# Multi-mode Microscopic Hyperspectral Imager for the Sensing of Biological Samples
## 2 Materials and Methods
### Epi-illumination and Trans-illumination  
Under the previous illumination pattern (lateral illumination), an objective with high magnification and short working distance (sub-millimeter level) may cause some ununiformed illumination or possible shadows covering of the focal plane [15]. Figure 2a describes the generation of some shadows, from which we can see part of the lateral illumination light is blocked by the objective or sample, causing the shadow in the FOV. In addition, the illumination intensity decreases gradually with the propagation distance of light, which also leads to ununiformed illumination of the sample plane. These drawbacks will affect the imaging quality and detection efficiency. To overcome the shadow issue, two illumination patterns were proposed to make the illumination and detection coaxial, while ensuring the uniformity of illumination within the FOV. Generally speaking, epi-illumination is sensitive to the scattered light and fluorescence from the surface [16], and thus is suitable for a reflection or fluorescence detection mode of opaque specimen, while trans-illumination is suitable for a transmission detection mode of translucent or transparent samples [17].  
In the epi-illumination pattern, the light source is coupled to a single-mode optical fiber and is outputted to an air-spaced doublet collimator (F810 SMA-780, Thorlabs, Newton, NJ, USA). The collimated light firstly passes through a doublet lens (f = 50 mm, DHC, Beijing, China), then is deflected by a beam splitter (BSX10R, Thorlabs, Newton, NJ, USA), and finally converges onto the back focal plane of the objective. After passing through the objective, a parallel light can be generated since the light source is emitted from the back focal point, and consequently uniformly-illuminated condition can be achieved in the focal plane of the objective. The reflected/scattered/fluorescence signal can be captured by the same objective and imaged onto the CMOS. A long pass filter (FELH0450, Thorlabs, Newton, NJ, USA) is adopted to eliminate the excited light when choosing the fluorescence detection mode. By selecting a broadband light source or blue-violet laser source, the reflection or fluorescence mode can be realized, respectively.  
In contrast to epi-illumination, the trans-illumination structure is relatively simple. A collimated light (uniformly-illuminated light) is incident from below and captured by the imager above after  
Figure 2: (**a**) Schematic diagram of shadow generation under the lateral illumination condition. (**b**) Illustration of multi-mode detections. Reflection mode and fluorescence mode use epi-illumination, while transmission mode employs trans-illumination. Schematic diagram of each component: 1. Single mode fiber, 2. Fiber collimator, 3. Doublet lens, 4. Beam splitter, 5. Long pass filter, 6. Objective, 7. Sample and motion stage, 8. Infinity-corrected hyperspectral imaging system, consisting of the imaging module (including the imaging lens and slit), the diffraction module (including the doublet lens and PGP structure) and the detection module (including the tube lens and CMOS).  
penetrating the sample. Thus, transmission imaging can be realized and the sample absorption characteristics can be obtained at the same time.  
By switching among different illumination patterns, multi-mode detection can be achieved for different samples and applications.