A. Basic concepts for Optical Microscopy: Magnification
- Magnification is the ratio of the size of an object seen under the microscope to the actual size observed with the naked eye or camera.
- The total magnification of a microscope is calculated by multiplying the magnifying power of the objective lens and the eyepiece (for naked-eye observation) or the camera tube (for camera or detector).
- In other words, magnification is also defined as the ratio between the image size and the object size. Although the wording is different from the previous definition, the resulted magnification must be the same.
- Understanding the concept of magnification is important when using a camera or detector to capture images from the specimen, as the imaging area size is greatly dependent on the magnification (and the CMOS of camera).
- In the current Calcium imaging setup, the camera tube and objective installed in fluorescence microscopy are WFA4102 and TL2X-SAP respectively. The total magnification would be 1X.
B. Determination of Imaging Area
- The imaging area is the dimension of area that capturing images by the camera with the highest resolution (i.e. without applying Region of Interests ROI).
- Several parameters have to be considered in order to determine the imaging area size, including the total magnification of microscope and camera sensor size. Mathematically, the size of the imaging area could be calculated as:
- The specification of the camera sensor is an important factor in Fluorescence microscopy and Imaging experiment. It determines the image resolution (Effective number of pixels), original imaging area size, max frame rate, and shutter type, etc.
- In the Calcium imaging setup, the camera installed in fluorescence microscopy is CS2100M-USB, which has the following specifications:
| Sensor Type: | Monochrome sCMOS |
| Maximum Image Resolution (Effective number of Pixels): | 1920 x 1080 |
| Original Imaging Area Size: | 9.6768 mm x 5.4432 mm |
| Maximum Frame rate at Full sensor: | 50 fps |
| Sensor Shutter Type: | Rolling |
- Currently, we have two camera tubes and two objectives in the Ephys laboratory, providing valuable total magnification and imaging area size. Detail information would be shown in the table below (Blod for current setup in Fluorescence microscopy):
| Camera Tube (Magnification) | Objective (Magnification) | Total Magnification | Actual Imaging Size |
| WFA4102 (0.5X) | TL2X-SAP (2.0X) | 1.0X | 9.6768 mm x 5.4432 mm |
| WFA4100 (1.0X) | TL2X-SAP (2.0X) | 2.0X | 4.8384 mm x 2.7216 mm |
| WFA4102 (0.5X) | MY5X-802 (5.0X) | 2.5X | 3.8707 mm x 2.1773 mm |
| WFA4100 (1.0X) | MY5X-802 (5.0X) | 5.0X | 1.9354 mm x 1.0886 mm |
- Since the size of the rat auditory cortex is roughly 5mm x 5mm, the combination of WFA4102 and TL2X-SAP (together with the ROIs setting) could well-cover the whole auditory cortex region for imaging. The detailed information on Region of Interest (ROIs) would be introduced in the Software Design chapter.