Abstract
We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (<15 mm in diameter). In this paper we describe the hardware and software modifications to TSLIM that allow for static and uniform light-sheet illumination with SI and HiLo image demodulation. The static light-sheet has a thickness of 3.2 µm; whereas, the scanned side has a light-sheet thickness of 4.2 µm. The scanned side images specimens with subcellular resolution (<1 µm lateral and <4 µm axial resolution) with a size up to 15 mm. SI and HiLo produce superior contrast compared to both the uniform static and scanned light-sheets. HiLo contrast was greater than SI and is faster and more robust than SI because as it produces images in two-thirds of the time and exhibits fewer intensity streaking artifacts.
Highlights
The thin-sheet laser imaging microscope (TSLIM) is a light-sheet microscope that was developed for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear and rat and mouse brain [1]
TSLIM was designed as a dual beam, light-sheet microscope with air mounted illuminating and observing objectives so that large specimens can be imaged
While the lateral resolution is mainly governed by the Abbe diffraction limit and the numerical aperture (NA) of the detection objective, the axial resolution is mainly determined by the thickness of the light-sheet
Summary
The thin-sheet laser imaging microscope (TSLIM) is a light-sheet microscope that was developed for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear and rat and mouse brain [1]. TSLIM was designed as a dual beam, light-sheet microscope with air mounted illuminating and observing objectives so that large specimens can be imaged. It can image transparent live specimens, it was optimized for observing fixed tissue that has been fluorophore labeled and chemically cleared to transparency. While the lateral resolution is mainly governed by the Abbe diffraction limit and the numerical aperture (NA) of the detection objective, the axial resolution is mainly determined by the thickness of the light-sheet. That is the reason why investigators like Neil [2] and Mertz [3] developed techniques using pattern illumination for enhancing image contrast and axial resolution independent of light-sheet thickness. In this paper we describe hardware and software modifications to TSLIM for a scanned light-sheet and patterned illumination. sTSLIM’s scanned laser side is similar to a digital scanned laser light-sheet fluorescence microscope [4], or DSLM with SI [5] but exhibits important differences such as air mounted objectives, the use of a single galvanometer mirror and the addition of HiLo modulation
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