Programmable Microscopy

 By introducing high-resolution light modulators into the imaging and/or illumination path of a conventional light microscope it is possible to considerably extend the imaging capabilities of the system. We apply this technique for the implementation of various imaging techniques and the overall improvement of the imaging quality.
 

 The general principle of using a spatial light modulator in microscopy was originally used in optical micromanipulation (holographic optical tweezers) and holographic micro-ablation.
A high resolution phase-only spatial light modulator (SLM) is placed in a plane conjugate to the illumination and/or imaging pupil of the optical system. Typically, the exit pupil of the imaging microscope objective lens is not accessible and, therefore, typically an additional imaging stage (e.g. using a Kepler telescope) is used. We have build several microscopes with such elements. Our current system uses two LED illumination modules (trans- and epi-illumination) and one laser for structured illumination. Half of the employed SLM (Holoeye Pluto HDTV) is employed for imaging and the other half for illumination.

With such a system it becomes possible to freely program the pupils of the illumination an the imaging and currently we implemented the following imaging techniques:

• aberration correction
• phase contrast microscopy
• (Dark Field, Zernike, DIC, Spiral phase contrast, VDIC, WDIC, quantitative phase contrast)
• stereo microscopy
• depth scanning microscopy and simplified confocal microscopy

Of course, a lot of other methods can be implemented as well. One can easily switch by software between the different methods or combine them. Compared to a conventional microscope the user gains the advantage of very high flexibility and a very high imaging quality even when using cheap objective lenses. The parameters of the imaging methods can be changed in video-realtime and digital combination of images obtained with different parameter settings is possible to achieve improved visualization of specimen

 Fig. 3 shows a comparison of the resolution of our system when using aberration correction compared to a High-End Zeiss Ergoplan. The ….
 

In Fig. 4  an example of simple Zernike-type filtering with different settings of the Filter parameters is shown and Fig. 5 shows a digital combination of differential interference contrast filtering and Zernike filtering. The hue of the final image is based on the Zernike output (which is proportional to the phase) whereas the intensity is based on the DIC output which varies with the gradient of the phase and therefore shows small structures very effectively.
 

Fig. 6 shows an example of a stereovision anaglyph. The main idea is to write two spatially separated gratings into the SLM (see Fig. 7). Thereby a pupil seperation is achieved.

 We think that this sort of programmable SLM-based microscopy is the future of research microscopy because the user has the full flexibility of changing all kind of filter parameters which are normally fixed in a conventional microscope and it is possible to easily switch between different imaging methods in video-realtime. Due to the aberration correction very good imaging quality can be achieved.  The fast method of operation allows one to digitally combine images obtained with different methods or parameters. Also, programmable microscopy is perfect for testing new imaging techniques.
On the downside, there is some loss of light due to the additional modulation by the SLM. For the imaging path it is about 50% and this is a limitation for low-light fluorescence applications. If the full field of the microscope is desired, two images have to be recorded and digitally combined.