice a fraction of the energy. For positions close to the surface, this overfilling is not a problem, as scattering has not yet removed a significant number of photons and we still have the high density that is required for two-photon excitation. If we focus deeper and deeper into the sample, scattering will cause an increasing loss of photons and the excitation efficiency will drop accordingly. At a certain position below the surface, the photon density will be too low to prime sufficient fluorescence for recording a decent image. Here, we would be better off sacrificing the resolution a little by narrowing the beam diameter which increases the number of photons passing through the pupil and reaching the focus position [3]. With the variable beam expander (VBE) from Leica Microsystems, we can look deeper into microscopic objects. The user can tune the beam to attain the deepest insight and finest detail (Figure 1).

An inherent benefit of exciting more than one type of fluorochrome by one multiphoton excitation line is that we can then know that all emitted photons are from the same location in the sample, no matter the color of the emitted light. However, for the more complex case of excitation with multiple colors, this condition is not guaranteed. Different infrared wavelengths are usually focused onto different positions when ordinary lenses are employed. To compensate for this effect and bring the excitation planes to the same position, we can introduce an appropriate divergence in the initial parallel laser beam. The optimal imaging solution would have all IR-laser ports equipped with an option for tuning the divergence. Such an option would ensure that all excitations coincide in the same focal plane and, hence, that all emissions radiate from the same plane, as well (Figure 2).

The variable beam expander, VBE, from Leica Microsystems offers a series of up to four laser ports, all equipped with both tunable beam expansion and divergence [4]

Spectral Non-Descanning Detection

Because the optical section in a multiphoton microscope is inherently generated by non-linear excitation, the beam path does not need a pinhole. Consequently, the emission from a scanned illumination spot must not necessarily pass through a descanning module (the scanning mirrors) to form a static beam, but can be collected directly from the sample. The signal is stored into the computer’s memory at all times with accurate x, y, and z values, as fluorescence emission can only radiate from the illuminated spot whose position is known. It is common to use “non-descanned” detectors, i.e., sensor devices that can be mounted directly under the sample (transmitted light non-descanned detector) or above the sample (reflected light non-descanned detector). These non-descanned detectors allow significantly more emitted light to be collected compared to the descanned path.

As always, this device must also perform for multiparameter fluorescence. Therefore, for that case, it is required to combine a number of sensors and associate the necessary elements in order to guide the desired fractions of the emission spectrum towards the sensors. Classically, these elements are beam splitting mirrors for separating the spectral bands (secondary beam splitters). For further refinement of the spectral range with the intent to avoid cross talk, glass barrier filters are mounted in front of each sensor. In the classical case, the filters and splitters have fixed spectral characteristics and need to be changed manually.

For truly professional operation, we wish to have a device that allows unrestricted, stepless adjustment of each spectral band, even during image acquisition. This adjustment should also be servo-controlled, without the need to manually remount any parts. It should be operated in the same way as the famous SP detector from Leica Microsystems. Just define a spectral band using the graphical interface and the instrument makes sure you get the desired signal with the highest efficiency and minimum of crosstalk, of course, for all four channels. The spectral non-descanned detector, 4Tune, by Leica Microsystems [5] performs free band selection for up to four non-descanning detection channels (Figure 3). It comprises an ingenious way to avoid spectral changes from the scan procedure [6]. 4Tune can easily adapt to any fluorochrome emission in the visible and near IR wavelengths. It replaces manually convertible filters and dichroic mirrors with fixed specifications.

And Speed Too

Last, but not least, speed matters when imaging a living sample. As we, meanwhile, have fluorescent sensors for many ions and biological metabolites, the fast dynamics in living objects become accessible. A resonant scanner producing 40 full frames per second is already satisfying most demands, still reducing the frame size will allow nearly 10 times faster imaging.

A long list of challenges and requirements. Leica Microsystems offers a versatile solution with the TCS SP8 multiphoton microscope and its features described above.