Lenslet array tunable snapshot imaging spectrometer (LATIS) for hyperspectral fluorescence microscopy.

Jason G Dwight,Tomasz S Tkaczyk

doi:10.1364/boe.8.001950

Abstract

Snapshot hyperspectral imaging augments pixel dwell time and acquisition speeds over existing scanning systems, making it a powerful tool for fluorescence microscopy. While most snapshot systems contain fixed datacube parameters (x,y,λ), our novel snapshot system, called the lenslet array tunable snapshot imaging spectrometer (LATIS), demonstrates tuning its average spectral resolution from 22.66 nm (80x80x22) to 13.94 nm (88x88x46) over 485 to 660 nm. We also describe a fixed LATIS with a datacube of 200x200x27 for larger field-of-view (FOV) imaging. We report <1 sec exposure times and high resolution fluorescence imaging with minimal artifacts.

Highlights

Fluorescence microscopy has become widespread in its use in biology over the last few decades [1]
Spectral resolution improvement is obtained by increasing the focal length of the refocusing lens by a factor of 2 and the focal length of the collimating lens by a factor of 1.5, giving a 33% percent improvement in the spectral resolution while increasing the spectral sampling from 22 to 46 pixels
3 images are necessary for calibration after alignment at each tuning setting

Summary

Introduction

Fluorescence microscopy has become widespread in its use in biology over the last few decades [1]. Hyperspectral Fluorescence Microscopy (HFM) acquires many spectral bands of a scene to create hyperspectral datacubes (x,y,λ), called lambda stacks, and can provide high spectral resolution for demanding fluorescence imaging applications [8,9]. They were first employed in astronomy [16] and remote sensing [17] These spectral imagers can be designed to be advantageous over existing scanning spectrometers due to longer pixel dwell times, permitting their use in dim applications, and higher speeds, which eliminate motion artifacts for dynamic scenes [18]. IFS with lenslet arrays continues to be used in the field of astronomy [30,31,32] and remote sensing [33,34] This technique has seen limited use in microscopy. To the best of our knowledge, this is the first implementation of lenslet arrays as an IFS for biological samples and the first snapshot spectral imager capable of tuning spectral resolution

Integral field spectroscopy with lenslet arrays

Tunability principle of LATIS

Spectral accuracy of LATIS

Bead imaging experiments with LATIS

Findings

Discussion