# Multi-mode Microscopic Hyperspectral Imager for the Sensing of Biological Samples
## 1 Introduction  
The appearance of microscopy opens the door for humans to explore the micro world, and spectral analysis provides the intrinsic absorption or fingerprint characteristics of specific substances [1]. Recently, the combination of hyperspectral technology and optical microscopic imaging in bio-sensing has been widely used in tumor cell detection [2], food detection [3], zooplankton classification [4] and other applications [5]. In these applications, the separation of a microscopic imaging module and a hyperspectral detection module may easily cause the detection errors and increase the cost of the system. Hyperspectral imaging (HSI) technology, with the advantage of acquiring two-dimensional image and corresponding spectrum of each pixel, has strong potential for, e.g., on-site monitoring, clinic diagnose and biomedicine [6; 7; 8]. Through single scanning, a hyperspectral cube containing both spatial and spectral information can be obtained. The spectral information can be used in point-to-point spectral analysis for interested regions in the 2D hyperspectral spatial image.  
At present, HSI technology is widely used in some remote sensing area, which is a macroscopic observation. Most of the commercial imaging spectrometers work in a single detection mode, e.g., collecting the reflection or transmission signal from the sample [9; 10], which greatly limits their ability to test a variety of samples. Microscopic applications are mainly limited by the detectionefficiency, spatial and spectral resolutions of the HSI system. Some experiments on cancerous tissues and tumor detection [2,8] show spatial and spectral resolutions of 30 \(\upmu\)m and 5 nm, respectively. Moreover, the dyeing steps for some samples also increase the complexity of experiments in microscopic applications.  
In this work, we present a multi-mode microscopic hyperspectral imager (MMHI) with 5 \(\upmu\)m spatial resolution and 3 nm spectral resolution that works in the transmission mode, reflection mode and fluorescence mode. To avoid possible shadows produced by the short working distance of a high magnification objective, epi-illumination [11] and trans-illumination [12] patterns are designed to ensure the co-axiality between the illumination and detection. By switching the illumination pattern or light source, an appropriate detection mode can be selected for samples with different characteristics. Here, three experimental detections on zebrafish (translucent sample), human fingerprint (opaque sample) and disaster-causing microalgae (fluorescence samples) were performed to verify the system's capability. Due to the realization of uniform-illumination of the whole focal plane, it is easy to exchange objectives with different magnification, meeting the demands of the corresponding resolution, amplification or field-of-view (FOV). As implemented, the imager has the advantages of low cost, high accuracy and good sensitivity, multiple application scenarios, and has the potential for on-site micro environment monitoring and clinical applications.  
The structure of this paper is as follows: Section 1 provides a brief introduction to the background of the HSI technology and the motivations for the MMHI system. Section 2 describes the construction and calibration of the MMHI system in detail, illustrates the shadow issue and demonstrates two illumination patterns to overcome the shadow issue. Section 3 introduces three experiments under different detection modes, to show the capability of the MMHI system. Section 4 summarizes the present work, and provides future prospects and the possible improvement of the MMHI system.