Light Sheet Microscopy

  • Speaker:             Malte Wachsmuth
  • Department:     Cell biology 
  • Location:            Erasmus MC
  • Date:                    23-02-2017
  • Author:               Katja Slangewal

Light-sheet microscopy is a microscopy technique optimized by a start-up company called Luxendo (part of EMBL). This technique is special because it enables live cell imaging for an increased amount of time with a resolution close to confocal resolution. The main idea behind light-sheet microscopy is the uncoupling of the illumination and detection light paths. The illuminating beam illuminates a thin sheet of the sample (2-8μm), which is in the focal plane of the detection lens. This enables full area detection with a single light sheet (figure 1, right). This creates an advantage over confocal microscopes, since they use point-wise raster scanning. This leads to the illumination of a quite large part of the sample over time (figure 1, left). So, light-sheet microscopy reduces the amount of light needed to image your sample. This reduces the photobleaching of your samples. Also, by imaging with a single light-sheet is becomes possible to capture your sample in one single shot. This makes light-sheet imaging ideal for the imaging of dynamic processes in live specimen. One disadvantage or possible problem could be the intensity drop of the illuminating beam. The amount of intensity drop is dependent on your sample and labeling, so it might not be a problem in most cases. However, there is a way to reduce the intensity drop. By using dual illumination (simultaneously from the front and back of the sample), the intensity drop can be reduced.

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Figure 1: Light-sheet microscopy (right) versus confocal microscopy (left). Light-sheet microscopy drastically reduces the amount of illumination of your sample, thereby decreasing photobleaching. Source: http://luxendo.eu/

The MuVi-SPIM (Multiview selective-plane illumination microscope) is one of the microscopes using light-sheet microscopy. The MuVi-SPIM is based on four objective lenses. Two lenses are used for illumination of the sample and two lenses are used for the detection of the fluorescent signal. The four lenses give four different views on your sample, which can be fused to one optimized image (figure 2). This way you can image larger living specimen without rotating them, thereby reducing the acquisition time.

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Figure 2: The MuVi-SPIM: by using four objective lenses and adding the four separate images an optimal image can be formed, thereby reducing noise and increasing the signal. Source: http://luxendo.eu/

Besides the MuVi-SPIM there are more possible geometries which are able to use the principles of light-sheet microscopy. One example is the InVi-SPIM (inverted view selective-plane illumination microscope). This geometry uses only two objective lenses, one for illumination and one for detection (figure 3). The InVi-SPIM has been developed for long-term 3D imaging of living specimen. It has an inverted microscope configuration, which makes it easier to access the sample chamber. According to Wachsmuth, your specimen will be able to stay alive for approximately 2 days in the chamber. The InVi-SPIM is for instance very useful for stem cell differentiation assays. This because of the lower amount of photobleaching compared to a confocal microscope, but with similar resolution. A small disadvantage is the inability to use dual illumination (illuminating your sample from the front and back simultaneously).

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Figure 3: The InVi-SPIM enables the imaging of living specimen for longer periods of time. Source: http://luxendo.eu/invi-spim

Light-sheet microscopy makes it possible to image two different parts of a sample at the same time. One could for instance image both the heart of a zebrafish and its blood flow in the tail. This is not possible with a confocal microscope, since the confocal microscope needs time to image by point-wise raster scanning. This together with the lower amount of photobleaching and higher imaging speed makes light-sheet microscopy a promising microscopy technique.

This seminar was different from the other seminars I have visited so far. This was mainly because Malte Wachsmuth was representing a company instead of a research group. This gave the talk a bit the appearance of a sales pitch. However, it was still very interesting. I hadn’t heard about light sheet microscopy before and I think it sounds like an interesting technique. Malte was a very enthusiastic speaker and he had a very clear talk.

 

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