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Species specific depth profiling for in situ 3D imaging – InFeRa

Published: 25 March 2021

The project InFeRa has been ongoing since 1 of January 2019 and is founded by the Swedish foundation for Strategic Research (SSF). InFeRa, a new imaging method, combines spatially gated InterFerometric imaging (InFe) with stimulated Raman scattering (Ra). InFeRa will provide label free, three dimensional (3D) in situ imaging of chemical and (bio)molecular processes in different materials.

The breakthrough we foresee within the duration of the project is the design of a single instrument delivering:

  • Simultaneous visualization of embedded structures and formations/deformations in 3D.
  • Information of biochemical processes.
  • Information on generation and abundance of specific species.
  • Information of structural changes caused by specific species.

A schematic of the optical principle of InFeRa is shown in Fig. 1.

Fig. 1) Sketch of principle for the proposed instrument showing the pump laser source (PLS), beamsplitters (BS), a spatial light modulator (SLM), the imaging light source (ILS), a dichroic mirror (DM), imaging lens and a piezo electric mirror (PZT). The components that are used for SRS (green square) and interferometric imaging (red square) have been highlighted.

In this project, the system will be tested on bioelectrochemical systems (BESs): BESs use microbes to catalyze different electrochemical reactions for diverse purposes. Experiments on BES of the bacterium Geobacter sulfurreducens have been performed to investigate direct electron transfer between the cathode and the bacteria. This research is in collaboration with Biochemical Process Engineering at the Department of Civil, Environmental and Natural Resources Engineering, see:

The Raman lines of cytochrome c, an electron donor protein, have been identified [1,2]. Control and characterization of 3D biofilm formation and the action of mitochondrial biomarkers will finally be performed by InFeRa.

Currently (spring 2021) we are working separately on the development of interferometric imaging and stimulated Raman scattering (SRS) combined with a spatial light modulator (SLM). Upcoming sections contains preliminary results of both parts.

Depth-resolved Speckle Correlation with Quasi-Incoherent interferometric imaging

The interferometric imaging part of InFeRa is depicted by the dashed red square in Fig. 1.  Fig. 2 shows a prototype setup that applies a rotating diffusor to the emitted light of an He-Ne laser, to generate an incoherent light source. Fig. 3 shows the effects of depth-gating, by having correlated the speckle pattern in the reference arm with that of the object arm at varying optical path differences. Fig. 4 shows an example of image reconstruction using phase shifting.

Fig. 2) Prototype of coherence gated interferometry system. A rotating diffusor was used to shorten the coherence length of the incoming light from a He-Ne laser. The object was imaged (magnification = 2.5) through a Michelson-interferometric scheme using an objective (Component-S 5.6/100, Schneider Kreuznach, Germany) and a digital camera (Genie Nano M2420, Teledyne Dalsa, Canada). One of the arms of the interferometer was attached to a piezo-electric element that enabled image reconstruction through phase-shifting (Component-S 5.6/100, Schneider Kreuznach, Germany).
Fig.3) Depth-resolved correlation map across the field-of-view. The correlation of the speckle patterns in object and reference arm depends upon the difference in optical path length. As the reference arm is displaced to match the optical path distance of the object arm the correlation is maximized. Where this occur, coherent information can be extracted.
Fig. 4) Image reconstruction from four phase shifted images. I1 – I4 (left-hand-side) shows the raw images of the highlighted measured area (top, right-hand-side), each consecutive image has been phase shifted by π\/2. The result after reconstruction shows that the information in the measured area can be extracted from the raw images (bottom, right-hand-side).

Spatial Control of Stimulated Raman Scattering using a Spatial light Modulator

The SRS process will only take place if two beams overlap in a material that has a Raman shift equal to the difference in wavelength of the two beams. In Fig. 5 a sketch of the current experimental setup is seen. The CH2 asymmetric stretching ethanol Raman band at 2934cm -1 is investigated. This part is included in the dashed green square of Fig. 1 that shows a prototype of the InFeRa setup. Fig. 6 illustrates how the SLM can be used to probe the sample at different locations in the glass cuvette and Fig. 7 shows the results obtained by imaging the SRS signal due to such motion.

Fig. 5) A schematic of the optical system. A Q-switched 1064nm Nd:YAG (Continuum PL 8000) was used to produce both the pump and Stokes beam. The frequency doubled532nm pump beam (green dotted lines) was guided through a rotator and two thin film polarizers (1) for seamless control of the light intensity. The pump light was expanded 3x by a Galilean telescope (2) to cover the active area of the SLM head 3a) (Hamamatzu X10468-04 LCOS-SLM). The SLM was connected to a control module, (3b), coupled to a computer (3c). The phase modulated light was focused into the sample with a 250mm lens (4). The frequency tripled 355nm Stokes light was guided into an OPO system (Continuum Sunlite EX OPO) and tuned to 630.45nm. The Stokes beam was expended 16x by a Galilean telescope (6) to then pass through a beam splitter (5) to a glass cuvette (35×70×180mm3) containing 99.7% ethanol (Solveco AB, Rosberg, Sweden) (7). The overlap of the beams in the sample generated a SRS signal that was guided through a 250mm lens (9) towards a PCO edge camera (11). To remove the pump light from the signal image a 532nm mirror (8) and two edge filters (10) were used. In front of the camera a neutral density filter OD 2.0, a RG 630 filter and a BG39 filter were mounted to improve the signal to noise ratio.
Fig. 6) The Stokes beam, red tinted area, is illuminating a larger area inside the sample depicted by a black square. The pump beam, green dotted line, is focused into the sample to coincide with the Stokes beam. A) The pump light is focused on the plane P1 centered at the z axis, B) the pump light is phase modulated and focused on the plane P1 at a different position, C) the pump light is phase modulated and focused in the plane P2 while centered at the z axis.
Fig. 7) A) The SRS gain (red dot), B) the SRS gain after a vertical movement (down) of the pump beam, C) the SRS gain after a horizontal movement (right) of the pump beam. The field of view is 10×8.4mm2.

Future plans

In the near future (fall 2021) the two imaging parts will be combined. First measurements will be carried out on polymer beads on a glass surface. After that, first experiments on BES will be performed.


This project is financially supported by the Swedish Foundation for Strategic Research (ITM17-0056) the Kempe Foundation and LTUs lab fund.


Mikael Sjödahl
Kerstin Ramser


[1]: Krige, A., et al., On-Line Raman Spectroscopic Study of Cytochromes’ Redox State of Biofilms in Microbial Fuel Cells.     Molecules, 2019. 24(3): p. 646.

[2]: Krige, A., et al., A New Approach for Evaluating Electron Transfer Dynamics by Using In Situ Resonance Raman Microscopy and Chronoamperometry in Conjunction with a Dynamic Model. Applied and Environmental Microbiology, 2020. 86(20).