Imaging Biological Samples with Optical and Confocal Microscopy

Overview

Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut

Optical microscopes have been around for centuries, and while they reached their theoretical limitation of resolution decades ago, new equipment and techniques, such as confocal and digital image processing, have created new niches within the field of optical imaging. The best optical microscopes will typically have a resolution down to 200 nm in ideal conditions. However, optical microscopes are limited by the diffraction of waves, a function of the wavelength, which is around 500 nm for visible light. While the resolution of optical microscopes does not reach that of electron microscopes, they are the most valuable tools in the imaging of biological macrostructures and are a staple in any biological lab.

In conventional light microscopes, the signal produced from the imaged object is from the full thickness of the specimen, which does not allow most of it to be in focus to the observer. This causes the image to have "out of focus blur". The confocal microscope, on the other hand, illuminates the sample through a pin-hole, and is therefore able to filter out the out-of-focus light from above and below the point of focus in the object.

This demonstration provides an introduction to image acquisition using optical and confocal microscopy methods. Here, a sectioned piece of mouse brain will be studied.  Image acquisition and analysis, including the tools to generate topographical maps and composite images, will be covered. The advantages and disadvantages of different imaging methods as they relate to resolution, depth of focus and sample type will also be discussed. The purpose of this demonstration is to provide more information on optical and confocal microscopes to determine if these microscopy modules are the best fit for a type of biological sample.

Procedure

1. Confocal Imaging

  1. Load the sample onto the stage. Center it beneath the lens. It should not exceed the weight limitation of the stage, which in this case is 5 kg. The sample should not be more than 100 mm thick.
  2. Open the imaging software and select "Create Job."
  3. Under the Topographies Column, choose the assistant button.
  4. Create an overview image at the lowest magnification, 2.5X. Before switching the magnifications, ensure that the sample is in focus by changin

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Results

The following images give an overview of results that can be obtained of a mouse brain using a confocal microscope. They show how varying levels of information can be obtained and how a topographical map of the results reveals the height of the sample.

Figure 5
Figure 5: Confocal images at

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Application and Summary

In this demonstration, the depth of focus, field of view and maximum resolution and magnification of optical and confocal microscopes were optimized to view biological samples. This demonstration was designed to help the participant decide which microscopy module is the best for a certain application. Both modes of microscopy have advantages in analyzing biological samples for their ease of preparation and high resolution composite images.

The applications for optical and confocal microscopy a

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Tags
MicroscopyOptical MicroscopyConfocal MicroscopyBiological SamplesImagingDetailed StructureSamplesLensesMagnifyCompound MicroscopeAntonie Van LeeuwenhoekBacteriaYeastRed Blood CellsCirculationCapillary VesselsScientific ContributionsMicroscopic AdvancementsResearchClinical SettingsMedical DiagnosisPinholeOptical ResolutionContrastOperating PrinciplesHigh Resolution ImagesAnalysisApplicationsBiomedical Engineering

跳至...

0:07

Overview

1:26

Principles of Confocal Microscopy

3:37

Confocal Imaging

6:09

Digital Optical Microscope

7:56

Results

9:05

Applications

10:16

Summary

此集合中的视频:

article

Now Playing

Imaging Biological Samples with Optical and Confocal Microscopy

Biomedical Engineering

35.5K Views

article

生物样品的SEM成像

Biomedical Engineering

23.4K Views

article

纳米药物载体的生物分布:SEM的应用

Biomedical Engineering

9.2K Views

article

腹大塔的高频超声成像

Biomedical Engineering

14.2K Views

article

腹部主动脉瘤的定量应变图谱

Biomedical Engineering

4.6K Views

article

红外主塔中图像血液和脂质的光声断层扫描

Biomedical Engineering

5.6K Views

article

心脏磁共振成像

Biomedical Engineering

14.6K Views

article

脑动脉瘤血流的计算流体动力学模拟

Biomedical Engineering

11.6K Views

article

腹主动脉瘤的近红外荧光成像

Biomedical Engineering

8.2K Views

article

非侵入性血压测量技术

Biomedical Engineering

11.7K Views

article

心电图(心电图)信号的采集与分析

Biomedical Engineering

103.1K Views

article

可吸收生物材料的拉伸强度

Biomedical Engineering

7.4K Views

article

小鼠脊髓的微CT成像

Biomedical Engineering

7.9K Views

article

大鼠非侵入性ACL损伤后膝关节退化的可视化

Biomedical Engineering

8.1K Views

article

结合 SPECT 和 CT 成像,使心脏功能可视化

Biomedical Engineering

10.9K Views

JoVE Logo

政策

使用条款

隐私

科研

教育

关于 JoVE

版权所属 © 2025 MyJoVE 公司版权所有,本公司不涉及任何医疗业务和医疗服务。