Molecular contrast on conventional microscope
Image: Molecular-contrast image of HeLa cells. [Takuro Ideguchi]
Japan-based researchers have unveiled a 'molecular-contrast unit' that allows standard microscopes to image where specific molecules are within a cell by searching for the vibration or heat signal of that molecule.
The attachable unit contains an amplitude-modulated mid-infrared laser that excites target molecules, causing them to vibrate - different molecules can be selected by using different infrared wavelengths.
As Professor Takuro Ideguchi from the University of Tokyo Institute for Photon Science and Technology and colleagues highlight, no specialised preparation of the cells, such as labelling with fluorescent dyes, is required when imaging with the unit.
“We believe the concept of upgrading existing, widespread standard optical microscopes to become molecular-sensitive will expand the research capabilities of various end users," says Ideguchi. "Also, since our method translates the local heating of the sample to the molecular contrast, it could also serve as a tool to probe the local thermal parameters within biological cells.”
While a benchtop microscope platform, equipped with say bright-field and phase-contrast modes, has proven critical to many researchers, such a set-up cannot acquire molecular contrast in a label-free manner.
With this in mind, Ideguchi and colleagues developed a simple add-on optical unit, to attach to a standard microscope platform.
The unit delivers the additional molecular contrast of the specimen on top of its conventional microscopic image, based on the principle of photothermal effect.
Image of two human cells was made by combining the molecular density information obtained by the molecular-contrast unit (MC, yellow) and the image taken by the conventional phase-contrast portion of a standard light microscope (PC, greyscale). [Takuro Ideguchi]
As the researchers write in Nature Scientific Reports, the amplitude-modulated mid-infrared beam is weakly focused onto the sample placed at the objective focus to cover the entire region of the specimen.
“This induces the molecular-vibrational photothermal effect at specific sites within the specimen where vibrationally resonant molecules exist, which is detected as temporal intensity-modulations in the time-series of the recorded phase contrast images,” says Ideguchi. “These photothermal signatures are computationally extracted to reveal and add the resonant molecular contrast to conventional phase contrast microscope images.”
The researchers went on to demonstrate high-speed label-free molecular-contrast phase-contrast imaging of silica-polystyrene microbeads mixture and molecular-vibrational spectroscopic imaging of HeLa cells.
"We were pleased to visualise the protein distribution in biological cells,” says Ideguchi. “The protein seemed to concentrate around the cells’ nuclei, which could represent the existence of some intracellular structures [involved in the synthesis and transport of proteins], such as the endoplasmic reticulum and Golgi apparatus.”
Research is published in Scientific Reports.