TILL Two-Photon iMIC

It is easy to combine imaging modalities in the Two-Photon iMIC!

The iMIC architecture with its stacked layers that each have several identical optical ports lends itself to incorporating any number of novel techniques. In the Two-Colour variant of the Two-Photon iMIC, which has three iMIC layers, only 5 of 9 ports are used (for epi-illumination condenser, camera, laser scanner, and the two detector modules). Each of the four open ports can be used >>> for an additional camera, a laser scanner, a point detector, or an additional light source. The laser scanning software Colibri is based on LabVIEW, therefore controlling custom hardware from within the microscope software is easy.

We encourage and support customers who want to integate novel extensions. To inspire your creativity, here are two example applications that the Two-Photon iMIC makes possible:

Neurons in a mouse brain preparation were bleached in a FRAP-like experiment. This stack reveals how thin the belached regin is within the 3D volume. The stack comprises a volume of 80x80x37µm. Bleaching was performed in an area of 20x20µm in a single optical section.

3D-uncaging / 3D-FRAP / 3D-photo-activation One very promising new application for two-photon microscopes is 3D-uncaging / 3D-FRAP / 3D-photo-activation. "3D" implies that one can achieve tremendously increased spatial definition in the 3D volume when pulsed IR laser light is used for photo-interaction-techniques like uncaging, FRAP, or photo-activation instead of UV flashes or visible lasers. The common principle in uncaging, FRAP, and photo-activation experiments is that light physically interacts and changes molecules within a sample. After locally defined irradiation, a time-lapse recording is done to visualize either the action of the uncaged agents, or the diffusion of unbleached dye molecules into the bleached region in a FRAP experiment, or the diffusion or cell migration in case of activated fluorescent particles.

In all photo-interaction-techniques, defining the target area for irradiation is critical. Lasers and laser scanners or UV flashes and masks are used to irradiate custom shapes in the sample plane. This works well in 2D; and 2D is good enough for single cells or other flat samples. In thick samples, however, the lack of definition along the depth axis becomes obvious. The whole column of tissue above and below the focal plane is uncaged/FRAPped/activated. This lack of definition in depth prohibits meaningful photo-interaction experiments in thicker samples, like tissues or live animals.

By uncaging/FRAPping/activating via two-photon absorption of pulsed IR light, one can achieve for the first time real depth definition of the desired effect. Observation of the effect can be done as before, by wide field camera imaging or spinning disc confocal imaging during/after IR irradiation.

The impact of this new dimension for the established light-interaction techniques is not entirely clear; it might be the next big thing! While it is still on the way to breakthrough, you can already buy the required hardware at TILL.

Two-photon-polarisation microscopy 2PPM

Polarisation microscopy is not new. Polarizers have been used for a hundred years in material microscopes for generating meaningful contrast in crystals and other double-refracting non-animate matter. Using polarisation filters for the detection of orientation of fluorescent biomarkers, however, is a rather new technique which has been hampered by the necessity to mechanically turn polarizers and analysers, and low contrast between the pairs of images created with different polarisation.

In the 2PPM iMIC, alignment of dye molecules is detected by contrasting successive pixel-wise measurements obtained with alternating polarisation of pulsed IR laser light. Each polarisation angle of the excitation light leads to selective excitation of dye molecules that are arranged with a certain angle relative to the light polarisation. In the 2PPM iMIC we turn the polarisation between acquisition of individual pixels with a Pockel's cell. All even pixels are illuminated with horizontally polarized light, all odd pixels are illuminated with vertically polarized light. This has a number of advantages over conventional methods in which polarisation is changed only between successive image acquisitions. We will dwell on this further down. Resulting images look like this:

GFP crystal needles imaged with alternating vertical and horizontal polarisation. Please note the column-structure of the image.

By separating the image column-wise into two images and calculating the ratio for each pixel, one can obtain a false-color image that reveals the polarisation of dye molecules (horizontal: red, vertical: green).

In the example images, even pixels were illuminated with horizontally polarized light, all odd pixels were illuminated with vertically polarized light. Depending on the prevalent orientation of dye molecules, either even or odd pixels are brighter. The example image shows needles of crystalline GFP, which is anisotropic. The dye molecules are oriented in parallel in the crystal, so there is a very strong anisotropy of the crystal needles. The brightness difference between neighbouring columns of pixels is easy to see also in the gray image. By calculating the brightness ratio from pairs of neighbouring pixels, one can produce false colour images which code for molecule orientation.

Time lapse movie showing the transmitter-evoked dissociation and re-association of a G-protein complex. Red and green colour indicate the trimeric state, yellow indicates the dissociated state. Total length of the video was 3.5 minutes.

Application of 2PPM Orientation of dye molecules in biological tissues can be probed in the same way. Dye molecules with fixed relation to the cytoskeleton or the cell membrane generate polarisation contrast. Lipidated GFP, or GFP bound to membrane-proteins are examples. Orientation of dye molecules can depend on the physiological state of the cell: in the example given, GFP is rigidly bound to a sub-unit of a G-protein complex. The protein sub-unit with the dye is fixed whenever the trimeric protein complex is complete. Now a stron polarisation contrast can be observed (movie at the left, saturated red and green show strong polarisation contrast). Dissociation leads to a dramatic decrease of the 2PPM signal (yellow colour). The image sequence, based on a time-lapse recording over three minutes, shows how a G-protein complex breaks up upon stimulation of the cell, and re-establishes later. Until now, such measurements would have required demanding genetics and a FRET measurement: one would express two separate dyes attached to two of the G-protein sub-units. The dyes would form a FRET-pair once the complex is formed. Compared to 2PPM, the resulting signal-to-noise would much lower than what we achieved easily with 2PPM [Lazar, Firestein et a., Nature Methods 2011].

If you want to learn more about two-photon polarization microscopy or if you want to find out if your experiments will benefit from it, please have a look at the partner sites from Innovative Bioimaging.